Publications

@article{noKey,

title = {A bacterial immunity protein directly senses two disparate phage proteins},

author = {Zhang, Tong},

url = {https://www.nature.com/articles/s41586-024-08039-y},

doi = {https://doi.org/10.1038/s41586-024-08039-y},

year  = {2024},

date = {2024-10-15},

journal = {Nature Conferences},

abstract = {Eukaryotic innate immune systems use pattern recognition receptors to sense infection by detecting pathogen-associated molecular patterns, which then triggers an immune response. Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators, known as bacteriophages1,2,3,4,5,6. Although different immunity proteins can recognize different phage-encoded triggers, individual bacterial immunity proteins have been found to sense only a single trigger during infection, suggesting a one-to-one relationship between bacterial pattern recognition receptors and their ligands7,8,9,10,11. Here we demonstrate that the antiphage defence protein CapRelSJ46 in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using the same sensory domain, with overlapping but distinct interfaces. Our results highlight the notable versatility of an immune sensory domain, which may be a common property of antiphage defence systems that enables them to keep pace with their rapidly evolving viral predators. We found that Bas11 phages harbour both trigger proteins that are sensed by CapRelSJ46 during infection, and we demonstrate that such phages can fully evade CapRelSJ46 defence only when both triggers are mutated. Our work shows how a bacterial immune system that senses more than one trigger can help prevent phages from easily escaping detection, and it may allow the detection of a broader range of phages. More generally, our findings illustrate unexpected multifactorial sensing by bacterial defence systems and complex coevolutionary relationships between them and their phage-encoded triggers.},

keywords = {NT8®},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {A single Leishmania adenylate forming enzyme of the ANL superfamily generates both acetyl- and acetoacetyl-CoA},

author = {J. Jezewski, Andrew},

url = {https://www.jbc.org/article/S0021-9258(24)02381-0/fulltext},

doi = {https://doi.org/10.1016/j.jbc.2024.107879},

year  = {2024},

date = {2024-10-08},

journal = {Journal of Biological Chemistry},

abstract = {Leishmania, a protozoan parasite, is responsible for significant morbidity and mortality worldwide, manifesting as cutaneous, mucocutaneous, and visceral leishmaniasis. These diseases pose a substantial burden, especially in impoverished regions with limited access to effective medical treatments. Current therapies are toxic, have low efficacy, and face growing resistance. Understanding the metabolic pathways of Leishmania, particularly those differing from its host, can unveil potential therapeutic targets. In this study, we investigated the acetyl-CoA synthetase (ACS) enzyme from Leishmania infantum (LiAcs1), which, unlike many organisms, also exhibits acetoacetyl-CoA synthetase (KBC) activity. This dual functionality is unique among ANL superfamily enzymes and crucial for the parasite's reliance on leucine catabolism, energy production and sterol biosynthesis. Our biochemical characterization of LiAcs1 revealed its ability to utilize both acetate and acetoacetate substrates. Additionally, LiAcs1 displayed a distinct CoA substrate inhibition pattern, partially alleviated by acetoacetate. Structural analysis provided insights into the substrate binding flexibility of LiAcs1, highlighting a more promiscuous substrate pocket compared to other ACS or KBC-specific enzymes. Substrate mimetics elucidated its ability to accommodate both small and large AMP-ester derivatives, contributing to its dual ACS/KBC functionality. These findings not only advance our understanding of Leishmania metabolism but also present LiAcs1 as a promising drug target. The dual functionality of LiAcs1 underscores the potential for developing selective inhibitors that could disrupt critical metabolic pathways across Leishmania spp. as it appears this enzyme is highly conserved across this genus. This paves the way for developing novel effective treatments against this devastating disease.},

keywords = {NT8®},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Targeting RSV-neutralizing B cell receptors with anti-idiotypic antibodies},

