1052 Citations
Menstrual toxic shock syndrome mTSS is a rare but severe disorder associated with the use of menstrual products such as high-absorbency tampons and is caused by Staphylococcus aureus strains that produce the toxic shock syndrome toxin- TSST- superantigen Herein we screened a library of small bioactive molecules for the ability to inhibit transcription of the TSST- gene without inhibiting growth of S aureus The dominant positive regulator of TSST- is the SaeRS two-component system TCS and we identified phenazopyridine hydrochloride PP-HCl that repressed production of TSST- by inhibiting the kinase function of SaeS PP-HCl competed with ATP for binding of ... More
Menstrual toxic shock syndrome (mTSS) is a rare but severe disorder associated with the use of menstrual products such as high-absorbency tampons and is caused by Staphylococcus aureus strains that produce the toxic shock syndrome toxin-1 (TSST-1) superantigen. Herein, we screened a library of 3920 small bioactive molecules for the ability to inhibit transcription of the TSST-1 gene without inhibiting growth of S. aureus. The dominant positive regulator of TSST-1 is the SaeRS two-component system (TCS), and we identified phenazopyridine hydrochloride (PP-HCl) that repressed production of TSST-1 by inhibiting the kinase function of SaeS. PP-HCl competed with ATP for binding of the kinase SaeS leading to decreased phosphorylation of SaeR and reduced expression of TSST-1 as well as several other secreted virulence factors known to be regulated by SaeRS. PP-HCl targets virulence of S. aureus, but it also decreases the impact of TSST-1 on human lymphocytes without affecting the healthy vaginal microbiota. Our findings demonstrate the promising potential of PP-HCl as a therapeutic strategy against mTSS. Less
Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy especially considering the anthropogenic effects in the global warming and natural resources depletion This includes the valorization of the hemicellulosic fraction of plant biomass aiming to produce prebiotic oligosaccharides widely explored in food and feed industries In the present work we conducted biochemical and biophysical characterization of two prokaryotic xylanases of family from Bacillus pumilus and Ruminococcus champanellensis and assessed their applicability for xylooligosaccharides production using alkaline pretreated corn cob and eucalyptus sawdust collected from a local market and ... More
Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy, especially considering the anthropogenic effects in the global warming and natural resources depletion. This includes the valorization of the hemicellulosic fraction of plant biomass, aiming to produce prebiotic oligosaccharides, widely explored in food and feed industries. In the present work, we conducted biochemical and biophysical characterization of two prokaryotic xylanases of family 30_8 from Bacillus pumilus and Ruminococcus champanellensis, and assessed their applicability for xylooligosaccharides production using alkaline pretreated corn cob and eucalyptus sawdust collected from a local market and a sawmill shop in Araraquara, respectively. Mass spectrometry and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) analysis revealed that RcXyn30A liberates mainly long monoglucuronylated xylooligosaccharides and proved to be highly inefficient in the cleavage of X4, X5 and X6, whereas BpXyn30A produces both linear and branched oligosaccharides. Crystallographic structure of BpXyn30A and RcXyn30A catalytic domain were solved and refined to 2.16 Å and 1.37Å resolution, respectively. Structural analysis of the enzymes binding cleft showed a conserved set of amino acids interacting with glucuronic acid substitution in the subsite -2b by several hydrogen bonds and ionic interactions, a characteristic shared between true glucunoxylanases. Furthermore, RcXyn30A has a larger β5-α5 loop as compared to other GH30 xylanases, which might be crucial for creating an additional aglycone subsite (+3). Finally, B. pumilus xylanase obtained higher conversion yields from pretreated biomasses than RcXyn30A, although the latter presents a specific activity against glucuronoxylan 9 times higher than the former. Less
Periplasmic solute-binding proteins SBPs are key ligand recognition components of bacterial ATP-binding cassette ABC transporters that allow bacteria to import nutrients and metabolic precursors from the environment Periplasmic SBPs comprise a large and diverse family of proteins of which only a small number have been empirically characterized In this work we identify a set of unique uncharacterized proteins within the SBP bac family that are found in conserved operons comprising genes encoding i ABC transport systems and ii putative amidases from the FmdA AmdA family From these uncharacterized SBP bac proteins we characterize a representative periplasmic SBP from Mesorhizobium sp ... More
Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from Mesorhizobium sp. A09 (MeAmi_SBP) and show that MeAmi_SBP binds l-amino acid amides but not the corresponding l-amino acids. An X-ray crystal structure of MeAmi_SBP bound to l-serinamide highlights the residues that impart distinct specificity for l-amino acid amides and reveals a structural Ca2+ binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca2+ binding are conserved amongst the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake and metabolism of l-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that MeAmi_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with l-amino acid amides and bacterial nitrogen utilization. Less
Hematopoietic stem cells HSCs develop from the hemogenic endothelium HE in the aorta- gonads-and mesonephros AGM region and reside within Intra-aortic hematopoietic clusters IAHC along with hematopoietic progenitors HPC The signalling mechanisms that distinguish HSCs from HPCs are unknown Notch signaling is essential for arterial specification IAHC formation and HSC activity but current studies on how Notch segregates these different fates are inconsistent We now demonstrate that Notch activity is highest in a subset of GFI HSC-primed HE cells and is gradually lost with HSC maturation We uncover that the HSC phenotype is maintained due to increasing levels of NOTCH ... More
Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium (HE) in the aorta- gonads-and mesonephros (AGM) region and reside within Intra-aortic hematopoietic clusters (IAHC) along with hematopoietic progenitors (HPC). The signalling mechanisms that distinguish HSCs from HPCs are unknown. Notch signaling is essential for arterial specification, IAHC formation and HSC activity, but current studies on how Notch segregates these different fates are inconsistent. We now demonstrate that Notch activity is highest in a subset of, GFI1 + , HSC-primed HE cells, and is gradually lost with HSC maturation. We uncover that the HSC phenotype is maintained due to increasing levels of NOTCH1 and JAG1 interactions on the surface of the same cell (cis) that renders the NOTCH1 receptor from being activated. Forced activation of the NOTCH1 receptor in IAHC activates a hematopoietic differentiation program. Our results indicate that NOTCH1-JAG1 cis-inhibition preserves the HSC phenotype in the hematopoietic clusters of the embryonic aorta. Less
Infected cell protein ICP is an immediate-early regulatory protein of herpes simplex virus HSV- that possesses E ubiquitin ligase activity ICP transactivates viral genes in part through its C-terminal dimer domain residues Deletion of this dimer domain results in reduced viral gene expression lytic infection and reactivation from latency Since ICP s dimer domain is associated with its transactivation activity and efficient viral replication we wanted to determine the structure of this specific domain The C-terminus of ICP was purified from bacteria and analyzed by X-ray crystallography to solve its structure Each subunit or monomer in the ICP dimer is ... More
Infected cell protein 0 (ICP0) is an immediate-early regulatory protein of herpes simplex virus 1 (HSV-1) that possesses E3 ubiquitin ligase activity. ICP0 transactivates viral genes, in part, through its C-terminal dimer domain (residues 555–767). Deletion of this dimer domain results in reduced viral gene expression, lytic infection, and reactivation from latency. Since ICP0’s dimer domain is associated with its transactivation activity and efficient viral replication, we wanted to determine the structure of this specific domain. The C-terminus of ICP0 was purified from bacteria and analyzed by X-ray crystallography to solve its structure. Each subunit or monomer in the ICP0 dimer is composed of nine β-strands and two α-helices. Interestingly, two adjacent β-strands from one monomer “reach” into the adjacent subunit during dimer formation, generating two β-barrel-like structures. Additionally, crystallographic analyses indicate a tetramer structure is formed from two β-strands of each dimer, creating a “stacking” of the β-barrels. The structural protein database searches indicate the fold or structure adopted by the ICP0 dimer is novel. The dimer is held together by an extensive network of hydrogen bonds. Computational analyses reveal that ICP0 can either form a dimer or bind to SUMO1 via its C-terminal SUMO-interacting motifs but not both. Understanding the structure of the dimer domain will provide insights into the activities of ICP0 and, ultimately, the HSV-1 life cycle. Less
ADP-ribosyltransferases PARP and PARP play a major role in DNA repair mechanism by detecting the DNA damage and inducing poly-ADP-ribosylation dependent chromatin relaxation and recruitment of repair proteins Catalytic PARP inhibitors are used as anticancer drugs especially in the case of tumors arising from sensitizing mutations Recently a study showed that Histone PARylation Factor HPF forms a joint active site with PARP The interaction of HPF with PARP alters the modification site from Aspartate Glutamate to Serine which has been shown to be a key ADP-ribosylation event in the context of DNA damage Therefore disruption of PARP -HPF interaction could ... More
ADP-ribosyltransferases PARP1 and PARP2 play a major role in DNA repair mechanism by detecting the DNA damage and inducing poly-ADP-ribosylation dependent chromatin relaxation and recruitment of repair proteins. Catalytic PARP inhibitors are used as anticancer drugs especially in the case of tumors arising from sensitizing mutations. Recently, a study showed that Histone PARylation Factor (HPF1) forms a joint active site with PARP1/2. The interaction of HPF1 with PARP1/2 alters the modification site from Aspartate/Glutamate to Serine, which has been shown to be a key ADP-ribosylation event in the context of DNA damage. Therefore, disruption of PARP1/2-HPF1 interaction could be an alternative strategy for drug development to block the PARP1/2 activity. In this study, we describe a FRET based high-throughput screening assay to screen inhibitor libraries against PARP-HPF1 interaction. We optimized the conditions for FRET signal and verified the interaction by competing the FRET pair in multiple ways. The assay is robust and easy to automate. Validatory screening showed the robust performance of the assay, and we discovered two compounds Dimethylacrylshikonin and Alkannin, with µM inhibition potency against PARP1/2-HPF1 interaction. The assay will facilitate the discovery of inhibitors against HPF1-PARP1/2 complex and to develop potentially new effective anticancer agents. Less
Primordial follicle activation PFA is a pivotal event in female reproductive biology coordinating the transition from quiescent to growing follicles This study employed comprehensive single-cell RNA sequencing to gain insights into the detailed regulatory mechanisms governing the synchronized dormancy and activation between granulosa cells GCs and oocytes with the progression of the PFA process Wntless Wls conditional knockout cKO mice served as a unique model suppressing the transition from pre-GCs to GCs and disrupting somatic cell-derived WNT signaling in the ovary Our data revealed immediate transcriptomic changes in GCs post-PFA in Wls cKO mice leading to a divergent trajectory while ... More
