Mutations in isocitrate dehydrogenase 1 (IDH1) drive most low-grade gliomas and secondary glioblastomas and many chondrosarcomas and acute myeloid leukemia cases. Most tumor-relevant IDH1 mutations are deficient in the normal oxidization of isocitrate to α-ketoglutarate (αKG), but gain the neomorphic activity of reducing αKG to D-2-hydroxyglutarate (D2HG), which drives tumorigenesis. We found previously that IDH1 mutants exhibit one of two reactivities: deficient αKG and moderate D2HG production (including commonly observed R132H and R132C) or moderate αKG and high D2HG production (R132Q). Here, we identify a third type of reactivity, deficient αKG and high D2HG production (R132L). We show that R132Q IDH1 has unique structural features and distinct reactivities towards mutant IDH1 inhibitors. Biochemical and cell-based assays demonstrate that while most tumor-relevant mutations were effectively inhibited by mutant IDH1 inhibitors, R132Q IDH1 had up to a 16 300-fold increase in IC50 versus R132H IDH1. Only compounds that inhibited wild-type (WT) IDH1 were effective against R132Q. This suggests that patients with a R132Q mutation may have a poor response to mutant IDH1 therapies. Molecular dynamics simulations revealed that near the NADP+/NADPH-binding site in R132Q IDH1, a pair of α-helices switches between conformations that are more wild-type-like or more mutant-like, highlighting mechanisms for preserved WT activity. Dihedral angle changes in the dimer interface and buried surface area charges highlight possible mechanisms for loss of inhibitor affinity against R132Q. This work provides a platform for predicting a patient’s therapeutic response and identifies a potential resistance mutation that may arise upon treatment with mutant IDH inhibitors.

Zika virus (ZIKV) has been linked to the development of microcephaly in newborns, as well as Guillain-Barré syndrome. There are currently no drugs available to treat ZIKV infection, and accordingly, there is an unmet medical need for the discovery of new therapies. High-throughput drug screening efforts focusing on indirect readouts of cell viability are prone to a higher frequency of false positives in cases where the virus is viable in the cell but the cytopathic effect (CPE) is reduced or delayed. Here, we describe a fast and label-free phenotypic high-content imaging assay to detect cells affected by the virus-induced CPE using automated imaging and analysis. Protection from the CPE correlates with a decrease in viral antigen production, as observed by immunofluorescence. We trained our assay using a collection of nucleoside analogues with activity against ZIKV; the previously reported antiviral activities of 2'--methylribonucleosides and ribavirin against the Zika virus in Vero cells were confirmed using our developed method. To validate the ability of our assay to reveal new anti-ZIKV compounds, we profiled a novel library of 24 natural product derivatives and found compound 1 to be an inhibitor of the ZIKV-induced cytopathic effect; the activity of the compound was confirmed in human fetal neural stem cells (NSCs). The described technique can be easily leveraged as a primary screening assay for profiling of the activities of large compound libraries against ZIKV and can be expanded to other ZIKV strains and other cell lines displaying morphological changes upon ZIKV infection.

Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate to α-ketoglutarate (αKG) to provide critical cytosolic substrates and drive NADPH-dependent reactions like lipid biosynthesis and glutathione regeneration. In biochemical studies, the forward reaction is studied at neutral pH, while the reverse reaction is typically characterized in more acidic buffers. This led us to question whether IDH1 catalysis is pH-regulated, which would have functional implications under conditions that alter cellular pH, like apoptosis, hypoxia, cancer, and neurodegenerative diseases. Here, we show evidence of catalytic regulation of IDH1 by pH, identifying a trend of increasing kcat values for αKG production upon increasing pH in the buffers we tested. To understand the molecular determinants of IDH1 pH sensitivity, we used the pHinder algorithm to identify buried ionizable residues predicted to have shifted pKa values. Such residues can serve as pH sensors, with changes in protonation states leading to conformational changes that regulate catalysis. We identified an acidic residue buried at the IDH1 dimer interface, D273, with a predicted pKa value upshifted into the physiological range. D273 point mutations had decreased catalytic efficiency and, importantly, loss of pH-regulated catalysis. Based on these findings, we conclude that IDH1 activity is regulated, at least in part, by pH. We show this regulation is mediated by at least one buried acidic residue ~12 Å from the IDH1 active site. By establishing mechanisms of regulation of this well-conserved enzyme, we highlight catalytic features that may be susceptible to pH changes caused by cell stress and disease.

