Inhibition of mutant IDH1 is being evaluated clinically as a promising treatment option for various cancers with hotspot mutation at Arg. Having identified an allosteric, induced pocket of IDH1, we have explored 3-pyrimidin-4-yl-oxazolidin-2-ones as mutant IDH1 inhibitors for modulation of 2-HG production and potential brain penetration. We report here optimization efforts toward the identification of clinical candidate (), a potent and selective mutant IDH1 inhibitor that has demonstrated brain exposure in rodents. Preclinical characterization of this compound exhibited correlation of 2-HG reduction and efficacy in a patient-derived IDH1 mutant xenograft tumor model. () has progressed into human clinical trials for the treatment of cancers with IDH1 mutation.

Nonstructural protein 5B (NS5B) is theviral RNA-dependent RNA polymerase thatcatalyzes the replication of the hepatitis C virusgenome. It is a major target for antiviral drugs,including nucleotide analogs (NAs) such as theprodrugs mericitabine and sofosbuvir, which getmetabolized to 2’-fluoro-2’-C-methylcytidine-5’-triphosphate and 2’fluoro-2’-C-methyluridine-5’-triphosphate, respectively. These analogs act aschain terminators after they are incorporated duringviral RNA synthesis. Recently, it has been shownthat NS5B can efficiently remove chain terminatorsby a nucleotide-mediated excision reaction thatrescues RNA synthesis. In this study, we usedtransient-state kinetics to study the efficiency ofNS5B inhibition by five NAs. We show that NS5Breadily incorporates CTP analogs into a growingprimer, but that these analogs are also efficientlyexcised. In contrast, although UMP analogs weremore slowly incorporated, UMP excision was alsoslow and inefficient, and modifications to the 2’Cof the UTP ribose ring further decreased excisionrates to an undetectable level. Taken together, theseresults suggest that the greater clinical effectivenessof the UMP analog sofosbuvir is largely due to itbeing intractable to nucleotide-mediated excisioncompared with similar NAs such as mericitabine.

Mutant isocitrate dehydrogenase 1 (IDH1) is an attractive therapeutic target for the treatment of various cancers such as AML, glioma, and glioblastoma. We have evaluated 3-pyrimidin-4-yl-oxazolidin-2-ones as mutant IDH1 inhibitors that bind to an allosteric, induced pocket of IDH1. This Letter describes SAR exploration focused on improving both the and metabolic stability of the compounds, leading to the identification of as a potent and selective mutant IDH1 inhibitor that has demonstrated brain penetration and excellent oral bioavailability in rodents. In a preclinical patient-derived IDH1 mutant xenograft tumor model study, efficiently inhibited the production of the biomarker 2-HG.

The hepatitis C virus RNA-dependent RNA polymerase NS5B is responsible for the replication of the viral genome. Previous studies have uncovered NTP-mediated excision mechanisms which may be responsible for aiding in maintaining fidelity (the frequency of incorrect incorporation events relative to correct), but little is known about the fidelity of NS5B. In this study, we used transient-state kinetics to examine the mechanistic basis for polymerase fidelity. We observe a wide range of efficiency for incorporation of various mismatched base pairs and have uncovered a mechanism in which the rate constant for pyrophosphate release is slowed for certain misincorporation events. This results in an increase in fidelity against these specific misincorporations. Furthermore, we discover that some mismatches are highly unfavorable and cannot be observed under the conditions used here. The calculated fidelity of NS5B ranges between 10-4-10-9 for different mismatches.

The Hepatitis C Virus (HCV) infects an estimated 71 million people worldwide. Currently, there is no vaccine available to treat or prevent HCV infections. Nucleoside analogs (NA) with a modification on the 2′‐carbon are a group of drugs that target the HCV RNA‐dependent RNA polymerase, NS5B. Among these nucleoside analogs are the prodrug Mericitabine and Sofosbuvir which get metabolized to 2′‐fluoro‐2′‐C‐methylcytidine‐5′‐triphosphate and 2′fluoro‐2′‐C‐methyluridine‐5′‐triphosphate, respectively. These compounds work by mimicking natural nucleotides and competing for binding and incorporation during the replication of the HCV genome. Once incorporated they act as chain terminators by preventing further elongation of the nascent RNA strand. Recently, it has been shown that NA can be excised by a nucleotide‐mediated excision reaction which rescues RNA synthesis. In this study we use transient‐state kinetics to understand the mechanism of inhibition for two CTP analogs and a UTP analog. The results demonstrate that while CTP analogs are readily incorporated and excised, UTP and the UTP analogs efficiently incorporated but are resistant to excision. Support or Funding Information This work was supported by funding from the NIH This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.