author = {C. Scharffenberger, Samuel},

url = {https://www.cell.com/cell-reports/fulltext/S2211-1247(24)01162-8},

doi = {https://doi.org/10.1016/j.celrep.2024.114811},

year  = {2024},

date = {2024-10-07},

journal = {Cell reports},

abstract = {Respiratory syncytial virus (RSV) causes lower respiratory tract infections with significant morbidity and mortality at the extremes of age. Vaccines based on the viral fusion protein are approved for adults over 60, but infant protection relies on passive immunity via antibody transfer or maternal vaccination. An infant vaccine that rapidly elicits protective antibodies would fulfill a critical unmet need. Antibodies arising from the VH3-21/VL1-40 gene pairing can neutralize RSV without the need for affinity maturation, making them attractive to target through vaccination. Here, we develop an anti-idiotypic monoclonal antibody (ai-mAb) immunogen that is specific for unmutated VH3-21/VL1-40 B cell receptors (BCRs). The ai-mAb efficiently engages B cells with bona fide target BCRs and does not activate off-target non-neutralizing B cells, unlike recombinant pre-fusion (preF) protein used in current RSV vaccines. These results establish proof of concept for using an ai-mAb-derived vaccine to target B cells hardwired to produce RSV-neutralizing antibodies.},

keywords = {NT8®},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Structure-Based Engineering of Monoclonal Antibodies for Improved Binding to Counteract the Effects of Fentanyl and Carfentanil},

author = {Rodarte, Justas},

url = {https://pubs.acs.org/doi/full/10.1021/acsomega.4c06617},

doi = {https://doi.org/10.1021/acsomega.4c06617},

year  = {2024},

date = {2024-10-06},

journal = {ACS publications},

abstract = {The opioid overdose epidemic is a growing and evolving public health crisis fueled by the widespread presence of fentanyl and fentanyl analogues (F/FAs) in both street mixtures and counterfeit pills. To expand current treatment options, drug-targeting monoclonal antibodies (mAbs) offer a viable therapeutic for both pre- and postexposure clinical scenarios. This study reports the isolation, in vitro characterization, and in vivo efficacy of two murine mAb families targeting fentanyl, carfentanil, or both. Because humanization of the mAbs by CDR grafting negatively impacted affinity for both fentanyl and carfentanil, crystal structures of mAbs in complex with fentanyl or carfentanil were analyzed to identify key residues involved in ligand binding in murine versus humanized structures, and site-directed mutagenesis was used to verify their functional importance. The structural analysis identified a framework residue, Tyr36, present in the murine germline sequence of two mAbs, which was critical for binding to fentanyl and carfentanil. These studies emphasize the importance of structural considerations in mAb engineering to optimize mAbs targeting small molecules including opioids and other drugs of public health interest.},

keywords = {NT8®},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Crystal structure of Alzheimer's disease phospholipase D3 provides a molecular basis for understanding its normal and pathological functions},

author = {Ishii, Kenta},

url = {https://febs.onlinelibrary.wiley.com/doi/full/10.1111/febs.17277},

doi = {https://doi.org/10.1111/febs.17277},

year  = {2024},

date = {2024-09-25},

journal = {The FEBS journal},

abstract = {Human 5′-3′ exonuclease PLD3, a member of the phospholipase D family of enzymes, has been validated as a therapeutic target for treating Alzheimer's disease. Here, we have determined the crystal structure of the luminal domain of the enzyme at 2.3 Å resolution, revealing a bilobal structure with a catalytic site located between the lobes. We then compared the structure with published crystal structures of other human PLD family members which revealed that a number of catalytic and lipid recognition residues, previously shown to be key for phospholipase activity, are not conserved or, are absent. This led us to test whether the enzyme is actually a phospholipase. We could not measure any phospholipase activity but the enzyme shows robust nuclease activity. Finally, we have mapped key single nucleotide polymorphisms onto the structure which reveals plausible reasons as to why they have an impact on Alzheimer's disease.},

keywords = {NT8®, Rock Imager®},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {The Human T-cell Leukemia Virus capsid protein is a potential drug target},