Primordial follicle activation (PFA) is a pivotal event in female reproductive biology, coordinating the transition from quiescent to growing follicles. This study employed comprehensive single-cell RNA sequencing to gain insights into the detailed regulatory mechanisms governing the synchronized dormancy and activation between granulosa cells (GCs) and oocytes with the progression of the PFA process. Wntless (Wls) conditional knockout (cKO) mice served as a unique model, suppressing the transition from pre-GCs to GCs, and disrupting somatic cell-derived WNT signaling in the ovary. Our data revealed immediate transcriptomic changes in GCs post-PFA in Wls cKO mice, leading to a divergent trajectory, while oocytes exhibited modest transcriptomic alterations. Subpopulation analysis identified the molecular pathways affected by WNT signaling on GC maturation, along with specific gene signatures linked to dormant and activated oocytes. Despite minimal evidence of continuous up-regulation of dormancy-related genes in oocytes, the loss of WNT signaling in (pre-)GCs impacted gene expression in oocytes even before PFA, subsequently influencing them globally. The infertility observed in Wls cKO mice was attributed to compromised GC-oocyte molecular crosstalk and the microenvironment for oocytes. Our study highlights the pivotal role of the WNT-signaling pathway and its molecular signature, emphasizing the importance of intercellular crosstalk between (pre-)GCs and oocytes in orchestrating folliculogenesis. Less
Osteoclasts are over-activated as we age which results in bone loss Src deficiency in mice leads to severe osteopetrosis due to a functional defect in osteoclasts indicating that Src function is essential in osteoclasts G-protein-coupled receptors GPCRs are the targets for of approved drugs but it is still unclear how GPCRs regulate Src kinase activity Here we reveal that GPR activation by its natural ligand Kisspeptin- Kp- causes Dusp to dephosphorylate Src at Tyr Mechanistically Gpr recruits both active Src and the Dusp phosphatase at its proline arginine-rich motif in its C terminus We show that Kp- binding to Gpr ... More
Osteoclasts are over-activated as we age, which results in bone loss. Src deficiency in mice leads to severe osteopetrosis due to a functional defect in osteoclasts, indicating that Src function is essential in osteoclasts. G-protein-coupled receptors (GPCRs) are the targets for ∼35% of approved drugs but it is still unclear how GPCRs regulate Src kinase activity. Here, we reveal that GPR54 activation by its natural ligand Kisspeptin-10 (Kp-10) causes Dusp18 to dephosphorylate Src at Tyr 416. Mechanistically, Gpr54 recruits both active Src and the Dusp18 phosphatase at its proline/arginine-rich motif in its C terminus. We show that Kp-10 binding to Gpr54 leads to the up-regulation of Dusp18. Kiss1, Gpr54 and Dusp18 knockout mice all exhibit osteoclast hyperactivation and bone loss, and Kp-10 abrogated bone loss by suppressing osteoclast activity in vivo. Therefore, Kp-10/Gpr54 is a promising therapeutic target to abrogate bone resorption by Dusp18-mediated Src dephosphorylation. Less
Melanocortin receptor MC -R antagonists are actively sought for treating cancer cachexia We determined the structures of complexes with PG- and SBL-MC- These peptides differ from SHU by substituting His with Pro and inserting Gly or Arg The structures revealed two subpockets at the TM -TM -TM domains separated by N Two peptide series based on the complexed peptides led to an antagonist activity and selectivity SAR study Most ligands retained the SHU potency but several SBL-MC- -derived peptides significantly enhanced MC -R selectivity over MC -R by - to -fold We also investigated MC -R coupling to the K ... More
Melanocortin 4 receptor (MC4-R) antagonists are actively sought for treating cancer cachexia. We determined the structures of complexes with PG-934 and SBL-MC-31. These peptides differ from SHU9119 by substituting His6 with Pro6 and inserting Gly10 or Arg10. The structures revealed two subpockets at the TM7-TM1-TM2 domains, separated by N2857.36. Two peptide series based on the complexed peptides led to an antagonist activity and selectivity SAR study. Most ligands retained the SHU9119 potency, but several SBL-MC-31-derived peptides significantly enhanced MC4-R selectivity over MC1-R by 60- to 132-fold. We also investigated MC4-R coupling to the K+ channel, Kir7.1. Some peptides activated the channel, whereas others induced channel closure independently of G protein coupling. In cell culture studies, channel activation correlated with increased feeding, while a peptide with Kir7.1 inhibitory activity reduced eating. These results highlight the potential for targeting the MC4-R:Kir7.1 complex for treating positive and restrictive eating disorders. Less
Dihydroorotate dehydrogenase DHODH is a mitochondrial enzyme that affects many aspects essential to cell proliferation and survival Recently DHODH has been identified as a potential target for acute myeloid leukemia therapy Herein we describe the identification of potent DHODH inhibitors through a scaffold hopping approach emanating from a fragment screen followed by structure-based drug design to further improve the overall profile and reveal an unexpected novel binding mode Additionally these compounds had low P-gp efflux ratios allowing for applications where exposure to the brain would be required
Memory encodes past experiences thereby enabling future plans The basolateral amygdala is a centre of salience networks that underlie emotional experiences and thus has a key role in long-term fear memory formation Here we used spatial and single-cell transcriptomics to illuminate the cellular and molecular architecture of the role of the basolateral amygdala in long-term memory We identified transcriptional signatures in subpopulations of neurons and astrocytes that were memory-specific and persisted for weeks These transcriptional signatures implicate neuropeptide and BDNF signalling MAPK and CREB activation ubiquitination pathways and synaptic connectivity as key components of long-term memory Notably upon long-term memory ... More
Memory encodes past experiences, thereby enabling future plans. The basolateral amygdala is a centre of salience networks that underlie emotional experiences and thus has a key role in long-term fear memory formation1. Here we used spatial and single-cell transcriptomics to illuminate the cellular and molecular architecture of the role of the basolateral amygdala in long-term memory. We identified transcriptional signatures in subpopulations of neurons and astrocytes that were memory-specific and persisted for weeks. These transcriptional signatures implicate neuropeptide and BDNF signalling, MAPK and CREB activation, ubiquitination pathways, and synaptic connectivity as key components of long-term memory. Notably, upon long-term memory formation, a neuronal subpopulation defined by increased Penk and decreased Tac expression constituted the most prominent component of the memory engram of the basolateral amygdala. These transcriptional changes were observed both with single-cell RNA sequencing and with single-molecule spatial transcriptomics in intact slices, thereby providing a rich spatial map of a memory engram. The spatial data enabled us to determine that this neuronal subpopulation interacts with adjacent astrocytes, and functional experiments show that neurons require interactions with astrocytes to encode long-term memory. Less
Exposure to environmental pollutants is linked to numerous toxic outcomes warranting concern about the effect of pollutants on human health To assess the threat of pollutant exposure it is essential to understand their biological activity Unfortunately gaps remain for many pollutants specific biological activity and molecular targets A superfamily of signaling proteins G-protein-coupled receptors GPCRs has been shown as potential targets for pollutant activity However research investigating the pollutant activity at the GPCRome is scarce This work explores pollutant activity across a library of human GPCRs by leveraging modern high-throughput screening techniques devised for drug discovery and pharmacology We designed ... More
Exposure to environmental pollutants is linked to numerous toxic outcomes, warranting concern about the effect of pollutants on human health. To assess the threat of pollutant exposure, it is essential to understand their biological activity. Unfortunately, gaps remain for many pollutants’ specific biological activity and molecular targets. A superfamily of signaling proteins, G-protein-coupled receptors (GPCRs), has been shown as potential targets for pollutant activity. However, research investigating the pollutant activity at the GPCRome is scarce. This work explores pollutant activity across a library of human GPCRs by leveraging modern high-throughput screening techniques devised for drug discovery and pharmacology. We designed and implemented a pilot screen of eight pollutants at 314 human GPCRs and discovered specific polychlorinated biphenyl (PCB) activity at sphingosine-1-phosphate and melatonin receptors. The method utilizes open-source resources available to academic and governmental institutions to enable future campaigns that screen large numbers of pollutants. Thus, we present a novel high-throughput approach to assess the biological activity and specific targets of pollutants. Less
Background Pseudomonas aeruginosa is the dominant pathogen causing lung infections in people with both cystic fibrosis CF and bronchiectasis associated with poorer outcomes Unlike CF bronchiectasis has been a neglected disease More extensive genomic studies of larger bronchiectasis patient cohorts and within patient sampling are needed to improve understanding of the evolutionary mechanisms underpinning P aeruginosa infections to guide novel and improved treatments Methods We have performed genome sequencing of P aeruginosa isolates from patients attending clinics worldwide to analyse the genomic diversity between and within patient infections Results We observed high genetic diversity between infections with low incidence of ... More
Background Pseudomonas aeruginosa is the dominant pathogen causing lung infections in people with both cystic fibrosis (CF) and bronchiectasis, associated with poorer outcomes. Unlike CF, bronchiectasis has been a neglected disease. More extensive genomic studies of larger bronchiectasis patient cohorts and within patient sampling are needed to improve understanding of the evolutionary mechanisms underpinning P. aeruginosa infections to guide novel and improved treatments.
Methods We have performed genome sequencing of 2,854 P. aeruginosa isolates from 180 patients attending clinics worldwide to analyse the genomic diversity between and within patient infections.
Results We observed high genetic diversity between infections with low incidence of highly transmissible strains. Our genomic data provide evidence for the mutational targets driving P. aeruginosa evolution in bronchiectasis. Some functions found to gain mutations were comparable to CF, including biofilm and iron acquisition, whilst others highlighted distinct evolutionary paths in bronchiectasis such as pyocin production and resistance, and a novel efflux pump gene (PA1874). We also show a high incidence of antimicrobial resistance-associated mutations and acquired resistance genes, in particular multidrug efflux and fluoroquinolone resistance mechanisms.
Conclusions Our findings highlight important differences between P. aeruginosa infections in bronchiectasis and CF and provide evidence of the relatively minor role transmissible strains play in bronchiectasis. Our study provides a 10-fold increase in the available genomic data for these infections and is a global resource to improve our knowledge and understanding, to facilitate better patient outcomes.
Summary The largest genomic study of Pseudomonas aeruginosa bronchiectasis isolates to-date, providing an unprecedented global genomic resource. We highlight important differences between bronchiectasis and cystic fibrosis, including key genes under selection. Less
Methods We have performed genome sequencing of 2,854 P. aeruginosa isolates from 180 patients attending clinics worldwide to analyse the genomic diversity between and within patient infections.
Results We observed high genetic diversity between infections with low incidence of highly transmissible strains. Our genomic data provide evidence for the mutational targets driving P. aeruginosa evolution in bronchiectasis. Some functions found to gain mutations were comparable to CF, including biofilm and iron acquisition, whilst others highlighted distinct evolutionary paths in bronchiectasis such as pyocin production and resistance, and a novel efflux pump gene (PA1874). We also show a high incidence of antimicrobial resistance-associated mutations and acquired resistance genes, in particular multidrug efflux and fluoroquinolone resistance mechanisms.
Conclusions Our findings highlight important differences between P. aeruginosa infections in bronchiectasis and CF and provide evidence of the relatively minor role transmissible strains play in bronchiectasis. Our study provides a 10-fold increase in the available genomic data for these infections and is a global resource to improve our knowledge and understanding, to facilitate better patient outcomes.