Zika virus (ZIKV), an emerging flavivirus that causes neurodevelopmental impairment to fetuses and has been linked to Guillain-Barré syndrome continues to threaten global health due to the absence of targeted prophylaxis or treatment. Nucleoside analogues are good examples of efficient anti-viral inhibitors, and prodrug strategies using phosphate masking groups (ProTides) have been employed to improve the bioavailability of ribonucleoside analogues. Here, we synthesized and tested a small library of 13 ProTides against ZIKV in human neural stem cells. Strong activity was observed for 2′-C-methyluridine and 2′-C-ethynyluridine ProTides with an aryloxyl phosphoramidate masking group. Substitution of a 2-(methylthio) ethyl phosphoramidate for the aryloxyl phosphoramidate ProTide group of 2′-C-methyluridine completely abolished antiviral activity of the compound. The aryloxyl phosphoramidate ProTide of 2′-C-methyluridine outperformed the hepatitis C virus (HCV) drug sofosbuvir in suppression of viral titers and protection from cytopathic effect, while the former compound’s triphosphate active metabolite was better incorporated by purified ZIKV NS5 polymerase over time. These findings suggest both a nucleobase and ProTide group bias for the anti-ZIKV activity of nucleoside analogue ProTides in a disease-relevant cell model.

Zika virus (ZIKV) has been linked to the development of microcephaly in newborns, as well as Guillain-Barré syndrome. There are currently no drugs available to treat infection, and accordingly there is an unmet medical need for discovery of new therapies. High-throughput drug screening efforts focusing on indirect readouts of cell viability are prone to a higher frequency of false positives in cases where the virus is viable in the cell but the cytopathic effect is reduced or delayed. Here, we describe a fast and label-free phenotypic high-content imaging assay used to detect cells affected by the viral-induced cytopathic effect (CPE) using automated imaging and analysis. Protection from CPE correlates with a decrease in viral antigen production as observed by immunofluorescence. We trained our assay using a collection of nucleoside analogues against ZIKV; the previously reported antiviral activities of 2’-C-methylribonucleosides and ribavirin against the Zika virus in Vero cells were confirmed using our developed method. Profiling of a novel library of 24 natural product derivatives using our assay revealed compound 1 as an inhibitor of ZIKV-induced cytopathic effect; activity of the compound was confirmed in human fetal neural stem cells (NSCs). The described technique can be easily leveraged as a primary screening assay for profiling large compound libraries against ZIKV, and can be expanded to other ZIKV strains and other cell lines displaying morphological changes upon ZIKV infection.

§This paper is dedicated to the memory of our dear colleague and friend, Michelle Evon Scott (1990Scott ( -2020. ABSTRACTIsocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate to α-ketoglutarate (α-KG) to provide critical cytosolic substrates and drive NADPH-dependent reactions like lipid biosynthesis and glutathione regeneration. In biochemical studies, the forward reaction is studied at neutral pH, while the reverse reaction is typically characterized in more acidic buffers. This led us to question whether IDH1 catalysis is pH-regulated, which would have functional implications under conditions that alter cellular pH, like apoptosis, hypoxia, cancer, and neurodegenerative diseases. Here, we show evidence of catalytic regulation of IDH1 by pH, identifying a trend of increasing kcat values for α-KG production upon increasing pH in the buffers we tested. To understand the molecular determinants of IDH1 pH sensitivity, we used the pHinder algorithm to identify buried ionizable residues predicted to have shifted pKa values. Such residues can serve as pH sensors, with changes in protonation states leading to conformational changes that regulate catalysis. We identified an acidic residue buried at the IDH1 dimer interface, D273, with a predicted pKa value upshifted into the physiological range. D273 point mutations had decreased catalytic efficiency and, importantly, loss of pH-regulated catalysis. Based on these findings, we conclude that IDH1 activity is regulated, at least in part, by pH. We show this regulation is mediated by at least one buried acidic residue ~12 Å from the IDH1 active site. By establishing mechanisms of regulation of this well-conserved enzyme, we highlight catalytic features that may be susceptible to pH changes caused by cell stress and disease.