author = {Yu, Ruijie},

url = {https://www.biorxiv.org/content/10.1101/2024.09.09.612167v1.abstract},

doi = {https://doi.org/10.1101/2024.09.09.612167},

year  = {2024},

date = {2024-09-09},

journal = {Preprint},

abstract = {Human T-cell Leukemia Virus type 1 (HTLV-1) is an untreatable retrovirus that causes lethal malignancies and degenerative inflammatory conditions. Effective treatments have been delayed by substantial gaps in our knowledge of the fundamental virology, especially when compared to the closely related virus, HIV. A recently developed and highly effective anti-HIV strategy is to target the virus with drugs that interfere with capsid integrity and interactions with the host. Importantly, the first in class anti-capsid drug approved, lenacapavir, can provide long-acting pre-exposure prophylaxis. Such a property would provide a means to prevent the transmission of HTLV-1, but its capsid has not previously been considered as a drug target. Here we describe the first high-resolution crystal structures of the HTLV-1 capsid protein, define essential lattice interfaces, and identify a previously unknown ligand-binding pocket. We show that this pocket is essential for virus infectivity, providing a potential target for future anti-capsid drug development.},

keywords = {NT8®},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Short CDRL1 in intermediate VRC01-like mAbs is not sufficient to overcome key glycan barriers on HIV-1 Env},

author = {Agrawal, Parul},

url = {https://journals.asm.org/doi/epub/10.1128/jvi.00744-24},

doi = {https://doi.org/10.1128/jvi.00744-24},

year  = {2024},

date = {2024-09-05},

journal = {Journal of Virology},

abstract = {VRC01-class broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV-1, but they have not yet been elicited by vaccination. They are extensively somatically mutated and sometimes accumulate CDRL1 deletions. Such indels may allow VRC01-class antibodies to accommodate the glycans expressed on a conserved N276 N-linked glycosylation site in loop D of the gp120 subunit. These glycans constitute a major obstacle in the development of VRC01-class antibodies, as unmutated antibody forms are unable to accommodate them. Although immunizations of knock-in mice expressing human VRC01-class B-cell receptors (BCRs) with specifically designed Env-derived immunogens lead to the accumulation of somatic mutations in VRC01-class BCRs, CDRL1 deletions are rarely observed, and the elicited antibodies display narrow neutralizing activities. The lack of broad neutralizing potential could be due to the absence of deletions, the lack of appropriate somatic mutations, or both. To address this point, we modified our previously determined prime-boost immunization with a germline-targeting immunogen nanoparticle (426c.Mod.Core), followed by a heterologous core nanoparticle (HxB2.WT.Core), by adding a final boost with a cocktail of various stabilized soluble Env trimers. We isolated VRC01-like antibodies with extensive somatic mutations and, in one case, a seven-amino acid CDRL1 deletion. We generated chimeric antibodies that combine the vaccine-elicited somatic mutations with CDRL1 deletions present in human mature VRC01 bnAbs. We observed that CDRL1 indels did not improve the neutralizing antibody activities. Our study indicates that CDRL1 length by itself is not sufficient for the broadly neutralizing phenotype of this class of antibodies.},

keywords = {NT8®},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Dynamic and structural insights into allosteric regulation on MKP5 a dual-specificity phosphatase},

author = {Skeens, Erin},

url = {https://www.biorxiv.org/content/10.1101/2024.09.05.611520v1.abstract},

doi = {https://doi.org/10.1101/2024.09.05.611520},

year  = {2024},

date = {2024-09-04},

journal = {Preprint},

abstract = {Dual-specificity mitogen-activated protein kinase (MAPK) phosphatases (MKPs) directly dephosphorylate and inactivate the MAPKs. Although the catalytic mechanism of dephosphorylation of the MAPKs by the MKPs is established, a complete molecular picture of the regulatory interplay between the MAPKs and MKPs still remains to be fully explored. Here, we sought to define the molecular mechanism of MKP5 regulation through an allosteric site within its catalytic domain. We demonstrate using crystallographic and NMR spectroscopy approaches that residue Y435 is required to maintain the structural integrity of the allosteric pocket. Along with molecular dynamics simulations, these data provide insight into how changes in the allosteric pocket propagate conformational flexibility in the surrounding loops to reorganize catalytically crucial residues in the active site. Furthermore, Y435 contributes to the interaction with p38 MAPK and JNK, thereby promoting dephosphorylation. Collectively, these results highlight the role of Y435 in the allosteric site as a novel mode of MKP5 regulation by p38 MAPK and JNK},

keywords = {NT8®},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {The C2 domain augments Ras GTPase Activating Protein catalytic activity},