Summary The largest genomic study of Pseudomonas aeruginosa bronchiectasis isolates to-date, providing an unprecedented global genomic resource. We highlight important differences between bronchiectasis and cystic fibrosis, including key genes under selection. Less
In the fields of human health and agricultural research low coverage whole-genome sequencing followed by imputation to a large haplotype reference panel has emerged as a cost-effective alternative to genotyping arrays for assaying large numbers of samples However a systematic comparison of library preparation methods tailored for low coverage sequencing remains absent in the existing literature In this study we evaluated one full sized kit from IDT and miniaturized and evaluated three Illumina-compatible library preparation kits the KAPA HyperPlus kit Roche the DNA Prep kit Illumina and an IDT kit using human DNA samples Metrics evaluated included imputation concordance with ... More
In the fields of human health and agricultural research, low coverage whole-genome sequencing followed by imputation to a large haplotype reference panel has emerged as a cost-effective alternative to genotyping arrays for assaying large numbers of samples. However, a systematic comparison of library preparation methods tailored for low coverage sequencing remains absent in the existing literature. In this study, we evaluated one full sized kit from IDT and miniaturized and evaluated three Illumina-compatible library preparation kits—the KAPA HyperPlus kit (Roche), the DNA Prep kit (Illumina), and an IDT kit—using 96 human DNA samples. Metrics evaluated included imputation concordance with high-depth genotypes, coverage, duplication rates, time for library preparation, and additional optimization requirements. Despite slightly elevated duplication rates in IDT kits, we find that all four kits perform well in terms of imputation accuracy, with IDT kits being only marginally less performant than Illumina and Roche kits. Laboratory handling of the kits was similar: thus, the choice of a kit will largely depend on (1) existing or planned infrastructure, such as liquid handling capabilities, (2) whether a specific characteristic is desired, such as the use of full-length adapters, shorter processing times, or (3) use case, for instance, long vs short read sequencing. Our findings offer a comprehensive resource for both commercial and research workflows of low-cost library preparation methods suitable for high-throughput low coverage whole genome sequencing. Less
The adenosine subfamily G protein-coupled receptors A AR and A BR have been identified as promising cancer immunotherapy candidates One of the A AR A BR dual antagonists AB has progressed to a phase II clinical trial to treat rectal cancer However the precise mechanism underlying its dual-antagonistic properties remains elusive Herein we report crystal structures of the A AR complexed with AB and a selective A AR antagonist The structures revealed a common binding mode on A AR wherein the ligands established extensive interactions with residues from the orthosteric and secondary pockets In contrast the cAMP assay and A ... More
The adenosine subfamily G protein-coupled receptors A2AR and A2BR have been identified as promising cancer immunotherapy candidates. One of the A2AR/A2BR dual antagonists, AB928, has progressed to a phase II clinical trial to treat rectal cancer. However, the precise mechanism underlying its dual-antagonistic properties remains elusive. Herein, we report crystal structures of the A2AR complexed with AB928 and a selective A2AR antagonist 2–118. The structures revealed a common binding mode on A2AR, wherein the ligands established extensive interactions with residues from the orthosteric and secondary pockets. In contrast, the cAMP assay and A2AR and A2BR molecular dynamics simulations indicated that the ligands adopted distinct binding modes on A2BR. Detailed analysis of their chemical structures suggested that AB928 readily adapted to the A2BR pocket, while 2–118 did not due to intrinsic differences. This disparity potentially accounted for the difference in inhibitory efficacy between A2BR and A2AR. This study serves as a valuable structural template for the future development of selective or dual inhibitors targeting A2AR/A2BR for cancer therapy. Less
Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels This process is mediated by the parasite s polymorphic erythrocyte membrane protein PfEMP adhesion proteins A subset of PfEMP variants that bind human endothelial protein C receptor EPCR through their CIDR domains is responsible for severe malaria pathogenesis A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP variants Here we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDR The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of CIDR domains representing five of ... More
Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels. This process is mediated by the parasite’s polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. A subset of PfEMP1 variants that bind human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here, we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDRα1. The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of 34 CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins as well as parasite sequestration in bioengineered 3D brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with two different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies likely represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria. Less
We herein describe the development and application of a modular technology platform which incorporates recent advances in plate-based microscale chemistry automated purification in situ quantification and robotic liquid handling to enable rapid access to high-quality chemical matter already formatted for assays In using microscale chemistry and thus consuming minimal chemical matter the platform is not only efficient but also follows green chemistry principles By reorienting existing high-throughput assay technology the platform can generate a full package of relevant data on each set of compounds in every learning cycle The multiparameter exploration of chemical and property space is hereby driven by ... More
We herein describe the development and application of a modular technology platform which incorporates recent advances in plate-based microscale chemistry, automated purification, in situ quantification, and robotic liquid handling to enable rapid access to high-quality chemical matter already formatted for assays. In using microscale chemistry and thus consuming minimal chemical matter, the platform is not only efficient but also follows green chemistry principles. By reorienting existing high-throughput assay technology, the platform can generate a full package of relevant data on each set of compounds in every learning cycle. The multiparameter exploration of chemical and property space is hereby driven by active learning models. The enhanced compound optimization process is generating knowledge for drug discovery projects in a time frame never before possible. Less
The transcription factor BCL A is a critical regulator of the switch from fetal hemoglobin HbF to adult hemoglobin HbA during development BCL A binds at a cognate recognition site TGACCA in the -globin gene promoter and represses its expression DNA-binding is mediated by a triple zinc finger domain designated ZnF Here we report comprehensive investigation of ZnF leveraging X-ray crystallography and NMR to determine the structures in both the presence and absence of DNA We delve into the dynamics and mode of interaction with DNA Moreover we discovered that the last zinc finger of BCL A ZnF plays a ... More
The transcription factor BCL11A is a critical regulator of the switch from fetal hemoglobin (HbF: α 2 γ 2 ) to adult hemoglobin (HbA: α 2 β 2 ) during development. BCL11A binds at a cognate recognition site (TGACCA) in the γ-globin gene promoter and represses its expression. DNA-binding is mediated by a triple zinc finger domain, designated ZnF456. Here, we report comprehensive investigation of ZnF456, leveraging X-ray crystallography and NMR to determine the structures in both the presence and absence of DNA. We delve into the dynamics and mode of interaction with DNA. Moreover, we discovered that the last zinc finger of BCL11A (ZnF6) plays a special role in DNA binding and γ-globin gene repression. Our findings help account for some rare γ-globin gene promoter mutations that perturb BCL11A binding and lead to increased HbF in adults (hereditary persistence of fetal hemoglobin). Comprehending the DNA binding mechanism of BCL11A opens avenues for the strategic, structure-based design of novel therapeutics targeting sickle cell disease and β-thalassemia. Less
Elevated bacterial sialidase activity in the female genital tract is strongly associated with poor health outcomes including preterm birth and bacterial vaginosis These negative effects may arise from sialidase-mediated degradation of the protective mucus layer in the cervicovaginal environment Prior biochemical studies of vaginal bacterial sialidases have focused solely on the bacterial vaginosis-associated organism Gardnerella vaginalis Despite their implications for sexual and reproductive health sialidases from other vaginal bacteria have not been characterized Here we show that vaginal Prevotella species produce active sialidases that possess variable activity toward mucin These sialidases are highly conserved across clades of Prevotella from different ... More
Elevated bacterial sialidase activity in the female genital tract is strongly associated with poor health outcomes including preterm birth and bacterial vaginosis. These negative effects may arise from sialidase-mediated degradation of the protective mucus layer in the cervicovaginal environment. Prior biochemical studies of vaginal bacterial sialidases have focused solely on the bacterial vaginosis-associated organism Gardnerella vaginalis. Despite their implications for sexual and reproductive health, sialidases from other vaginal bacteria have not been characterized. Here, we show that vaginal Prevotella species produce active sialidases that possess variable activity toward mucin. These sialidases are highly conserved across clades of Prevotella from different geographies, hinting at their importance globally. Finally, we find that Prevotella sialidases, including mucin-degrading enzymes from Prevotella timonensis, are highly prevalent and abundant in human vaginal metagenomes and metatranscriptomes, Together, our results identify Prevotella as a critical source of sialidases in the vaginal microbiome, improving our understanding of this detrimental bacterial activity. Less
To identify starting points for therapeutics targeting SARS-CoV- the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate-binding pocket Of soaked fragments a total of hits bound either in the active site hits a remote binding pocket three hits or at crystal-packing interfaces two hits Notably two fragments with a pose that was sterically incompatible with a more occluded crystal form were identified Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue The structures ... More
To identify starting points for therapeutics targeting SARS-CoV-2, the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease. Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate-binding pocket. Of 631 soaked fragments, a total of 29 hits bound either in the active site (24 hits), a remote binding pocket (three hits) or at crystal-packing interfaces (two hits). Notably, two fragments with a pose that was sterically incompatible with a more occluded crystal form were identified. Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue. The structures also revealed a surprisingly strong influence of the crystal form on the binding pose of three published fragments used as positive controls, with implications for fragment screening by crystallography. Less
The tripartite ATP-independent periplasmic TRAP transporters use an extra cytoplasmic substrate binding protein SBP to transport a wide variety of substrates in bacteria and archaea The SBP can adopt an open- or closed state depending on the presence of substrate The two transmembrane domains of TRAP transporters form a monomeric elevator whose function is strictly dependent on the presence of a sodium ion gradient Insights from experimental structures structural predictions and molecular modeling have suggested a conformational coupling between the membrane elevator and the substrate binding protein Here we use a disulfide engineering approach to lock the TRAP transporter HiSiaPQM ... More
The tripartite ATP-independent periplasmic (TRAP) transporters use an extra cytoplasmic substrate binding protein (SBP) to transport a wide variety of substrates in bacteria and archaea. The SBP can adopt an open- or closed state depending on the presence of substrate. The two transmembrane domains of TRAP transporters form a monomeric elevator whose function is strictly dependent on the presence of a sodium ion gradient. Insights from experimental structures, structural predictions and molecular modeling have suggested a conformational coupling between the membrane elevator and the substrate binding protein. Here, we use a disulfide engineering approach to lock the TRAP transporter HiSiaPQM from Haemophilus influenzae in different conformational states. The SBP, HiSiaP, is locked in its substrate-bound form and the transmembrane elevator, HiSiaQM, is locked in either its assumed inward- or outward-facing states. We characterize the disulfide-locked constructs and use single-molecule total internal reflection fluorescence (TIRF) microscopy to study their interactions. Our experiments demonstrate that the SBP and the transmembrane elevator are indeed conformationally coupled, meaning that the open and closed state of the SBP recognize specific conformational states of the transporter and vice versa. Less
Monoolein-based liquid crystal phases are established media that are researched for various biological applications including drug delivery While water is the most common solvent for self-assembly some ionic liquids ILs can support lipidic self-assembly However currently there is limited knowledge of IL-lipid phase behavior in ILs In this study the lyotropic liquid crystal phase behavior of monoolein was investigated in six protic ILs known to support amphiphile self-assembly namely ethylammonium nitrate ethanolammonium nitrate ethylammonium formate ethanolammonium formate ethylammonium acetate and ethanolammonium acetate These ILs were selected to identify specific ion effects on monoolein self-assembly specifically increasing the alkyl chain length ... More
