Biology subjects

Harki, D. A.

Publications and source records attributed to Harki, D. A..

7 recordsLinked to original sources

bioRxiv2024

Development of Allosteric Small Molecule APOBEC3B Inhibitors from In Silico Screening

The APOBEC3 (A3) family of enzymes are zinc metalloenzymes that catalyze the conversion of 2'- deoxycytidine to 2'-deoxyuridine in single stranded DNA. APOBEC3B (A3B), a member of the A3 family, has emerged as a key driver of genomic instability in many cancer types by mutating host DNA to drive tumorigenesis and therapy resistance. Small molecule inhibitors of A3B would extend the durability of current therapies by limiting mutations that promote tumor escape and therapy resistance. Consequently, a computer aided drug discovery (CADD) campaign was employed to identify inhibitors of A3B. Through molecular dynamics (MD) simulations and computational solvent mapping analysis, we identified a novel putative allosteric pocket on the c-terminal domain of A3B and virtually screened the ChemBridge Diversity Set ([~]110,000 small molecules) against both the active and predicted allosteric sites. High scoring compounds were selected for in vitro testing and triage, resulting in 13 candidate inhibitors. Using cysteine reactive probes, one of the original hit compounds was mapped to the computationally identified allosteric pocket. However, after resynthesis of representative chemotypes and further investigation using in vitro assays, none of the chemotypes retained inhibitory activity. Further analysis revealed that most of the inhibition observed in the primary assay was due to metal contamination in the screening sample, which resulted in the removal of the catalytic zinc from the enzyme active site. Although validated A3B inhibitors were not discovered in this study, we report a ligandable allosteric site on A3B and several cautionary insights for researchers developing small molecule inhibitors of zinc metalloenzymes. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/591187v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@b7e577org.highwire.dtl.DTLVardef@13a1a43org.highwire.dtl.DTLVardef@192309eorg.highwire.dtl.DTLVardef@b60a31_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Rapid resistance profiling of SARS-CoV-2 protease inhibitors

Resistance to nirmatrelvir (Paxlovid) has been shown by multiple groups and may already exist in clinical SARS-CoV-2 isolates. Here a panel of SARS-CoV-2 main protease (Mpro) variants and a robust cell-based assay are used to compare the resistance profiles of nirmatrelvir, ensitrelvir, and FB2001. The results reveal distinct resistance mechanisms ("fingerprints") and indicate that these next-generation drugs have the potential to be effective against nirmatrelvir-resistant variants and vice versa.

microbiology↗

bioRxiv2022

Interfering with nucleotide excision by the coronavirus 3'-to-5' exoribonuclease

Some of the most efficacious antiviral therapeutics are ribonucleos(t)ide analogs. The presence of a 3-to-5 proofreading exoribonuclease (ExoN) in coronaviruses diminishes the potency of many ribonucleotide analogs. The ability to interfere with ExoN activity will create new possibilities for control of SARS-CoV-2 infection. ExoN is formed by a 1:1 complex of nsp14 and nsp10 proteins. We have purified and characterized ExoN using a robust, quantitative system that reveals determinants of specificity and efficiency of hydrolysis. Double-stranded RNA is preferred over single-stranded RNA. Nucleotide excision is distributive, with only one or two nucleotides hydrolyzed in a single binding event. The composition of the terminal basepair modulates excision. A stalled SARS-CoV-2 replicase in complex with either correctly or incorrectly terminated products prevents excision, suggesting that a mispaired end is insufficient to displace the replicase. Finally, we have discovered several modifications to the 3-RNA terminus that interfere with or block ExoN-catalyzed excision. While a 3-OH facilitates hydrolysis of a nucleotide with a normal ribose configuration, this substituent is not required for a nucleotide with a planar ribose configuration such as that present in the antiviral nucleotide produced by viperin. Design of ExoN-resistant, antiviral ribonucleotides should be feasible.

biochemistry↗

bioRxiv2022

Development of allosteric, selective cyclin-dependent kinase 2 (CDK2) inhibitors that are negatively cooperative with cyclin binding and show potential as contraceptive agents

Compared to most ATP-site kinase inhibitors, small molecules that target an allosteric pocket have the potential for improved selectivity due to the often observed lower structural similarity at these distal sites. Despite their promise, relatively few examples of structurally confirmed, high-affinity allosteric kinase inhibitors exist. Cyclin-dependent kinase 2 (CDK2) is a target for many therapeutic indications, including non-hormonal contraception.1 However, an inhibitor against this kinase with exquisite selectivity has not reached the market because of the structural similarity between CDKs.1-2 In this paper, we describe the development and mechanism of action of new type III inhibitors that bind CDK2 with nanomolar affinity, making them the highest affinity, structurally confirmed allosteric CDK inhibitors reported. Notably, these anthranilic acid inhibitors exhibit a strong negative cooperative relationship with cyclin binding, which remains an underexplored mechanism for CDK2 inhibition. Furthermore, the binding profile of these compounds in both biophysical and cellular assays demonstrate the promise of this series for further development into a therapeutic selective for CDK2 over highly similar kinases like CDK1. The potential of these inhibitors as efficacious contraceptive agents is seen by incubation with mouse testicular explants, where they recapitulate Cdk2-/- and Spdya-/- phenotypes.