author = {E. Paul, Maxum},

url = {https://www.biorxiv.org/content/10.1101/2024.08.29.609784v1.abstract},

doi = {https://doi.org/10.1101/2024.08.29.609784},

year  = {2024},

date = {2024-08-28},

journal = {Preprint},

abstract = {Regulation of Ras GTPases by GTPase activating proteins (GAP) is essential for their normal signaling. Nine of the ten GAPs for Ras contain a C2 domain immediately proximal to their canonical GAP domain, and in RasGAP (p120GAP, p120RasGAP; RASA1) mutation of this domain is associated with vascular malformations in humans. Here, we show that the C2 domain of RasGAP is required for full catalytic activity towards Ras. Analysis of the RasGAP C2-GAP crystal structure, AlphaFold models, and sequence conservation reveal direct C2 domain interaction with the Ras allosteric lobe. This is achieved by an evolutionarily conserved surface centered around RasGAP residue R707, point mutation of which impairs the catalytic advantage conferred by the C2 domain in vitro. In mice, R707C mutation phenocopies the vascular and signaling defects resulting from constitutive disruption of the RASA1 gene. In SynGAP, mutation of the equivalent conserved C2 domain surface impairs catalytic activity. Our results indicate that the C2 domain is required to achieve full catalytic activity of Ras GTPase activating proteins.},

keywords = {NT8®},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Bimodal substrate binding in the active site of the glycosidase BcX},

author = {Saberi, Mahin},

url = {https://febs.onlinelibrary.wiley.com/doi/full/10.1111/febs.17251},

doi = {https://doi.org/10.1111/febs.17251},

year  = {2024},

date = {2024-08-25},

journal = {The FEBS journal},

abstract = {Bacillus circulans xylanase (BcX) from the glycoside hydrolase family 11 degrades xylan through a retaining, double-displacement mechanism. The enzyme is thought to hydrolyze glycosidic bonds in a processive manner and has a large, active site cleft, with six subsites allowing the binding of six xylose units. Such an active site architecture suggests that oligomeric xylose substrates can bind in multiple ways. In the crystal structure of the catalytically inactive variant BcX E78Q, the substrate xylotriose is observed in the active site, as well as bound to the known secondary binding site and a third site on the protein surface. Nuclear magnetic resonance (NMR) titrations with xylose oligomers of different lengths yield nonlinear chemical shift trajectories for active site nuclei resonances, indicative of multiple binding orientations for these substrates for which binding and dissociation are in fast exchange on the NMR timescale, exchanging on the micro- to millisecond timescale. Active site binding can be modeled with a 2 : 1 model with dissociation constants in the low and high millimolar range. Extensive mutagenesis of active site residues indicates that tight binding occurs in the glycon binding site and is stabilized by Trp9 and the thumb region. Mutations F125A and W71A lead to large structural rearrangements. Binding at the glycon site is sensed throughout the active site, whereas the weak binding mostly affects the aglycon site. The interactions with the two active site locations are largely independent of each other and of binding at the secondary binding site.},

keywords = {NT8®},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Borrelia burgdorferi BB0346 is an Essential, Structurally Variant LolA Homolog that is Primarily Required for Homeostatic Localization of Periplasmic Lipoproteins},

author = {T. Murphy, Bryan},

url = {https://www.biorxiv.org/content/10.1101/2024.08.06.606844v1.abstract},

doi = {https://doi.org/10.1101/2024.08.06.606844},

year  = {2024},

date = {2024-08-06},

journal = {Preprint},

abstract = {In diderm bacteria, the Lol pathway canonically mediates the periplasmic transport of lipoproteins from the inner membrane (IM) to the outer membrane (OM) and therefore plays an essential role in bacterial envelope homeostasis. After extrusion of modified lipoproteins from the IM via the LolCDE complex, the periplasmic chaperone LolA carries lipoproteins through the periplasm and transfers them to the OM lipoprotein insertase LolB, itself a lipoprotein with a LolA-like fold. Yet, LolB homologs appear restricted to ψ-proteobacteria and are missing from spirochetes like the tick-borne Lyme disease pathogen Borrelia burgdorferi, suggesting a different hand-off mechanism at the OM. Here, we solved the crystal structure of the B. burgdorferi LolA homolog BB0346 (LolABb) at 1.9 Å resolution. We identified multiple structural deviations in comparative analyses to other solved LolA structures, particularly a unique LolB-like protruding loop domain. LolABb failed to complement an Escherichia coli lolA knockout, even after codon optimization, signal I peptide adaptation, and a C-terminal chimerization which had allowed for complementation with an α-proteobacterial LolA. Analysis of a conditional B. burgdorferi lolA knockout strain indicated that LolABb was essential for growth. Intriguingly, protein localization assays indicated that initial depletion of LolABb led to an emerging mislocalization of both IM and periplasmic OM lipoproteins, but not surface lipoproteins. Together, these findings further support the presence of two separate primary secretion pathways for periplasmic and surface OM lipoproteins in B. burgdorferi and suggest that the distinct structural features of LolABb allow it to function in a unique LolB-deficient lipoprotein sorting system.},