Monoolein-based liquid crystal phases are established media that are researched for various biological applications, including drug delivery. While water is the most common solvent for self-assembly, some ionic liquids (ILs) can support lipidic self-assembly. However, currently, there is limited knowledge of IL-lipid phase behavior in ILs. In this study, the lyotropic liquid crystal phase behavior of monoolein was investigated in six protic ILs known to support amphiphile self-assembly, namely ethylammonium nitrate, ethanolammonium nitrate, ethylammonium formate, ethanolammonium formate, ethylammonium acetate, and ethanolammonium acetate. These ILs were selected to identify specific ion effects on monoolein self-assembly, specifically increasing the alkyl chain length of the cation or anion, the presence of a hydroxyl group in the cation, and varying the anion. The lyotropic liquid crystal phases with 20–80 wt. % of monoolein were characterized over a temperature range from 25 to 65 °C using synchrotron small angle x-ray scattering and cross-polarized optical microscopy. These results were used to construct partial phase diagrams of monoolein in each of the six protic ILs, with inverse hexagonal, bicontinuous cubic, and lamellar phases observed. Protic ILs containing the ethylammonium cation led to monoolein forming lamellar and bicontinuous cubic phases, while those containing the ethanolammonium cation formed inverse hexagonal and bicontinuous cubic phases. Protic ILs containing formate and acetate anions favored bicontinuous cubic phases across a broader range of protic IL concentrations than those containing the nitrate anion. Less
Transcription factors of the bHLH-PAS family play vital roles in animal development physiology and disease Two members of the family require binding of low-molecular weight ligands for their activity the vertebrate aryl hydrocarbon receptor AHR and the insect juvenile hormone receptor JHR In the fly Drosophila melanogaster the paralogous proteins GCE and MET constitute the ligand-binding component of JHR complexes Whilst GCE MET and AHR are phylogenetically heterologous their mode of action is similar JHR is targeted by several synthetic agonists that serve as insecticides disrupting the insect endocrine system AHR is an important regulator of human endocrine homeostasis and ... More
Transcription factors of the bHLH-PAS family play vital roles in animal development, physiology, and disease. Two members of the family require binding of low-molecular weight ligands for their activity: the vertebrate aryl hydrocarbon receptor (AHR) and the insect juvenile hormone receptor (JHR). In the fly Drosophila melanogaster, the paralogous proteins GCE and MET constitute the ligand-binding component of JHR complexes. Whilst GCE/MET and AHR are phylogenetically heterologous, their mode of action is similar. JHR is targeted by several synthetic agonists that serve as insecticides disrupting the insect endocrine system. AHR is an important regulator of human endocrine homeostasis and it responds to environmental pollutants and endocrine disruptors. Whether AHR signaling is affected by compounds that can activate JHR has not been reported. To address this question, we screened a chemical library of 50,000 compounds to identify 93 novel JHR agonists in a reporter system based on Drosophila cells. Of these compounds, 26% modulated AHR signaling in an analogous reporter assay in a human cell line, indicating a significant overlap in the agonist repertoires of the two receptors. To explore the structural features of agonist-dependent activation of JHR and AHR, we compared the ligand-binding cavities and their interactions with selective and common ligands of AHR and GCE. Molecular dynamics modeling revealed ligand-specific as well as conserved side chains within the respective cavities. Significance of predicted interactions was supported through site-directed mutagenesis. The results have indicated that synthetic insect juvenile hormone agonists might interfere with AHR signaling in human cells. Less
Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold They are generally described as electron carriers that rely on a T copper centre coordinated by four ligands provided by the folded polypeptide The discovery of novel cupredoxins demonstrates the high diversity of this family with variations in terms of copper-binding ligands copper centre geometry redox potential as well as biological function AcoP is a periplasmic cupredoxin belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans AcoP presents original features including high resistance to acidic pH and a constrained green-type copper centre of high ... More
Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper centre coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in terms of copper-binding ligands, copper centre geometry, redox potential, as well as biological function. AcoP is a periplasmic cupredoxin belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans. AcoP presents original features, including high resistance to acidic pH and a constrained green-type copper centre of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structures, including native reduced AcoP at 1.65 Å resolution, unveil a typical cupredoxin fold. The presence of extended loops, never observed in previously characterized cupredoxins, might account for the interaction of AcoP with physiological partners. The Cu-ligand distances, determined by both X-ray diffraction and EXAFS, show that the AcoP metal centre seems to present both T1 and T1.5 features, in turn suggesting that AcoP might not fit well to the coupled distortion model. The crystal structures of two AcoP mutants confirm that the active centre of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active centre rigidity due to the presence of an extensive hydrogen bond network. Finally, we show that other cupredoxins do not perfectly follow the coupled distortion model as well, raising the suspicion that further alternative models to describe copper centre geometries need to be developed, while the importance of rack-induced contributions should not be underestimated. Less
Immunomodulatory imide drugs IMiDs degrade specific C H zinc finger degrons in transcription factors making them effective against certain cancers SALL a cancer driver contains seven C H zinc fingers in four clusters including an IMiD degron in zinc finger cluster two ZFC Surprisingly IMiDs do not inhibit growth of SALL expressing cancer cells To overcome this limit we focused on a non-IMiD degron SALL zinc finger cluster four ZFC By combining AlphaFold and the ZFC -DNA crystal structure we identified a potential ZFC drug pocket Utilizing an in silico docking algorithm and cell viability assays we screened chemical libraries ... More
Immunomodulatory imide drugs (IMiDs) degrade specific C2H2 zinc finger degrons in transcription factors, making them effective against certain cancers. SALL4, a cancer driver, contains seven C2H2 zinc fingers in four clusters, including an IMiD degron in zinc finger cluster two (ZFC2). Surprisingly, IMiDs do not inhibit growth of SALL4 expressing cancer cells. To overcome this limit, we focused on a non-IMiD degron, SALL4 zinc finger cluster four (ZFC4). By combining AlphaFold and the ZFC4-DNA crystal structure, we identified a potential ZFC4 drug pocket. Utilizing an in silico docking algorithm and cell viability assays, we screened chemical libraries and discovered SH6, which selectively targets SALL4-expressing cancer cells. Mechanistic studies revealed that SH6 degrades SALL4 protein through the CUL4A/CRBN pathway, while deletion of ZFC4 abolished this activity. Moreover, SH6 led to significant 62% tumor growth inhibition of SALL4+ xenografts in vivo and demonstrated good bioavailability in pharmacokinetic studies. In summary, these studies represent a new approach for IMiD independent drug discovery targeting C2H2 transcription factors in cancer. Less
TP is the most frequently mutated gene in human cancer While it is well understood that the ability of p to act as a transcription factor is required for tumor suppression the key target genes downstream of p required for tumor suppression are still incompletely understood We first set out to characterize a rare African-specific germline variant of TP in the DNA binding domain Tyr His Y H Although we find that Y H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p target genes Y H mice develop spontaneous cancers ... More
TP53 is the most frequently mutated gene in human cancer. While it is well understood that the ability of p53 to act as a transcription factor is required for tumor suppression, the key target genes downstream of p53 required for tumor suppression are still incompletely understood. We first set out to characterize a rare, African-specific, germline variant of TP53 in the DNA binding domain, Tyr107His (Y107H). Although we find that Y107H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p53 target genes, Y107H mice develop spontaneous cancers and metastases. We identified the p53 target gene, PADI4, to be exquisitely sensitive to p53 mutation, and loss of PADI4 is seen in Y107H and other transcriptionally competent p53 hypomorphs. PADI4 is a regulator of histone modification and gene transcription via citrullination, which is the process of deiminating arginine to the non-natural amino acid citrulline. Our TCGA analysis reveals PADI4 is downregulated or mutated in multiple human cancers. Surprisingly, we show that PADI4 is sufficient to suppress tumor growth and sensitize wild-type p53 cells to chemotherapeutics. We further show that PADI4 is potently tumor suppressive in vivo, and complete tumor suppression by PADI4 requires an intact immune system. We find PADI4 enhances the transactivation of p53 targets and genes involved in immune activation. In addition, we identify a p53–PADI4 gene signature that is predictive of survival and the efficacy of immune-checkpoint inhibitors. We have further found that PADI4 interacts and modifies p53 via citrullination at key residues within the oligomerization and C-terminal domain of p53. PADI4 colocalizes with p53 on chromatin at non-canonical p53 target genes and genes devoid of a p53 response element. Citrullination of p53 may alter p53 function through enhanced oligomerization or binding of p53 to DNA. The findings from this study reveal PADI4 as not only a key target gene of p53, but a core regulator of p53 activity and target specificity through a novel protein modification. This work highlights the need to reassess the role of PADI4 in cancer, and provides insight into critical downstream target genes important for tumor suppression by p53. Less
Predictive drug testing of patient-derived tumor organoids PDTOs holds promise for personalizing treatment of metastatic colorectal cancer mCRC but prospective data are limited to chemotherapy regimens with conflicting results We describe a unified framework for PDTO-based predictive testing across standard-of-care chemotherapy and biologic and targeted therapy options In an Australian community cohort PDTO predictions based on treatment-naive patients n and response rates from first-line mCRC clinical trials achieve accuracy for forecasting responses in patients receiving palliative treatments patients treatments Similar assay accuracy is achieved in a prospective study of third-line or later mCRC treatment AGITG FORECAST- n patients Resistant predictions ... More
Predictive drug testing of patient-derived tumor organoids (PDTOs) holds promise for personalizing treatment of metastatic colorectal cancer (mCRC), but prospective data are limited to chemotherapy regimens with conflicting results. We describe a unified framework for PDTO-based predictive testing across standard-of-care chemotherapy and biologic and targeted therapy options. In an Australian community cohort, PDTO predictions based on treatment-naive patients (n = 56) and response rates from first-line mCRC clinical trials achieve 83% accuracy for forecasting responses in patients receiving palliative treatments (18 patients, 29 treatments). Similar assay accuracy is achieved in a prospective study of third-line or later mCRC treatment, AGITG FORECAST-1 (n = 30 patients). “Resistant” predictions are associated with inferior progression-free survival; misclassification rates are similar by regimen. Liver metastases are the optimal site for sampling, with testing achievable within 7 weeks for 68.8% cases. Our findings indicate that PDTO drug panel testing can provide predictive information for multifarious standard-of-care therapies for mCRC. Less
Membrane proteins are indispensable for every living organism yet their structural organization remains underexplored Despite the recent advancements in single-particle cryogenic electron microscopy and cryogenic electron tomography which have significantly increased the structural coverage of membrane proteins across various kingdoms certain scientific methods such as time-resolved crystallography still mostly rely on crystallization techniques such as lipidic cubic phase LCP or in meso crystallization In this study we present an open-access blueprint for a humidity control chamber designed for LCP in meso crystallization experiments using a Gryphon crystallization robot Using this chamber we have obtained crystals of a transmembrane aspartate transporter ... More
Membrane proteins are indispensable for every living organism, yet their structural organization remains underexplored. Despite the recent advancements in single-particle cryogenic electron microscopy and cryogenic electron tomography, which have significantly increased the structural coverage of membrane proteins across various kingdoms, certain scientific methods, such as time-resolved crystallography, still mostly rely on crystallization techniques, such as lipidic cubic phase (LCP) or in meso crystallization. In this study, we present an open-access blueprint for a humidity control chamber designed for LCP/in meso crystallization experiments using a Gryphon crystallization robot. Using this chamber, we have obtained crystals of a transmembrane aspartate transporter GltTk from Thermococcus kodakarensis in a lipidic environment using in meso crystallization. The data collected from these crystals allowed us to perform an analysis of lipids bound to the transporter. With this publication of our open-access design of a humidity chamber, we aim to improve the accessibility of in meso protein crystallization for the scientific community. Less
StayGold is an exceptionally bright and stable fluorescent protein that is highly resistant to photobleaching Despite favorable fluorescence properties use of StayGold as a fluorescent tag is limited because it forms a natural dimer Here we report the structure of StayGold and generate a derivative mStayGold that retains the brightness and photostability of the original protein while being fully monomeric
Objective Due to the challenge for intratumoral administration innate agonists have not made it beyond preclinical studies for efficacy testing in most of tumor types Pancreatic ductal adenocarcinoma PDAC has a T-cell excluded or deserted tumor microenvironment Innate agonist treatments may serve as a T cell priming mechanism to sensitize PDACs to anti-PD- antibody a-PD- treatment Design Using a transplant murine model with spontaneously formed liver metastasis and also the genetically engineered KPC mouse model that spontaneously develops PDAC we compared the antitumor efficacy between intrahepatic intratumoral and intramuscular systemic administration of BMS- a next-generation STING agonist Flow cytometry Nanostring ... More