pharmacology and toxicology↗

bioRxiv2022

Targeting N-Myc in Neuroblastoma with Selective Aurora Kinase A Degraders

Summary ParagraphMYCN amplification is the most frequent genetic driver in high-risk neuroblastoma (NB) and strongly associated with poor prognosis.1,2 The N-Myc transcription factor, which is encoded by MYCN, is a mechanistically validated, yet challenging target for NB therapy development.3,4 In normal neuronal progenitors, N-Myc undergoes rapid degradation, while in MYCN-amplified NB cells, Aurora kinase A (Aurora-A) binds to and stabilizes N-Myc, resulting in elevated protein levels.5,6 Allosteric Aurora-A inhibitors that displace N-Myc from binding can promote N-Myc degradation, but with limited efficacy.7-10 Here, we report a chemical approach to decrease N-Myc levels through the targeted protein degradation of Aurora-A. A first-in-class Aurora-A/N-Myc degrader, HLB-0532259 (compound 4), was developed from a novel Aurora-A-binding ligand that engages the Aurora-A/N-Myc complex. HLB-0532259 promotes the degradation of both Aurora-A and N-Myc with nanomolar potency and excellent selectivity and surpasses the cellular efficacy of established allosteric Aurora-A inhibitors. HLB-0532259 exhibits favorable pharmacokinetics properties and elicits tumor reduction in murine xenograft NB models. More broadly, this study delineates a novel strategy for targeting "undruggable" proteins that are reliant on accessory proteins for cellular stabilization.

cancer biology↗

bioRxiv2020

Induced copy-back RNA synthesis as a novel therapeutic mechanism against RNA viruses

The viral RNA-dependent RNA polymerase (RdRp) is a well-established target for development of broad-spectrum antiviral therapeutics. Incorporation of ribonucleotide analogues by the RdRp will either cause termination of RNA synthesis or mutagenesis of the RNA product. We demonstrated recently that incorporation of a pyrazine-carboxamide ribonucleotide into nascent RNA leads to pausing and backtracking of the elongating RdRp. Here, we provide evidence for the single-stranded RNA product of backtracking serving as an intermediate in RdRp-catalyzed, template-switching reactions. This intermediate is used for both intramolecular template-switching (copy-back RNA synthesis) and intermolecular template-switching (homologous RNA recombination). The use of a magnetic-tweezers platform to monitor RdRp elongation dynamics permitted direct observation of copy-back synthesis and illuminated properties of the RdRp that promote copy-back synthesis, including stability of the RdRp-nascent-RNA complex and the dimensions of the RdRp nucleic-acid-binding channel. In cells, recombination was stimulated by the presence of a pyrazine-carboxamide ribonucleotide. The effect of the drug on recombination was diminished for a recombination-defective virus, but this virus was not resistant to the drug. The discovery that a ribonucleotide analogue can induce copy-back RNA synthesis suggests that this third mechanistic class of compounds may function by promoting formation of defective viral genomes. This study identifies RdRp-catalyzed intra- and intermolecular template switching as a viable new mechanistic target with potentially broad-spectrum appeal.

microbiology↗

bioRxiv2019

Active site plasticity and possible modes of chemical inhibition of the human DNA deaminase APOBEC3B

The single-stranded DNA cytosine deaminase APOBEC3B (A3B) functions in innate immunity against viruses, but is also strongly implicated in eliciting mutations in cancer genomes. Because of the critical role of A3B in promoting virus and tumor evolution, small molecule inhibitors are desirable. However, there is no reported structure for any of the APOBEC3-family enzymes in complex with a small molecule bound in the active site, which hampers the development of small molecules targeting A3B. Here we report high-resolution structures of an active A3B catalytic domain chimera with loop 7 residues exchanged with those from the corresponding region of APOBEC3G (A3G). The structures reveal novel open conformations lacking the catalytically essential zinc ion, with the highly conserved active site residues extensively rearranged. These inactive conformations are stabilized by 2-pyrimidone or an iodide ion bound in the active site. Molecular dynamics simulations corroborate the remarkable plasticity of the engineered active site and identify key interactions that stabilize the native A3B active site. These data provide insights into A3B active site dynamics and suggest possible modes of its inhibition by small molecules, which would aid in rational design of selective A3B inhibitors for constraining virus and tumor evolution.

biochemistry↗