Biology subjects

Klima, M.

Publications and source records attributed to Klima, M..

8 recordsLinked to original sources

bioRxiv2026

Branched C7-Substituted 7-Deaza-SAH Analogues Occupy the Entire SAM-Binding Pocket of Mpox Virus VP39 and Dengue Virus NS5 Methyltransferases

Viral RNA-cap MTases are attractive targets for antiviral drug development. We previously identified C7-substituted 7-deaza-SAH analogues as potent inhibitors of the mpox virus 2'-O-MTase VP39. Here, we used structure-guided design to develop branched C7-substituted analogues intended to engage multiple hydrophobic regions of the VP39 SAM-binding pocket. The synthesized compounds were characterized using biochemical and crystallographic approaches. Several analogues effectively inhibited VP39, with the most potent compound displaying an IC50 in the tens-of-nanomolar range. Crystal structures of VP39 in complex with STM1187 and STM1189 confirmed that the branched aromatic substituents extend towards hydrophobic regions adjacent to the SAM binding site. The precise ligand conformations were strongly influenced by linker geometry and the branching groups mode of attachment. STM1078 also inhibited DENV3 NS5 MTase with submicromolar potency, and the complexs structure revealed a conserved binding mode of the SAH-like core accompanied by conformational adaptability of the branched substituent. These results demonstrate how three-dimensional expansion from the 7-deaza position can generate potent inhibitors capable of binding structurally distinct viral MTases.

molecular biology↗

bioRxiv2026

Catalytic and Structural Insights into Neil3-Dependent Unhooking of Endogenous Abasic DNA Crosslink

Abasic (Ap) sites arise frequently in genomic DNA and can form interstrand crosslinks (Ap-ICLs) that block DNA replication and threaten genome stability. The DNA glycosylase NEIL3 is required for replication-coupled repair of Ap-ICLs, yet its catalytic mechanism has remained unclear, as biochemical studies report lyase-dependent strand cleavage whereas cellular systems indicate incision-free unhooking. Here, we show that the catalytic outcome of NEIL3 is determined by the N-terminal processing of its NEI domain. Using biochemically and structurally defined NEI variants, we demonstrate that a native-like processed form (V2M), in which valine 2 is replaced by an initiating methionine, efficiently unhooks Ap-ICLs by releasing the crosslinked strand without generating toxic DNA strand breaks, and without {beta}- or {delta}-elimination. In contrast, an unprocessed form (M1) exhibits elevated Ap-lyase activity and generates strand breaks. Time-resolved Schiff-base trapping in the presence of a reducing agent reveals distinct high-molecular-weight intermediates during Ap-ICL unhooking. A crystal structure of NEIL3 bound to native-like substrate in form of a single-stranded DNA identifies features underlying its preference for fork-like substrates. Together, these findings reconcile previously conflicting models of NEIL3 function and define a mechanistic framework for replication-coupled repair of endogenous crosslinks, Ap-ICL, preserving fork integrity.

biochemistry↗

bioRxiv2025

Structure of the Hibernating Francisella tularensis Ribosome and Mechanistic Insights into Its Inhibition by Antibiotics

Francisella tularensis is the causative agent of tularemia, a zoonotic disease named after the city of Tulare, California. Symptoms include sudden fever, chills, fatigue, and swollen lymph nodes, among others, and without treatment it is very serious or even fatal. In addition, F. tularensis is considered a potential bioterrorism threat due to its high infectivity and lethality. Ribosomes are key targets for many classes of antibiotics. In this study, we examined the F. tularensis ribosome and determined its structure at 2.8[A] resolution using cryo-electron microscopy. Notably, we observed the stress-induced ribosome-associated inhibitor A (RaiA) protein bound to the ribosome. RaiA functions as a molecular hibernation factor, inhibiting bacterial translation in response to stress or nutrient deprivation. This mechanism parallels that described in the model organism Escherichia coli and in several pathogenic bacteria, such as Staphylococcus aureus. Furthermore, we solved structures of the antibiotics chloramphenicol and gentamicin bound to the F. tularensis ribosome. Collectively, these results provide structural insights that highlight previously unexplored opportunities for therapeutic intervention.

microbiology↗

bioRxiv2023

Drug Discovery in Low Data Regimes: Leveraging a Computational Pipeline for the Discovery of Novel SARS-CoV-2 Nsp14-MTase Inhibitors

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has led to significant global morbidity and mortality. A crucial viral protein, the non-structural protein 14 (nsp14), catalyzes the methylation of viral RNA and plays a critical role in viral genome replication and transcription. Due to the low mutation rate in the nsp region among various SARS-CoV-2 variants, nsp14 has emerged as a promising therapeutic target. However, discovering potential inhibitors remains a challenge. In this work, we introduce a computational pipeline for the rapid and efficient identification of potential nsp14 inhibitors by leveraging virtual screening and the NCI open compound collection, which contains 250,000 freely available molecules for researchers worldwide. The introduced pipeline provides a cost-effective and efficient approach for early-stage drug discovery by allowing researchers to evaluate promising molecules without incurring synthesis expenses. Our pipeline successfully identified seven promising candidates after experimentally validating only 40 compounds. Notably, we discovered NSC620333, a compound that exhibits a strong binding affinity to nsp14 with a dissociation constant of 427 {+/-} 84 nM. In addition, we gained new insights into the structure and function of this protein through molecular dynamics simulations. We identified new conformational states of the protein and determined that residues Phe367, Tyr368, and Gln354 within the binding pocket serve as stabilizing residues for novel ligand interactions. We also found that metal coordination complexes are crucial for the overall function of the binding pocket. Lastly, we present the solved crystal structure of the nsp14-MTase complexed with SS148 (PDB:8BWU), a potent inhibitor of methyltransferase activity at the nanomolar level (IC50 value of 70 {+/-} 6 nM). Our computational pipeline accurately predicted the binding pose of SS148, demonstrating its effectiveness and potential in accelerating drug discovery efforts against SARS-CoV-2 and other emerging viruses.