Objective: Due to the challenge for intratumoral administration, innate agonists have not made it beyond preclinical studies for efficacy testing in most of tumor types. Pancreatic ductal adenocarcinoma (PDAC) has a T-cell excluded or deserted tumor microenvironment. Innate agonist treatments may serve as a T cell priming mechanism to sensitize PDACs to anti-PD-1 antibody (a-PD-1) treatment. Design: Using a transplant murine model with spontaneously formed liver metastasis and also the genetically engineered KPC mouse model that spontaneously develops PDAC, we compared the antitumor efficacy between intrahepatic/intratumoral and intramuscular systemic administration of BMS-986301, a next-generation STING agonist. Flow cytometry, Nanostring, and cytokine assays were used to evaluate local and systemic immune responses. Results: The study demonstrated that administration of STING agonist systemically via intramuscular injection is equivalent or potentially superior to its intratumoral injection in inducing both effector T cell response and antitumor efficacy. Compared to intratumoral administration, T cell exhaustion and immunosuppressive signals induced by systemic administration were attenuated. Nonetheless, either local or systemic treatment of STING agonist was associated with increased expression of CTLA-4 in the tumors. However, the combination of a-PD-1 and anti-CTLA-4 antibody with systemic STING agonist demonstrated the antitumor efficacy in the KPC mouse spontaneous PDAC model. Our study also demonstrated the feasibility and antitumor efficacy of systemic administration of BMS-986299, a new NLRP3 agonist. Conclusion: For the first time, our study supports the clinical development of innate agonists via systemic administration, instead of local administration, for treating PDAC. Less
Microbial natural products are specialized metabolites that are sources of many bioactive compounds including antibiotics antifungals antiparasitics anticancer agents and probes of biology The assembly of libraries of producers of natural products has traditionally been the province of the pharmaceutical industry This sector has gathered significant historical collections of bacteria and fungi to identify new drug leads with outstanding outcomes - upwards of of drug scaffolds originate from such libraries Despite this success the repeated rediscovery of known compounds and the resultant diminishing chemical novelty contributed to a pivot from this source of bioactive compounds toward more tractable synthetic compounds ... More
Microbial natural products are specialized metabolites that are sources of many bioactive
compounds including antibiotics, antifungals, antiparasitics, anticancer agents, and probes of
biology. The assembly of libraries of producers of natural products has traditionally been the
province of the pharmaceutical industry. This sector has gathered significant historical
collections of bacteria and fungi to identify new drug leads with outstanding outcomes - upwards of 60% of drug scaffolds originate from such libraries. Despite this success, the repeated rediscovery of known compounds and the resultant diminishing chemical novelty contributed to a pivot from this source of bioactive compounds toward more tractable synthetic compounds in the drug industry. The advent of advanced mass spectrometry tools, along with rapid whole genome sequencing and in silico identification of biosynthetic gene clusters that encode the machinery necessary for the synthesis of specialized metabolites, offers the opportunity to revisit microbial natural product libraries with renewed vigor. Assembling a suitable library of microbes and extracts for screening requires the investment of resources and the development of methods that have customarily been the proprietary purview of large pharmaceutical companies. Here, we
report a perspective on our efforts to assemble a library of natural product-producing microbes
and the establishment of methods to extract and fractionate bioactive compounds using resources available to most academic labs. We validate the library and approach through a series of screens for antimicrobial and cytotoxic agents. This work serves as a blueprint for establishing libraries of microbial natural product producers and bioactive extract fractions suitable for screens of bioactive compounds. Less
compounds including antibiotics, antifungals, antiparasitics, anticancer agents, and probes of
biology. The assembly of libraries of producers of natural products has traditionally been the
province of the pharmaceutical industry. This sector has gathered significant historical
collections of bacteria and fungi to identify new drug leads with outstanding outcomes - upwards of 60% of drug scaffolds originate from such libraries. Despite this success, the repeated rediscovery of known compounds and the resultant diminishing chemical novelty contributed to a pivot from this source of bioactive compounds toward more tractable synthetic compounds in the drug industry. The advent of advanced mass spectrometry tools, along with rapid whole genome sequencing and in silico identification of biosynthetic gene clusters that encode the machinery necessary for the synthesis of specialized metabolites, offers the opportunity to revisit microbial natural product libraries with renewed vigor. Assembling a suitable library of microbes and extracts for screening requires the investment of resources and the development of methods that have customarily been the proprietary purview of large pharmaceutical companies. Here, we
report a perspective on our efforts to assemble a library of natural product-producing microbes
and the establishment of methods to extract and fractionate bioactive compounds using resources available to most academic labs. We validate the library and approach through a series of screens for antimicrobial and cytotoxic agents. This work serves as a blueprint for establishing libraries of microbial natural product producers and bioactive extract fractions suitable for screens of bioactive compounds. Less
NADH cytochrome b oxidoreductase Ncb or is a cytosolic ferric reductase implicated in diabetes and neurological conditions Ncb or comprises cytochrome b b and cytochrome b reductase b R domains separated by a CHORD-Sgt CS linker domain Ncb or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme While full-length human Ncb or has proven resistant to crystallization we have succeeded in obtaining high-resolution atomic structures of the b domain and a construct containing the CS and b R domains CS b R Ncb or also contains an N-terminal intrinsically ... More
NADH cytochrome b5 oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b5 (b5) and cytochrome b5 reductase (b5R) domains separated by a CHORD-Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full-length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high-resolution atomic structures of the b5 domain and a construct containing the CS and b5R domains (CS/b5R). Ncb5or also contains an N-terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L34MDWIRL40 motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to a b5 domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N-terminal region naturally fused to the b5 domain, we were able to obtain a high-resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25–129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp120 (corresponding to invariant Trp37 in Ncb5or) to be part of an 11-residue α-helix (S116QMDWLKLTRT126) packing against two of the four helices in the b5 domain that surround heme (α2 and α5). The Trp120 side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr85 and Tyr88 (α2), Cys124 (α5), and Leu47 in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp37 in nucleating the formation of the N-terminal helix, whose location in the N/b5 module suggests a role in regulating the function of this multi-domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b5 module as reflected by its existence in a class of cytochrome b5 proteins from three kingdoms among eukaryotes. Less
Flavins such as flavin mononucleotide or flavin adenine dinucleotide are bound by diverse proteins yet have very similar spectra when in the oxidized state Recently we developed new variants of flavin-binding protein CagFbFP exhibiting notable blue Q V or red I V A Q shifts of fluorescence emission maxima Here we use time-resolved and low-temperature spectroscopy to show that whereas the chromophore environment is static in Q V an additional protein-flavin hydrogen bond is formed upon photoexcitation in the I V A Q variant Consequently in Q V excitation emission and phosphorescence spectra are shifted whereas in I V A ... More
Flavins such as flavin mononucleotide or flavin adenine dinucleotide are bound by diverse proteins, yet have very similar spectra when in the oxidized state. Recently, we developed new variants of flavin-binding protein CagFbFP exhibiting notable blue (Q148V) or red (I52V A85Q) shifts of fluorescence emission maxima. Here, we use time-resolved and low-temperature spectroscopy to show that whereas the chromophore environment is static in Q148V, an additional protein-flavin hydrogen bond is formed upon photoexcitation in the I52V A85Q variant. Consequently, in Q148V, excitation, emission, and phosphorescence spectra are shifted, whereas in I52V A85Q, excitation and low-temperature phosphorescence spectra are relatively unchanged, while emission spectrum is altered. We also determine the x-ray structures of the two variants to reveal the flavin environment and complement the spectroscopy data. Our findings illustrate two distinct color-tuning mechanisms of flavin-binding proteins and could be helpful for the engineering of new variants with improved optical properties. Less
For optimal bioperformance the drug in an amorphous solid dispersion ASD should ideally not undergo crystallization in the solid dosage form during storage or from the supersaturated solution generated upon dissolution Incomplete processing during hot melt extrusion HME can lead to residual crystallinity Commonly residual crystallinity is evaluated using techniques such as powder X-ray diffraction pXRD However residual crystallinity at levels below the detection limit of pXRD can be detrimental to the ASD performance The goal of this study was to evaluate the impact of different levels of residual crystallinity in an ASD containing the fast-crystallizing drug posaconazole PCZ and ... More
For optimal bioperformance, the drug in an amorphous solid dispersion (ASD) should ideally not undergo crystallization in the solid dosage form during storage or from the supersaturated solution generated upon dissolution. Incomplete processing during hot melt extrusion (HME) can lead to residual crystallinity. Commonly, residual crystallinity is evaluated using techniques such as powder X-ray diffraction (pXRD). However, residual crystallinity at levels below the detection limit of pXRD can be detrimental to the ASD performance. The goal of this study was to evaluate the impact of different levels of residual crystallinity in an ASD containing the fast-crystallizing drug posaconazole (PCZ) and hydroxypropyl methylcellulose acetate succinate (HPMCAS) on dissolution and additional crystallization. ASDs with and without residual crystallinity at 10, 25, and 50 wt % drug loadings were prepared using HME, processing at temperatures below and above the critical temperature, which was calculated using the Flory–Huggins theory. Some of the ASDs contained levels of residual crystallinity that were below the quantification limit of pXRD, requiring the use of second harmonic generation (SHG) imaging. The impact of residual crystallinity on dissolution was studied by using two-stage dissolution. Additional characterization in support of dissolution measurements included SHG imaging and particle size evolution with focused beam reflectance measurement (FBRM) using pH-shift experiments. The 10 wt % ASD processed below the critical solution temperature contained residual crystallinity of 0.3%, which promoted rapid crystallization when the ASD was in a solution environment. Real-time monitoring of both the solid and solution phases revealed that PCZ in ASDs containing residual crystals underwent crystallization both in the matrix and from solution. The study supports the need to select a sufficiently sensitive crystallinity estimation technique, a suitable discriminatory dissolution technique, and appropriate HME processing conditions in order to optimize and achieve successful performance of ASDs of fast-crystallizing drugs. Less
Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection It is responsible for US deaths annually Neutrophils are an integral part of the innate immune response and rapidly clear pathogens from circulation using neutrophil extracellular traps NETs which are released through a process called NETosis NETs prevent dissemination of pathogens by entrapment in externalized chromatin containing deactivating enzymes While we have learned much about the mechanisms underlying NETosis we are yet to translate it to improved therapies or patient outcomes This gap may be attributable to the models used to study NETosis Current models used ... More
Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection (1). It is responsible for ~370,000 US deaths annually (2). Neutrophils are an integral part of the innate immune response and rapidly clear pathogens from circulation using neutrophil extracellular traps (NETs), which are released through a process called NETosis (3). NETs prevent dissemination of pathogens by entrapment in externalized chromatin containing deactivating enzymes. While we have learned much about the mechanisms underlying NETosis, we are yet to translate it to improved therapies or patient outcomes. This gap may be attributable to the models used to study NETosis. Current models used to investigate NETosis are limited and routinely employ unnatural triggers such as phorbol 12-myristate 13-acetate (PMA). PMA is not a physiological trigger present in the immune system and may bypass the natural pathways that regulate NETs production. Mouse models that use isolated neutrophils and neutrophil-like cells induced from immortalized cell lines do not completely reflect the complex cellular and molecular biology underlying neutrophil activation and NETosis, especially in a whole-blood environment. Therefore, it is crucial to study how specific factors, known to be upregulated in disease, interact and potentially induce NETosis. Here we use high-throughput screening and natural NETosis triggers to develop a more biologically relevant ex vivo NETosis (Synthetic-Sepsis™) model.