keywords = {NT8®},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {The crystal structure of Acinetobacter baumannii bacterioferritin reveals a heteropolymer of bacterioferritin and ferritin subunits},

author = {Yao, Huili},

url = {https://www.nature.com/articles/s41598-024-69156-2},

doi = {https://doi.org/10.1038/s41598-024-69156-2},

year  = {2024},

date = {2024-08-05},

journal = {Nature Scientific Reports},

abstract = {Iron storage proteins, e.g., vertebrate ferritin, and the ferritin-like bacterioferritin (Bfr) and bacterial ferritin (Ftn), are spherical, hollow proteins that catalyze the oxidation of Fe2+ at binuclear iron ferroxidase centers (FOC) and store the Fe3+ in their interior, thus protecting cells from unwanted Fe3+/Fe2+ redox cycling and storing iron at concentrations far above the solubility of Fe3+. Vertebrate ferritins are heteropolymers of H and L subunits with only the H subunits having FOC. Bfr and Ftn were thought to coexist in bacteria as homopolymers, but recent evidence indicates these molecules are heteropolymers assembled from Bfr and Ftn subunits. Despite the heteropolymeric nature of vertebrate and bacterial ferritins, structures have been determined only for recombinant proteins constituted by a single subunit type. Herein we report the structure of Acinetobacter baumannii bacterioferritin, the first structural example of a heteropolymeric ferritin or ferritin-like molecule, assembled from completely overlapping Ftn homodimers harboring FOC and Bfr homodimers devoid of FOC but binding heme. The Ftn homodimers function by catalyzing the oxidation of Fe2+ to Fe3+, while the Bfr homodimers bind a cognate ferredoxin (Bfd) which reduces the stored Fe3+ by transferring electrons via the heme, enabling Fe2+ mobilization to the cytosol for incorporation in metabolism.},

keywords = {NT8®},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {The structure of a NEMO construct engineered for screening reveals novel determinants of inhibition},

author = {E. Kennedy, Amy},

url = {chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/https://www.biorxiv.org/content/10.1101/2024.07.18.604176v1.full.pdf},

doi = {https://doi.org/10.1101/2024.07.18.604176},

year  = {2024},

date = {2024-07-21},

journal = {Preprint},

abstract = {NEMO is an essential component in the activation of the canonical NFκ B pathway and exerts its function by recruiting the I κ B kinases (IKK) to the IKK complex. Inhibition of the NEMO/IKKs interaction is an attractive therapeutic paradigm for diseases related to NFκ B mis-regulation, but a difficult endeavor because of the extensive protein-protein interface. Here we report the design and characterization of novel engineered constructs of the IKK-binding domain of NEMO, programmed to render this difficult protein domain amenable to NMR and X-ray characterization, while preserving the biological function. ZipNEMO binds IKK β with nanomolar affinity, is amenable to heteronuclear NMR techniques and structure determination by X-ray crystallography. We show that NMR spectra of zipNEMO allow to detect inhibitor binding in solution and resonance assignment. The X-ray structure of zipNEMO highlights a novel ligand binding motif and the adaptability of the binding pocket and inspired the design of new peptide inhibitors.},

keywords = {NT8®},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Rational exploration of 2,4-diaminopyrimidines as DHFR inhibitors active against Mycobacterium abscessus and Mycobacterium avium, two emerging human pathogens},

author = {A. Meirelles, Matheus},

url = {https://chemrxiv.org/engage/chemrxiv/article-details/66913826c9c6a5c07a16eeee},

