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Biology subjects

Klima, M.

Publications and source records attributed to Klima, M..

5 recordsLinked to original sources

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↗

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↗

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↗

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↗

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↗