Whole blood was collected from healthy donors and aliquoted into a 384 well plate using a Formulatrix Mantis liquid handler. This plate contained small molecules associated with neutrophils or NETosis activation, such as interleukins: Il-1b, IL-5, IL-6, IL-8, IL-15, IL-17, IL-18 and other molecules TNF- α, LT-α, IFN-γ, G-CSF, GM-CSF, E-selectin, PAF-16, CXCL1, CXCL2, LTB4, CXCL5, CCL2, CCL3, fMLP, Ferritin, HMGB1, C5a and LPS. We used a combinatorial pooling strategy designed using JMP software to identify which combinations of small molecules could stimulate NET formation. NETosis was assessed using Sytox green intercalation at 5 minute intervals for up to 24 hours using a Molecular Devices plate reader. PMA was utilized as a positive control for NETosis induction at varying concentrations.
Using our combinatorial pooling approach of the various factors, we successfully induced NETosis in an ex vivo whole blood system using naturally occurring cytokines and chemokines at physiologically relevant concentrations. We found that different combinations of factors evoke distinct neutrophil responses both in the time of NET generation and/or magnitude of NET-associated intercalation signal. We observed inter-donor variability in response time and amplitude however, similar small molecule pools induced consistent responses across donors. Furthermore, our findings suggest that at least four naturally occurring factors are necessary to induce NETosis in our system. Although some factors activate similar pathways, they are unable to induce a signal alone and as the number of factors increased beyond four, there was an enhanced NET response. Interestingly, we found either TNF-α or LT-α was required to cause a NETosis response, underlining the potentially significant roles these factors play in inflammatory disease. These results suggest an underlying master regulatory mechanism, such that certain factors are essential but not individually sufficient to trigger NETosis.
To our knowledge, we report the first ex-vivo model using naturally occurring cytokines and chemokines to induce NETosis in whole blood. These findings emphasize the importance of expanding our understanding of neutrophil physiology in a biologically relevant context with physiological triggers to induce NETosis. This approach could reveal new dimensions in our understanding of disease pathology and risk factors and might unearth potential therapeutic targets providing novel strategies for disease intervention and treatment. Further investigation of these factors is underway to further understand the release of NETs in natural and pathological states. Less
Whole blood was collected from healthy donors and aliquoted into a 384 well plate using a Formulatrix Mantis liquid handler. This plate contained small molecules associated with neutrophils or NETosis activation, such as interleukins: Il-1b, IL-5, IL-6, IL-8, IL-15, IL-17, IL-18 and other molecules TNF- α, LT-α, IFN-γ, G-CSF, GM-CSF, E-selectin, PAF-16, CXCL1, CXCL2, LTB4, CXCL5, CCL2, CCL3, fMLP, Ferritin, HMGB1, C5a and LPS. We used a combinatorial pooling strategy designed using JMP software to identify which combinations of small molecules could stimulate NET formation. NETosis was assessed using Sytox green intercalation at 5 minute intervals for up to 24 hours using a Molecular Devices plate reader. PMA was utilized as a positive control for NETosis induction at varying concentrations.
Using our combinatorial pooling approach of the various factors, we successfully induced NETosis in an ex vivo whole blood system using naturally occurring cytokines and chemokines at physiologically relevant concentrations. We found that different combinations of factors evoke distinct neutrophil responses both in the time of NET generation and/or magnitude of NET-associated intercalation signal. We observed inter-donor variability in response time and amplitude however, similar small molecule pools induced consistent responses across donors. Furthermore, our findings suggest that at least four naturally occurring factors are necessary to induce NETosis in our system. Although some factors activate similar pathways, they are unable to induce a signal alone and as the number of factors increased beyond four, there was an enhanced NET response. Interestingly, we found either TNF-α or LT-α was required to cause a NETosis response, underlining the potentially significant roles these factors play in inflammatory disease. These results suggest an underlying master regulatory mechanism, such that certain factors are essential but not individually sufficient to trigger NETosis.
To our knowledge, we report the first ex-vivo model using naturally occurring cytokines and chemokines to induce NETosis in whole blood. These findings emphasize the importance of expanding our understanding of neutrophil physiology in a biologically relevant context with physiological triggers to induce NETosis. This approach could reveal new dimensions in our understanding of disease pathology and risk factors and might unearth potential therapeutic targets providing novel strategies for disease intervention and treatment. Further investigation of these factors is underway to further understand the release of NETs in natural and pathological states. Less
The phospholipase D PLD family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids PLD is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes yielding a soluble active form The deficiency of PLD leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors While the mechanism of PLD phospholipase activity has been extensively characterized not much is known about how PLDs bind and hydrolyze nucleic acids Here we determined the high-resolution crystal structure of the luminal N-glycosylated domain of ... More
The phospholipase D (PLD) family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids. PLD3 is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes, yielding a soluble active form. The deficiency of PLD3 leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors. While the mechanism of PLD phospholipase activity has been extensively characterized, not much is known about how PLDs bind and hydrolyze nucleic acids. Here, we determined the high-resolution crystal structure of the luminal N-glycosylated domain of human PLD3 in its apo- and single-stranded DNA-bound forms. PLD3 has a typical phospholipase fold and forms homodimers with two independent catalytic centers via a newly identified dimerization interface. The structure of PLD3 in complex with an ssDNA-derived thymidine product in the catalytic center provides insights into the substrate binding mode of nucleic acids in the PLD family. Our structural data suggest a mechanism for substrate binding and nuclease activity in the PLD family and provide the structural basis to design immunomodulatory drugs targeting PLD3. Less
The pathogen Paracoccidioides lutzii Pb is found in South America countries Colombia Ecuador Venezuela and Brazil especially in the central west and north regions of the latter It belongs to the Ajellomycetaceae family Onygenales order and is typically thermodimorphic presenting yeast cells when it grows in animal tissues but mycelia when in the environment where it produces the infectious propagule This fungus is one of the etiologic agents of Paracoccidioidomycosis PCM the most important endemic fungal infection in Latin America Investigations on its genome have contributed to a better understanding about its metabolism and revealed the complexity of several metabolic ... More
The pathogen Paracoccidioides lutzii (Pb01) is found in South America countries Colombia, Ecuador, Venezuela and Brazil, especially in the central, west, and north regions of the latter. It belongs to the Ajellomycetaceae family, Onygenales order, and is typically thermodimorphic, presenting yeast cells when it grows in animal tissues, but mycelia when in the environment, where it produces the infectious propagule. This fungus is one of the etiologic agents of Paracoccidioidomycosis (PCM), the most important endemic fungal infection in Latin America. Investigations on its genome have contributed to a better understanding about its metabolism and revealed the complexity of several metabolic glycolytic pathways. Glyceraldehyde-3-Phosphate Dehydrogenase from Paracoccidioides lutzii (PlGAPDH) is considered a moonlighting protein and participates in several biological processes of this pathogen. The enzyme was expressed and purified, as seen in SDS-PAGE gel, crystallized and had its three dimensional structure (3D) determined in complex with NAD+, a sulphate ion and d-galactonic acid, therefore, a type of ‘GAA site’. It is the first GAPDH structure to show this chemical type in this site and how this protein can bind an acid derived from oxidation of a linear hexose. Less
The efficacy of aminoglycoside antibiotics is waning due to the acquisition of diverse resistance mechanisms by bacteria Among the most prevalent are aminoglycoside acetyltransferases AACs that inactivate the antibiotics through acetyl coenzyme A-mediated modification Most AACs are members of the GCN superfamily of acyltransferases which lack conserved active site residues that participate in catalysis ApmA is the first reported AAC belonging to the left-handed -helix superfamily These enzymes are characterized by an essential active site histidine that acts as an active site base Here we show that ApmA confers broad-spectrum aminoglycoside resistance with a molecular mechanism that diverges from other ... More
The efficacy of aminoglycoside antibiotics is waning due to the acquisition of diverse resistance mechanisms by bacteria. Among the most prevalent are aminoglycoside acetyltransferases (AACs) that inactivate the antibiotics through acetyl coenzyme A-mediated modification. Most AACs are members of the GCN5 superfamily of acyltransferases which lack conserved active site residues that participate in catalysis. ApmA is the first reported AAC belonging to the left-handed β-helix superfamily. These enzymes are characterized by an essential active site histidine that acts as an active site base. Here we show that ApmA confers broad-spectrum aminoglycoside resistance with a molecular mechanism that diverges from other detoxifying left-handed β-helix superfamily enzymes and canonical GCN5 AACs. We find that the active site histidine plays different functions depending on the acetyl-accepting aminoglycoside substrate. This flexibility in the mechanism of a single enzyme underscores the plasticity of antibiotic resistance elements to co-opt protein catalysts in the evolution of drug detoxification. Less
This study aimed to identify inhibitors of the translocated intimin receptor Tir of enteropathogenic Escherichia coli EPEC EPEC is an intestinal pathogen that causes diarrhea and is a major health concern worldwide Because Tir is a key virulence factor involved in EPEC pathogenesis inhibiting its function is a potential strategy for controlling EPEC infections Virtual screening was applied to chemical libraries to search for compounds that inhibit Tir-mediated bacterial adherence to host cells Three sites were targeted using the cocrystal structure published earlier A selection of compounds was then assessed in a cell-based infection model and fluorescence microscopy assay The ... More
This study aimed to identify inhibitors of the translocated intimin receptor (Tir) of enteropathogenic Escherichia coli (EPEC). EPEC is an intestinal pathogen that causes diarrhea and is a major health concern worldwide. Because Tir is a key virulence factor involved in EPEC pathogenesis, inhibiting its function is a potential strategy for controlling EPEC infections. Virtual screening was applied to chemical libraries to search for compounds that inhibit Tir-mediated bacterial adherence to host cells. Three sites were targeted using the cocrystal structure published earlier. A selection of compounds was then assessed in a cell-based infection model and fluorescence microscopy assay. The results of this study provide a basis for further optimization and testing of Tir inhibitors as potential therapeutic agents for EPEC infections. Less
Gonorrhea a prevalent sexually transmitted infection affecting millions annually worldwide is caused by Neisseria gonorrhoeae Ngo a superbug resistant to all antibiotic classes Compounding the challenge the absence of protective immunity upon infection allows for reinfection and a viable vaccine against gonococcal infection remains elusive In response to infection the human host deploys nutritional immunity sequestering essential metals like iron from invading bacteria using metal binding proteins To overcome this Ngo employs outer-membrane TonB-dependent transporters TdTs like HpuAB to acquire iron from host metal binding proteins such as hemoglobin Hb Part of our study focused on HpuA the lipoprotein component ... More
Gonorrhea, a prevalent sexually transmitted infection affecting millions annually worldwide, is caused by Neisseria gonorrhoeae (Ngo), a "superbug" resistant to all antibiotic classes. Compounding the challenge, the absence of protective immunity upon infection allows for reinfection, and a viable vaccine against gonococcal infection remains elusive.
