The cytochrome P450 enzymes (P450s or CYPs) form a large family of heme proteins involved in drug metabolism and in the biosynthesis of steroids, lipids, vitamins and natural products. Their remarkable ability to catalyze the insertion of oxygen into non-activated C-H bonds has attracted the interest of chemists for several decades. Very few chemical methods exist that directly hydroxylate aliphatic or aromatic C-H bonds, and most of them are not selective or of limited scope. Biocatalysts such as P450s represent a promising alternative: however, their applications have been limited by substrate specificity, low activity, poor stability and the need for cofactors. This review covers the attempts to overcome these limitations using approaches such as mutagenesis, chemical modifications, conditions engineering and immobilization.
Ataxia telangiectasia and Rad3-related (ATR) kinase protects genome integrity during DNA replication. RP-3500 is a novel, orally bioavailable clinical-stage ATR kinase inhibitor (NCT04497116). RP-3500 is highly potent with IC 50 values of 1.0 and 0.33 nM in biochemical and cell-based assays, respectively. RP-3500 is highly selective for ATR with 30-fold selectivity over mTOR and >2,000-fold selectivity over ATM, DNA-PK, and PI3Kkinases. In vivo, RP-3500 treatment results in potent single-agent efficacy and/or tumor regression in multiple xenograft models at minimum effective doses (MED) of 5-7 mg/kg once daily. Pharmacodynamic assessments validate target engagement, with dose-proportional tumor pCHK1 inhibition (IC 80 = 18.6 nM) and induction of -H2AX, pDNA-PKcs, and pKAP1. RP-3500 exposure at MED indicates that circulating free plasma levels above the in vivo tumor IC 80 for 10-12 hours are sufficient for efficacy on a continuous schedule. However, short-duration intermittent (weekly 3 days on/4 days off) dosing schedules as monotherapy or given concomitantly with reduced doses of olaparib or niraparib, maximize tumor growth inhibition while minimizing the impact on red blood cell depletion, emphasizing the reversible nature of erythroid toxicity with RP-3500 and demonstrating superior efficacy compared with sequential treatment. These results provide a strong preclinical rationale to support ongoing clinical investigation of the novel ATR inhibitor, RP-3500, on an intermittent schedule as a monotherapy and in combination with PARP inhibitors as a potential means of maximizing clinical benefit.
P450 enzymes have attracted the attention of chemists for decades because of their impressive ability to catalyze the hydroxylation of inactivated C--H bonds. However, their use for synthesis in aqueous systems is limited. We report here a survey of the activity of purified human CYP3A4 in the presence of organic solvents or ionic liquids. We show that CYP3A4 tolerates only small amounts (<15 %) of water-miscible organic cosolvents or ionic liquids before its activity toward testosterone drops below detection. [BMIM][PF(6)] in a biphasic system was less detrimental to enzyme activity, with 20 % of the activity remaining in the presence of 15 % of this ionic liquid. CYP3A4 activity in the absence of buffer was only >or=10 % in solvents of the alkane series, with a minimum of 0.85 % water, and with the addition of sucrose and testosterone before enzyme lyophilization. Biphasic solvent systems were more promising, with approximately 85 % of the activity retained.
DNA polymerase theta (Polθ)
is an attractive synthetic lethal
target for drug discovery, predicted to be efficacious against breast
and ovarian cancers harboring BRCA-mutant alleles. Here, we describe
our hit-to-lead efforts in search of a selective inhibitor of human
Polθ (encoded by POLQ). A high-throughput screening campaign
of 350,000 compounds identified an 11 micromolar hit, giving rise
to the N2-substituted fused pyrazolo series, which was validated by
biophysical methods. Structure-based drug design efforts along with
optimization of cellular potency and ADME ultimately led to the identification
of RP-6685: a potent, selective, and orally bioavailable
Polθ inhibitor that showed in vivo efficacy in an HCT116 BRCA2–/– mouse tumor xenograft model.
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