biochemistry↗

bioRxiv2023

Fluorinated cGAMP analogs, which act as STING agonists and are not cleavable by poxins: structural basis of their function

The Stimulator of Interferon Genes (STING) plays a crucial role in the cGAS-STING pathway of innate immunity, detecting DNA in the cytoplasm and defending against certain cancers, viruses, and bacteria. We designed and synthesized fluorinated carbocyclic cGAMP analogs, MD1203 and MD1202D (MDs), to enhance their stability against nucleases and their affinity for STING. These compounds demonstrated exceptional activity against wild-type STING and all its allelic variations, including the hard-to-target REF isoform. Despite their distinct chemical modifications relative to the canonical CDNs, such as the substitution of guanine with hypoxanthine and the fluorination of the (pseudo)ribose ring, crystallographic analysis revealed a consistent binding mode with STING. Importantly, these compounds were resistant to cleavage by viral poxin nucleases. The crystallographic analysis of poxin/MD complexes unveiled their binding mode at the interface of poxin monomers, with dynamic adenine base orientations. Interestingly, MDs-bound poxin adopted an unliganded-like conformation, distinct from the conformation of cGAMP-bound poxin. Moreover, when MDs were in complex with poxin, they exhibited a different conformation than cGAMP when bound to poxin; in fact, it closely resembled the conformation observed when MDs were bound to STING. In conclusion, the development of MD1203 and MD1202D, showcases their potential as potent STING activators with remarkable stability against poxin-mediated degradation--a crucial characteristic for future development of antivirals.

molecular biology↗

bioRxiv2023

Structural and functional insights in flavivirus NS5 proteins gained by the structure of Ntaya virus polymerase and methyltransferase

Flaviviruses are single-stranded positive-sense RNA (+RNA) viruses that are responsible for several (re)emerging diseases such as Yellow, Dengue or West Nile fevers. The Zika epidemic highlighted their dangerousness when a relatively benign virus known since the 1950s turned into a deadly pathogen. The central protein for their replication is NS5 (non-structural protein 5), which is composed of the N-terminal methyltransferase (MTase) domain and the C-terminal RNA-dependent RNA-polymerase (RdRp) domain. It is responsible for both, RNA replication and installation of the 5 RNA cap. We structurally and biochemically analyzed the Ntaya virus MTase and RdRp domains and we compared their properties to other flaviviral NS5s. The enzymatic centers are well conserved across Flaviviridae, suggesting that the development of drugs targeting all flaviviruses is feasible. However, the enzymatic activities of the isolated proteins were significantly different for the MTase domains.

molecular biology↗

bioRxiv2022

The structure of monkeypox virus 2'-O-ribose methyltransferase VP39 in complex with sinefungin provides the foundation for inhibitor design

Monkeypox is an emerging, rapidly spreading disease with pandemic potential. It is caused by the monkeypox virus (MPXV), a dsDNA virus from the Poxviridae family, that replicates in the cytoplasm and must encode for its own RNA processing machinery including the capping machinery. Here, we present the crystal structure of its 2-O-RNA methyltransferase (MTase) VP39 in complex with the pan-MTase inhibitor sinefungin. A comparison of this 2-O RNA MTase with enzymes from unrelated ssRNA viruses (SARS-CoV-2 and Zika) reveals a surprisingly conserved sinefungin binding mode implicating that a single inhibitor could be used against unrelated viral families.

microbiology↗

bioRxiv2021

The mycobacterial guaB1 gene encodes a guanosine 5'-monophosphate reductase with a cystathione-β-synthase domain

Purine metabolism plays a pivotal role in bacterial life cycle, however, regulation of the de novo and purine salvage pathways have not been extensively detailed in mycobacteria. By gene knockout, biochemical and structural analyses, we identified Mycobacterium smegmatis (Msm) and Mycobacterium tuberculosis (Mtb) guaB1 gene product as a novel type of guanosine 5-monophosphate reductase (GMPR), which recycles guanosine monophosphate to inosine monophosphate within the purine salvage pathway and contains cystathione {beta}-synthase (CBS) domains with atypical orientation in the octamer. CBS domains share a much larger interacting area with a conserved catalytic domain in comparison with the only known CBS containing protozoan GMPR and closely related inosine monophosphate dehydrogenase structures. Our results revealed essential effect of pH on allosteric regulation of Msm GMPR activity and oligomerization with adenine and guanosine nucleotides binding to CBS domains.Bioinformatic analysis indicated the presence of GMPRs containing CBS domains across the entire Actinobacteria phylum.

biochemistry↗