Zika virus (ZIKV), an emerging flavivirus which causes neurodevelopmental impairment 22 to fetuses and has been linked to Guillain-Barré syndrome, continues to threaten global health due 23 to the absence of targeted prophylaxis or treatment. Nucleoside analogues are good examples of 24 efficient anti-viral inhibitors, and prodrug strategies using phosphate masking groups (ProTides) 25 have been employed to improve the bioavailability of ribonucleoside analogues. Here, we 26 synthesized and tested a library of 13 ProTides against ZIKV in human neural stem cells. Strong 27 activity was observed for 2'-C-methyluridine and 2'-C-ethynyluridine ProTides with an aryloxyl 28 phosphoramidate masking group. Conversion of the aryloxyl phosphoramidate ProTide group of 29 2'-C-methyluridine to a 2-(methylthio)ethyl phosphoramidate completely abolished antiviral 30 activity of the compound. The aryloxyl phosphoramidate ProTide of 2'-C-methyluridine 31 outperformed the hepatitis C virus (HCV) drug sofosbuvir in suppression of viral titers and 32 protection from cytopathic effect, while the former compound's triphosphate active metabolite was 33 better incorporated by purified ZIKV NS5 polymerase over time. Molecular superpositioning 34 revealed different orientations of residues opposite the 2'-fluoro group of sofosbuvir. These findings 35 suggest both a nucleobase and ProTide group bias for the anti-ZIKV activity of nucleoside analogue 36 ProTides in a disease-relevant cell model. 37 38 cells, RNA-dependent RNA polymerase 39 40 1. Introduction 41The explosive spread of Zika virus (ZIKV) during the 2015-2016 epidemics in Latin America 42 attracted worldwide attention to this previously neglected disease. The lack of effective vaccines or 43 small molecules to prevent or treat this infection remains a cause for concern and emphasizes the 44 urgent need for new therapeutic options [1]. ZIKV, an emerging flavivirus infection, causes several 48 manifestations of the disease, and in rare cases, ZIKV infection has been linked to the 49 neuroinflammatory Guillain-Barré syndrome [2]. 50 Viral polymerases remain attractive drug targets for the development of selective antiviral 51 therapies [3-5]. Generally, clinically-approved inhibitors that target these proteins fall into two broad 52 classes [6,7]. The first class consists of nucleoside analogues that mimic the natural substrate of the 53 enzyme. Upon analogue incorporation by the virally-encoded polymerase, DNA or RNA synthesis 54 is abrogated by preventing further nucleotide incorporation (chain termination), thereby arresting 55 viral replication. The second class is known as non-nucleoside inhibitors, which bind allosterically 56 and arrest viral nucleic acid synthesis by distorting the polymerase active site geometry to interfere 57 with nucleotide binding or nucleotide incorporation. 58 A common prodrug strategy used for antiviral ribonucleoside analogues involves the chemical 59 synthesis of nucleoside analogue monophosphates with metabolically-removable masking groups 60 [8...

Isocitrate dehydrogenase 1 (IDH1) is a metabolic enzyme responsible for catalysis of isocitrate to α‐ketoglutarate using NADP+ as a cofactor. Cancerous mutations of IDH1 yield the neomorphic conversion of α‐ketoglutarate to D‐2‐hydroxyglutarate, a proposed oncometabolite. Various cancers such as gliomas, glioblastomas, acute myeloid leukemias, and bone cancers, are driven by IDH1 mutations. Although it has been established that IDH1 is an enzymatic driver of disease, more research is necessary to elucidate the molecular mechanisms of its regulation. Post‐translational modifications (PTMs), such as acetylation, serve as effective methods of protein regulation. Here, we hypothesize that acetylation may have a significant impact on IDH1 efficiency. To investigate this, we treated wild type IDH1 with acetyl group donors and generated wild type IDH1 mutants (K81Q, K224Q, K321Q) to mimic acetylation in a physiologically relevant setting. We then employed steady state enzyme kinetics to measure the rate of the normal reaction yielding α‐ketoglutarate. We report that acetylation has a notable effect on IDH1 catalytic efficiency in both the acetylation mimics and acetyl‐treated IDH1. Our data suggests that acetylation may be an essential post‐translational modification for regulating IDH1 catalytic efficiency, thus helping us understand pathways relevant to IDH1 activity. Support or Funding Information This work was funded by a Research Scholar Grant, RSG‐19‐075‐01‐TBE, from the American Cancer Society (C.D.S.), National Institutes of Health R00 CA187594 (C.D.S.), U54CA132384 (SDSU) & U54CA132379 (UC San Diego), MARC 5T34GM008303 (SDSU), and IMSD 5R25GM058906 (SDSU), as well as the California Metabolic Research Foundation (SDSU).