In response to infection, the human host deploys nutritional immunity, sequestering essential metals like iron from invading bacteria, using metal binding proteins. To overcome this, Ngo employs outer-membrane TonB-dependent transporters (TdTs), like HpuAB, to acquire iron from host metal binding proteins, such as hemoglobin (Hb).
Part of our study focused on HpuA, the lipoprotein component of the HpuAB system. Mutations targeting hydrophobic residues crucial for Hb interaction were studied. Results demonstrated that without HpuB, strains failed to grow, emphasizing HpuB's role in iron internalization. Notably, when HpuB is produced, deletion and insertion mutations in loop 2 of HpuA affected growth and binding to Hb.
Further investigations into HpuB, the transmembrane protein of the HpuAB system, uncovered essential loop regions for binding and growth on Hb as a sole iron source. Deletion mutations in loops 2, 3, and 4 facilitated binding and growth independently of HpuA production. Intriguingly, mutations in loop 7 abrogated binding and impaired growth in the absence of HpuA, but partial growth and full binding recovery occurred when HpuA was present. This highlighted the importance of loop 7 in iron acquisition and suggested a potential role for both HpuA and HpuB in the binding Hb.
As a combination of non-binding TdT mutants is hypothesized to have the potential to improve vaccine efficacy and provide protection, identifying non-binding HpuB mutants could be important. In summary, this research sheds light on the intricacies of the HpuAB system, contributing valuable insights that could inform the development of an effective gonorrhea vaccine. Less
In response to infection, the human host deploys nutritional immunity, sequestering essential metals like iron from invading bacteria, using metal binding proteins. To overcome this, Ngo employs outer-membrane TonB-dependent transporters (TdTs), like HpuAB, to acquire iron from host metal binding proteins, such as hemoglobin (Hb).
Part of our study focused on HpuA, the lipoprotein component of the HpuAB system. Mutations targeting hydrophobic residues crucial for Hb interaction were studied. Results demonstrated that without HpuB, strains failed to grow, emphasizing HpuB's role in iron internalization. Notably, when HpuB is produced, deletion and insertion mutations in loop 2 of HpuA affected growth and binding to Hb.
Further investigations into HpuB, the transmembrane protein of the HpuAB system, uncovered essential loop regions for binding and growth on Hb as a sole iron source. Deletion mutations in loops 2, 3, and 4 facilitated binding and growth independently of HpuA production. Intriguingly, mutations in loop 7 abrogated binding and impaired growth in the absence of HpuA, but partial growth and full binding recovery occurred when HpuA was present. This highlighted the importance of loop 7 in iron acquisition and suggested a potential role for both HpuA and HpuB in the binding Hb.
As a combination of non-binding TdT mutants is hypothesized to have the potential to improve vaccine efficacy and provide protection, identifying non-binding HpuB mutants could be important. In summary, this research sheds light on the intricacies of the HpuAB system, contributing valuable insights that could inform the development of an effective gonorrhea vaccine. Less
MbtI from Mycobacterium tuberculosis Mtb is a Mg -dependent salicylate synthase belonging to the chorismate-utilizing enzyme CUE family As a fundamental player in iron acquisition MbtI promotes the survival and pathogenicity of Mtb in the infected host Hence it has emerged in the last decade as an innovative potential target for the anti-virulence therapy of tuberculosis In this context -phenylfuran- -carboxylic acids have been identified as potent MbtI inhibitors The first co-crystal structure of MbtI in complex with a member of this class was described in showing the enzyme adopting an open configuration Due to the high mobility of the ... More
MbtI from Mycobacterium tuberculosis (Mtb) is a Mg2+-dependent salicylate synthase, belonging to the chorismate-utilizing enzyme (CUE) family. As a fundamental player in iron acquisition, MbtI promotes the survival and pathogenicity of Mtb in the infected host. Hence, it has emerged in the last decade as an innovative, potential target for the anti-virulence therapy of tuberculosis. In this context, 5-phenylfuran-2-carboxylic acids have been identified as potent MbtI inhibitors. The first co-crystal structure of MbtI in complex with a member of this class was described in 2020, showing the enzyme adopting an open configuration. Due to the high mobility of the loop adjacent to the binding pocket, large portions of the amino acid chain were not defined in the electron density map, hindering computational efforts aimed at structure-driven ligand optimization. Herein, we report a new, high-resolution co-crystal structure of MbtI with a furan-based derivative, in which the closed configuration of the enzyme allowed tracing the entirety of the active site pocket in the presence of the bound inhibitor. Moreover, we describe a new crystal structure of MbtI in open conformation and in complex with the known inhibitor methyl-AMT, suggesting that in vitro potency is not related to the observed enzyme conformation. These findings will prove fundamental to enhance the potency of this series via rational structure-based drug-design approaches. Less
Norovirus is the leading cause of viral gastroenteritis worldwide and there are no approved vaccines or therapeutic treatments for chronic or severe norovirus infections The structural characterisation of the norovirus protease and drug development has predominantly focused upon GI noroviruses despite most global outbreaks being caused by GII noroviruses Here we determined the crystal structures of the GII Sydney ligand-free norovirus protease at and at with a covalently bound high-affinity IC M protease inhibitor NV- We show that the active sites of the ligand-free protease structure are present in both open and closed conformations as determined by their Arg side ... More
Norovirus is the leading cause of viral gastroenteritis worldwide, and there are no approved vaccines or therapeutic treatments for chronic or severe norovirus infections. The structural characterisation of the norovirus protease and drug development has predominantly focused upon GI.1 noroviruses, despite most global outbreaks being caused by GII.4 noroviruses. Here, we determined the crystal structures of the GII.4 Sydney 2012 ligand-free norovirus protease at 2.79 Å and at 1.83 Å with a covalently bound high-affinity (IC50 = 0.37 µM) protease inhibitor (NV-004). We show that the active sites of the ligand-free protease structure are present in both open and closed conformations, as determined by their Arg112 side chain orientation. A comparative analysis of the ligand-free and ligand-bound protease structures reveals significant structural differences in the active site cleft and substrate-binding pockets when an inhibitor is covalently bound. We also report a second molecule of NV-004 non-covalently bound within the S4 substrate binding pocket via hydrophobic contacts and a water-mediated hydrogen bond. These new insights can guide structure-aided drug design against the GII.4 genogroup of noroviruses. Less
Kinases are important therapeutic targets and their inhibitors are classified according to their mechanism of action which range from blocking ATP binding to covalent inhibition Here a mechanism of inhibition is highlighted by capturing p -activated kinase PAK in an intermediate state of activation using an Affimer reagent that binds in the P pocket PAK was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells Silencing of PAK resulted in reduced cell number G S arrest and enlargement of cells suggesting it to be important in urothelial cancer cell line survival and proliferation Affimer reagents were isolated ... More
Kinases are important therapeutic targets, and their inhibitors are classified according to their mechanism of action, which range from blocking ATP binding to covalent inhibition. Here, a mechanism of inhibition is highlighted by capturing p21-activated kinase 5 (PAK5) in an intermediate state of activation using an Affimer reagent that binds in the P+1 pocket. PAK5 was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells. Silencing of PAK5 resulted in reduced cell number, G1/S arrest, and enlargement of cells, suggesting it to be important in urothelial cancer cell line survival and proliferation. Affimer reagents were isolated to identify mechanisms of inhibition. The Affimer PAK5-Af17 recapitulated the phenotype seen with siRNA. Co-crystallization revealed that PAK5-Af17 bound in the P+1 pocket of PAK5, locking the kinase into a partial activation state. This mechanism of inhibition indicates that another class of kinase inhibitors is possible. Less
Poly ADP-ribose polymerase PARP inhibitors are used in the clinic to treat BRCA-deficient breast ovarian and prostate cancers As their efficacy is potentiated by loss of the nucleotide salvage factor DNPH there is considerable interest in the development of highly specific small molecule DNPH inhibitors Here we present X-ray crystal structures of dimeric DNPH bound to its substrate hydroxymethyl deoxyuridine monophosphate hmdUMP Direct interaction with the hydroxymethyl group is important for substrate positioning while conserved residues surrounding the base facilitate target discrimination Glycosidic bond cleavage is driven by a conserved catalytic triad and proceeds via a two-step mechanism involving formation ... More
Poly(ADP-ribose) polymerase (PARP) inhibitors are used in the clinic to treat BRCA-deficient breast, ovarian and prostate cancers. As their efficacy is potentiated by loss of the nucleotide salvage factor DNPH1 there is considerable interest in the development of highly specific small molecule DNPH1 inhibitors. Here, we present X-ray crystal structures of dimeric DNPH1 bound to its substrate hydroxymethyl deoxyuridine monophosphate (hmdUMP). Direct interaction with the hydroxymethyl group is important for substrate positioning, while conserved residues surrounding the base facilitate target discrimination. Glycosidic bond cleavage is driven by a conserved catalytic triad and proceeds via a two-step mechanism involving formation and subsequent disruption of a covalent glycosyl-enzyme intermediate. Mutation of a previously uncharacterised yet conserved glutamate traps the intermediate in the active site, demonstrating its role in the hydrolytic step. These observations define the enzyme’s catalytic site and mechanism of hydrolysis, and provide important insights for inhibitor discovery. Less
Transglutaminases TGases are a family of calcium-dependent enzymes primarily known for their ability to cross-link proteins Transglutaminase TG is one isozyme in this family whose role is multifaceted TG can act not only as a typical transamidase through its catalytic core but also as a G-protein via its GTP binding site These two discrete activities are tightly regulated by both environmental stimuli and redox reactions Ubiquitously expressed in humans TG has been implicated in numerous disease pathologies that require extensive investigation The catalytic activity of TG can be monitored through various mechanisms including hydrolysis transamidation or cleavage of isopeptide bonds ... More