doi = {https://orcid.org/0000-0003-1847-5090},

year  = {2024},

date = {2024-07-14},

journal = {Biological and Medicinal Chemistry},

abstract = {Nontuberculous mycobacteria (NTM) are emerging human pathogens linked to severe pulmonary diseases. Current treatments involve the prolonged use of multiple drugs and are often ineffective. Bacterial dihydrofolate reductase (DHFR) is a key enzyme targeted by antibiotics in Gram-negative bacterial infections. However, existing DHFR inhibitors designed for Gram-negative bacteria often fail against mycobacterial DHFRs. Here, we detail the rational design of NTM DHFR inhibitors based on P218, a malarial DHFR inhibitor. We identified 8, a 2,4-diaminopyrimidine exhibiting improved pharmacological properties and activity against purified DHFR and whole cell cultures of two predominant NTM species: Mycobacterium avium and Mycobacterium abscessus. This study underscores the potential of 8 as a promising candidate for the in vivo validation of DHFR as an effective treatment against NTM infections.},

keywords = {NT8®},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Structural insight into the functional regulation of Elongation factor Tu by reactive oxygen species in Synechococcus elongatus PCC 7942},

author = {Cheng, Chen},

url = {https://www.sciencedirect.com/science/article/pii/S0141813024044374},

doi = {https://doi.org/10.1016/j.ijbiomac.2024.133632},

year  = {2024},

date = {2024-07-03},

journal = {International Journal of Biological Macromolecules},

abstract = {In cyanobacteria, Elongation factor Tu (EF-Tu) plays a crucial role in the repair of photosystem II (PSII), which is highly susceptible to oxidative stress induced by light exposure and regulated by reactive oxygen species (ROS). However, the specific molecular mechanism governing the functional regulation of EF-Tu by ROS remains unclear. Previous research has shown that a mutated EF-Tu, where C82 is substituted with a Ser residue, can alleviate photoinhibition, highlighting the important role of C82 in EF-Tu photosensitivity. In this study, we elucidated how ROS deactivate EF-Tu by examining the crystal structures of EF-Tu in both wild-type and mutated form (C82S) individually at resolutions of 1.7 Å and 2.0 Å in Synechococcus elongatus PCC 7942 complexed with GDP. Specifically, the GDP-bound form of EF-Tu adopts an open conformation with C82 located internally, making it resistant to oxidation. Coordinated conformational changes in switches I and II create a tunnel that positions C82 for ROS interaction, revealing the vulnerability of the closed conformation of EF-Tu to oxidation. An analysis of these two structures reveals that the precise spatial arrangement of C82 plays a crucial role in modulating EF-Tu's response to ROS, serving as a regulatory element that governs photosynthetic biosynthesis.},

keywords = {NT8®},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Structural and biochemical characterization of USP28 inhibition by small molecule inhibitors},

author = {Karal Nair, Radhika},

url = {https://opus.bibliothek.uni-wuerzburg.de/frontdoor/index/index/docId/28174},