Transglutaminases (TGases) are a family of calcium-dependent enzymes primarily known for their ability to cross-link proteins. Transglutaminase 2 (TG2) is one isozyme in this family whose role is multifaceted. TG2 can act not only as a typical transamidase through its catalytic core but also as a G-protein via its GTP binding site. These two discrete activities are tightly regulated by both environmental stimuli and redox reactions. Ubiquitously expressed in humans, TG2 has been implicated in numerous disease pathologies that require extensive investigation. The catalytic activity of TG2 can be monitored through various mechanisms, including hydrolysis, transamidation, or cleavage of isopeptide bonds. Activity assays are required to monitor the activity of this isozyme not only for studying its transamidation reaction but also for validation of therapeutics designed to abolish this activity. Herein, we present the design, synthesis, and evaluation of a new TG2 activity substrate based on a previously optimized inhibitor scaffold. The substrate APH7 exhibits excellent affinity, selectivity, and reactivity with TG2 (KM = 3.0 μM). Furthermore, its application also allowed the discovery of unique hysteresis at play within the catalytic activity and inhibition reactivity of TG2. Less
Acute gastroenteritis caused by human noroviruses HuNoVs is a significant global health and economic burden and is without licensed vaccines or antiviral drugs The GII HuNoV causes most epidemics worldwide This virus undergoes epochal evolution with periodic emergence of variants with new antigenic profiles and altered specificity for histo-blood group antigens HBGA the determinants of cell attachment and susceptibility hampering the development of immunotherapeutics Here we show that a llama-derived nanobody M neutralizes multiple GII variants with high potency in human intestinal enteroids The crystal structure of M complexed with the protruding domain of the GII capsid protein VP revealed ... More
Acute gastroenteritis caused by human noroviruses (HuNoVs) is a significant global health and economic burden and is without licensed vaccines or antiviral drugs. The GII.4 HuNoV causes most epidemics worldwide. This virus undergoes epochal evolution with periodic emergence of variants with new antigenic profiles and altered specificity for histo-blood group antigens (HBGA), the determinants of cell attachment and susceptibility, hampering the development of immunotherapeutics. Here, we show that a llama-derived nanobody M4 neutralizes multiple GII.4 variants with high potency in human intestinal enteroids. The crystal structure of M4 complexed with the protruding domain of the GII.4 capsid protein VP1 revealed a conserved epitope, away from the HBGA binding site, fully accessible only when VP1 transitions to a “raised” conformation in the capsid. Together with dynamic light scattering and electron microscopy of the GII.4 VLPs, our studies suggest a mechanism in which M4 accesses the epitope by altering the conformational dynamics of the capsid and triggering its disassembly to neutralize GII.4 infection. Less
Objectives In order to treat a rat model of rotator cuff rupture this work concentrated on the expression of TNMD and RUNX followed by rotator cuff repair and secretome-hMSCs Methods A total of thirty -weeks-old male Sprague Dawley rats were separated into five groups randomly RC on week lesion treated with a rotator cuff repair and saline RC NaCl group n for and weeks and lesion treated with a rotator cuff repair and secretome-hMSCs RC secretome-hMSC group n for and weeks The supraspinatus and infraspinatus muscle tendon units were obtained for histological and biomechanical investigation at and weeks following injury ... More
Objectives: In order to treat a rat model of rotator cuff rupture, this work concentrated on the expression
of TNMD and RUNX2, followed by rotator cuff repair and secretome-hMSCs.
Methods: A total of thirty 10-weeks-old male Sprague–Dawley rats were separated into five groups randomly, RC
on week 0, lesion treated with a rotator cuff repair and saline (RC + NaCl group, n = 6) for 2 and 8 weeks, and lesion
treated with a rotator cuff repair and secretome-hMSCs (RC + secretome-hMSC group, n = 6) for 2 and 8 weeks.
The supraspinatus and infraspinatus muscle–tendon units were obtained for histological and biomechanical
investigation at 0, 2 and 8 weeks following injury.
Results: The findings showed that, in comparison with the RC + NaCl group, secretome-hMSCs significantly
improved tendon repair by upregulating TNMD and RUNX2 expression and histology score.
Conclusion: Combining Secretome-hypoxia MSCs with RC healing may help rats with rotator cuff tears.
Level of evidence: IV Less
of TNMD and RUNX2, followed by rotator cuff repair and secretome-hMSCs.
Methods: A total of thirty 10-weeks-old male Sprague–Dawley rats were separated into five groups randomly, RC
on week 0, lesion treated with a rotator cuff repair and saline (RC + NaCl group, n = 6) for 2 and 8 weeks, and lesion
treated with a rotator cuff repair and secretome-hMSCs (RC + secretome-hMSC group, n = 6) for 2 and 8 weeks.
The supraspinatus and infraspinatus muscle–tendon units were obtained for histological and biomechanical
investigation at 0, 2 and 8 weeks following injury.
Results: The findings showed that, in comparison with the RC + NaCl group, secretome-hMSCs significantly
improved tendon repair by upregulating TNMD and RUNX2 expression and histology score.
Conclusion: Combining Secretome-hypoxia MSCs with RC healing may help rats with rotator cuff tears.
Level of evidence: IV Less
Multidrug efflux is a well-established mechanism of drug resistance in bacterial pathogens like Salmonella Typhi styMdtM locus name STY is a multidrug efflux transporter of the major facilitator superfamily expressed in S Typhi Functional assays identified several residues important for its transport activity Here we used an AlphaFold model to identify additional residues for analysis by mutagenesis Mutation of peripheral residue Cys had no effect on the structure or function of the transporter However substitution of channel-lining residues Tyr and Tyr completely abolished transport function Finally mutation of Gln which faces peripheral helices of the transporter resulted in the loss ... More
Multidrug efflux is a well-established mechanism of drug resistance in bacterial pathogens like Salmonella Typhi. styMdtM (locus name; STY4874) is a multidrug efflux transporter of the major facilitator superfamily expressed in S. Typhi. Functional assays identified several residues important for its transport activity. Here, we used an AlphaFold model to identify additional residues for analysis by mutagenesis. Mutation of peripheral residue Cys185 had no effect on the structure or function of the transporter. However, substitution of channel-lining residues Tyr29 and Tyr231 completely abolished transport function. Finally, mutation of Gln294, which faces peripheral helices of the transporter, resulted in the loss of transport of some substrates. Crystallization studies yielded diffraction data for the wild-type protein at 4.5 Å resolution and allowed the unit cell parameters to be established as a = b = 64.3 Å, c = 245.4 Å, α = β = γ = 90°, in space group P4. Our studies represent a further stepping stone towards a mechanistic understanding of the clinically important multidrug transporter styMdtM. Less
The thermophilic bacterium Clostridium thermocellum efficiently degrades polysaccharides into oligosaccharides The metabolism of - -linked cello-oligosaccharides is initiated by three enzymes i e the cellodextrin phosphorylase Cdp the cellobiose phosphorylase Cbp and the -glucosidase A BglA in C thermocellum In comparison how the oligosaccharides containing other kinds of linkage are utilized is rarely understood In this study we found that BglA could hydrolyze the - -disaccharide laminaribiose with much higher activity than that against the - -disaccharide cellobiose The structural basis of the substrate specificity was analyzed by crystal structure determination and molecular docking Genetic deletions of BglA and Cbp ... More
The thermophilic bacterium Clostridium thermocellum efficiently degrades polysaccharides into oligosaccharides. The metabolism of β-1,4-linked cello-oligosaccharides is initiated by three enzymes, i.e., the cellodextrin phosphorylase (Cdp), the cellobiose phosphorylase (Cbp), and the β-glucosidase A (BglA), in C. thermocellum. In comparison, how the oligosaccharides containing other kinds of linkage are utilized is rarely understood. In this study, we found that BglA could hydrolyze the β-1,3-disaccharide laminaribiose with much higher activity than that against the β-1,4-disaccharide cellobiose. The structural basis of the substrate specificity was analyzed by crystal structure determination and molecular docking. Genetic deletions of BglA and Cbp, respectively, and enzymatic analysis of cell extracts demonstrated that BglA is the key enzyme responsible for laminaribiose metabolism. Furthermore, the deletion of BglA can suppress the expression of Cbp and the deletion of Cbp can up-regulate the expression of BglA, indicating that BglA and Cbp have cross-regulation and BglA is also critical for cellobiose metabolism. These insights pave the way for both a fundamental understanding of metabolism and regulation in C. thermocellum and emphasize the importance of the degradation and utilization of polysaccharides containing β-1,3-linked glycosidic bonds in lignocellulose biorefinery. Less
Septins are membrane-associated GTP-binding proteins that are present in most eukaryotes They polymerize to play important roles as scaffolds and or diffusion barriers as part of the cytoskeleton -Helical coiled-coil domains are believed to contribute to septin assembly and those observed in both human SEPT and SEPT form antiparallel homodimers These are not compatible with their parallel heterodimeric organization expected from the current model for protofilament assembly but they could explain the interfilament cross-bridges observed by microscopy Here the first structure of a heterodimeric septin coiled coil is presented that between SEPT and SEPT the former is a SEPT SEPT ... More
Septins are membrane-associated, GTP-binding proteins that are present in most eukaryotes. They polymerize to play important roles as scaffolds and/or diffusion barriers as part of the cytoskeleton. α-Helical coiled-coil domains are believed to contribute to septin assembly, and those observed in both human SEPT6 and SEPT8 form antiparallel homodimers. These are not compatible with their parallel heterodimeric organization expected from the current model for protofilament assembly, but they could explain the interfilament cross-bridges observed by microscopy. Here, the first structure of a heterodimeric septin coiled coil is presented, that between SEPT14 and SEPT7; the former is a SEPT6/SEPT8 homolog. This new structure is parallel, with two long helices that are axially shifted by a full helical turn with reference to their sequence alignment. The structure also has unusual knobs-into-holes packing of side chains. Both standard seven-residue (heptad) and the less common 11-residue (hendecad) repeats are present, creating two distinct regions with opposite supercoiling, which gives rise to an overall straight coiled coil. Part of the hendecad region is required for heterodimerization and therefore may be crucial for selective septin recognition. These unconventional sequences and structural features produce a metastable heterocomplex that nonetheless has enough specificity to promote correct protofilament assembly. For instance, the lack of supercoiling may facilitate unzipping and transitioning to the antiparallel homodimeric state. Less