doi = {https://doi.org/10.25972/OPUS-28174},

year  = {2024},

date = {2024-07-02},

journal = {Thesis/ Dessertation},

abstract = {Ubiquitination is an important post-translational modification that maintains cellular homeostasis by regulating various biological processes. Deubiquitinases (DUBs) are enzymes that reverse the ubiquitination process by catalyzing the removal of ubiquitin from a substrate. Abnormal expression or function of DUBs is often associated with the onset and progression of various diseases, including cancer. Ubiquitin specific proteases (USPs), which constitute the largest family of DUBs in humans, have become the center of interest as potential targets in cancer therapy as many of them display increased activity or are overexpressed in a range of malignant tumors or the tumor microenvironment.  Two related members of the USP family, USP28 and USP25, share high sequence identities but play diverse biological roles. USP28 regulates cell proliferation, oncogenesis, DNA damage repair and apoptosis, whereas USP25 is involved in the anti-viral response, innate immunity and ER-associated degradation in addition to carcinogenesis. USP28 and USP25 also exhibit different oligomeric states – while USP28 is a constitutively active dimer, USP25 assumes an auto-inhibited tetrameric structure. The catalytic domains of both USP28 and USP25 comprise the canonical, globular USP-domain but contain an additional, extended insertion site called USP25/28 catalytic domain inserted domain (UCID) that mediates oligomerization of the proteins. Disruption of the USP25 tetramer leads to the formation of an activated dimeric protein. However, it is still not clear what triggers its activation.  Due to their role in maintaining and stabilizing numerous oncoproteins, USP28 and USP25 have emerged as interesting candidates for anti-cancer therapy. Recent advances in smallmolecular inhibitor development have led to the discovery of relatively potent inhibitors of USP28 and USP25. This thesis focuses on the structural elucidation of USP28 and the biochemical characterization of USP28/USP25, both in complex with representatives of three out of the eight compound classes reported as USP28/USP25-specific inhibitors. The crystal structures of USP28 in complex with the AZ compounds, Vismodegib and FT206 reveal that all three inhibitor classes bind into the same allosteric pocket distant from the catalytic center, located between the palm and the thumb subdomains (the S1-site). Intriguingly, this binding pocket is identical to the UCID-tip binding interface in the USP25 tetramer, rendering the protein in a locked, inactive conformation. Formation of the binding pocket in USP28 requires a shift in the helix α5, which induces conformational changes and local distortion of the binding channel that typically accommodates the C-terminal tail of I Ubiquitin, thus preventing catalysis and abrogating USP28 activity. The key residues of the USP28-inhibitor binding pocket are highly conserved in USP25. Mutagenesis studies of these residues accompanied by biochemical and biophysical assays confirm the proposed mechanism of inhibition and similar binding to USP25.  This work provides valuable insights into the inhibition mechanism of the small molecule compounds specifically for the DUBs USP28 and USP25. The USP28-inhibitor complex structures offer a framework to develop more specific and potent inhibitors.},

keywords = {NT8®},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Structure-Guided Design of Potent Coronavirus Inhibitors with a 2‑Pyrrolidone Scaffold: Biochemical, Crystallographic, and Virological Studies},

author = {S. Dampalla, Chamandi},

url = {https://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.4c00551},

doi = {https://doi.org/10.1021/acs.jmedchem.4c00551},

year  = {2024},

date = {2024-07-01},

journal = {Journal of Medicinal Chemistry},

abstract = {Zoonotic coronaviruses are known to produce

severe infections in humans and have been the cause of significant

morbidity and mortality worldwide. SARS-CoV-2 was the largest

and latest contributor of fatal cases, even though MERS-CoV has

the highest case-fatality ratio among zoonotic coronaviruses. These

infections pose a high risk to public health worldwide warranting

efforts for the expeditious discovery of antivirals. Hence, we hereby

describe a novel series of inhibitors of coronavirus 3CLpro

embodying an N-substituted 2-pyrrolidone scaffold envisaged to

exploit favorable interactions with the S3−S4 subsites and

connected to an invariant Leu-Gln P2−P1 recognition element.

Several inhibitors showed nanomolar antiviral activity in enzyme and cell-based assays, with no significant cytotoxicity. Highresolution

crystal structures of inhibitors bound to the 3CLpro were determined to probe and identify the molecular determinants

associated with binding, to inform the structure-guided optimization of the inhibitors, and to confirm the mechanism of action of the

inhibitors.},

keywords = {NT8®},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Synthesis and biological characterization of an orally bioavailable lactate dehydrogenase-A inhibitor against pancreatic cancer},

author = {Sharma, Horrick},

url = {https://www.sciencedirect.com/science/article/pii/S0223523424004781},

doi = {https://doi.org/10.1016/j.ejmech.2024.116598},

year  = {2024},

date = {2024-06-25},

journal = {European Journal of Medicinal Chemistry},

abstract = {Lactate dehydrogenase-A (LDHA) is the major isoform of lactate dehydrogenases (LDH) that is overexpressed and linked to poor survival in pancreatic ductal adenocarcinoma (PDAC). Despite some progress, current LDH inhibitors have poor structural and physicochemical properties or exhibit unfavorable pharmacokinetics that have hampered their development. The present study reports the synthesis and biological evaluation of a novel class of LDHA inhibitors comprising a succinic acid monoamide motif. Compounds 6 and 21 are structurally related analogs that demonstrated potent inhibition of LDHA with IC50s of 46 nM and 72 nM, respectively. We solved cocrystal structures of compound 21-bound to LDHA that showed that the compound binds to a distinct allosteric site between the two subunits of the LDHA tetramer. Inhibition of LDHA correlated with reduced lactate production and reduction of glycolysis in MIA PaCa-2 pancreatic cancer cells. The lead compounds inhibit the proliferation of human pancreatic cancer cell lines and patient-derived 3D organoids and exhibit a synergistic cytotoxic effect with the OXPHOS inhibitor phenformin. Unlike current LDHA inhibitors, 6 and 21 have appropriate pharmacokinetics and ligand efficiency metrics, exhibit up to 73% oral bioavailability, and a cumulative half-life greater than 4 h in mice.},

keywords = {NT8®},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Pseudomonas aeruginosa gene PA4880 encodes a Dps-like protein with a Dps fold, bacterioferritin-type ferroxidase centers, and endonuclease activity},

author = {Rajapaksha, Nimesha},

url = {https://www.frontiersin.org/articles/10.3389/fmolb.2024.1390745/full},

doi = {https://doi.org/10.3389/fmolb.2024.1390745},

year  = {2024},

date = {2024-05-21},

journal = {Frontiers in Molecular Biosciences},

abstract = {We report the biochemical, structural, and functional characterization of the protein coded by gene PA4880 in the P. aeruginosa PAO1 genome. The PA4880 gene had been annotated as coding a probable bacterioferritin. Our structural work shows that the product of gene PA4880 is a protein that adopts the Dps subunit fold, which oligomerizes into a 12-mer quaternary structure. Unlike Dps, however, the ferroxidase di-iron centers and iron coordinating ligands are buried within each subunit, in a manner identical to that observed in the ferroxidase center of P. aeruginosa bacterioferritin. Since these structural characteristics correspond to Dps-like proteins, we term the protein as P. aeruginosa Dps-like, or Pa DpsL. The ferroxidase centers in Pa DpsL catalyze the oxidation of Fe2+ utilizing O2 or H2O2 as oxidant, and the resultant Fe3+ is compartmentalized in the interior cavity. Interestingly, incubating Pa DpsL with plasmid DNA results in efficient nicking of the DNA and at higher concentrations of Pa DpsL the DNA is linearized and eventually degraded. The nickase and endonuclease activities suggest that Pa DpsL, in addition to participating in the defense of P. aeruginosa cells against iron-induced toxicity, may also participate in the innate immune mechanisms consisting of restriction endonucleases and cognate methyl transferases.},

keywords = {NT8®},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Macrocyclic Azapeptide Nitriles: Structure-Based Discovery of Potent SARS-CoV‑2 Main Protease Inhibitors as Antiviral Drugs},

author = {Breidenbach, Julian},

url = {https://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.4c00053},

doi = {https://doi.org/10.1021/acs.jmedchem.4c00053},

year  = {2024},

date = {2024-05-15},

journal = {Journal of Medicinal Chemistry},

abstract = {Given the crucial role of the main protease (Mpro) in the replication cycle of SARS-CoV-2, this viral cysteine protease constitutes a high-profile drug target. We investigated peptidomimetic azapeptide nitriles as auspicious, irreversibly acting inhibitors of Mpro. Our systematic approach combined an Mpro active-site scanning by combinatorially assembled azanitriles with structure-based design. Encouraged by the bioactive conformation of open-chain inhibitors, we conceptualized the novel chemotype of macrocyclic azanitriles whose binding mode was elucidated by cocrystallization. This strategy provided a favorable entropic contribution to target binding and resulted in the development of the extraordinarily potent Mpro inhibitor 84 with an IC50 value of 3.23 nM and a second-order rate constant of inactivation, kinac/Ki, of 448,000 M–1s–1. The open-chain Mpro inhibitor 58, along with the macrocyclic compounds 83 and 84, a broad-spectrum anticoronaviral agent, demonstrated the highest antiviral activity with EC50 values in the single-digit micromolar range. Our findings are expected to promote the future development of peptidomimetic Mpro inhibitors as anti-SARS-CoV-2 agents.},

keywords = {NT8®},

pubstate = {published},

tppubtype = {article}

}