Design and Optimization of Novel Competitive, Non-peptidic, SARS-CoV-2 Mpro Inhibitors.

The SARS-CoV-2 main protease (Mpro) has been proven to be a highly effective target for therapeutic intervention, yet only one drug currently holds FDA approval status for this target. We were inspired by a series of publications emanating from the Jorgensen and Anderson groups describing the design of potent, non-peptidic, competitive SARS-CoV-2 Mpro inhibitors, and we saw an opportunity to make several design modifications to improve the overall pharmacokinetic profile of these compounds without losing potency. To this end, we created a focused virtual library using reaction-based enumeration tools in the Schrödinger suite. These compounds were docked into the Mpro active site and subsequently prioritized for synthesis based upon relative binding affinity values calculated by FEP+. Fourteen compounds were selected, synthesized, and evaluated both biochemically and in cell culture. Several of the synthesized compounds proved to be potent, competitive Mpro inhibitors with improved metabolic stability profiles.

Development of a Dihydroquinoline-Pyrazoline GluN2C/2D-Selective Negative Allosteric Modulator of the N-Methyl-d-aspartate Receptor.

Subunit-selective inhibition of N-methyl-d-aspartate receptors (NMDARs) is a promising therapeutic strategy for several neurological disorders, including epilepsy, Alzheimer's and Parkinson's disease, depression, and acute brain injury. We previously described the dihydroquinoline-pyrazoline (DQP) analogue 2a (DQP-26) as a potent NMDAR negative allosteric modulator with selectivity for GluN2C/D over GluN2A/B. However, moderate (<100-fold) subunit selectivity, inadequate cell-membrane permeability, and poor brain penetration complicated the use of 2a as an in vivo probe. In an effort to improve selectivity and the pharmacokinetic profile of the series, we performed additional structure-activity relationship studies of the succinate side chain and investigated the use of prodrugs to mask the pendant carboxylic acid. These efforts led to discovery of the analogue (S)-(-)-2i, also referred to as (S)-(-)-DQP-997-74, which exhibits >100- and >300-fold selectivity for GluN2C- and GluN2D-containing NMDARs (IC50 0.069 and 0.035 µM, respectively) compared to GluN2A- and GluN2B-containing receptors (IC50 5.2 and 16 µM, respectively) and has no effects on AMPA, kainate, or GluN1/GluN3 receptors. Compound (S)-(-)-2i is 5-fold more potent than (S)-2a. In addition, compound 2i shows a time-dependent enhancement of inhibitory actions at GluN2C- and GluN2D-containing NMDARs in the presence of the agonist glutamate, which could attenuate hypersynchronous activity driven by high-frequency excitatory synaptic transmission. Consistent with this finding, compound 2i significantly reduced the number of epileptic events in a murine model of tuberous sclerosis complex (TSC)-induced epilepsy that is associated with upregulation of the GluN2C subunit. Thus, 2i represents a robust tool for the GluN2C/D target validation. Esterification of the succinate carboxylate improved brain penetration, suggesting a strategy for therapeutic development of this series for NMDAR-associated neurological conditions.

Discovery of 5'-Substituted 5-Fluoro-2'-deoxyuridine Monophosphate Analogs: A Novel Class of Thymidylate Synthase Inhibitors.

5-Fluorouracil and 5-fluorouracil-based prodrugs have been used clinically for decades to treat cancer. Their anticancer effects are most prominently ascribed to inhibition of thymidylate synthase (TS) by metabolite 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). However, 5-fluorouracil and FdUMP are subject to numerous unfavorable metabolic events that can drive undesired systemic toxicity. Our previous research on antiviral nucleotides suggested that substitution at the nucleoside 5'-carbon imposes conformational restrictions on the corresponding nucleoside monophosphates, rendering them poor substrates for productive intracellular conversion to viral polymerase-inhibiting triphosphate metabolites. Accordingly, we hypothesized that 5'-substituted analogs of FdUMP, which is uniquely active at the monophosphate stage, would inhibit TS while preventing undesirable metabolism. Free energy perturbation-derived relative binding energy calculations suggested that 5'(R)-CH3 and 5'(S)-CF3 FdUMP analogs would maintain TS potency. Herein, we report our computational design strategy, synthesis of 5'-substituted FdUMP analogs, and pharmacological assessment of TS inhibitory activity.

Expanding the toolbox of metabolically stable lipid prodrug strategies.

Nucleoside- and nucleotide-based therapeutics are indispensable treatment options for patients suffering from malignant and viral diseases. These agents are most commonly administered to patients as prodrugs to maximize bioavailability and efficacy. While the literature provides a practical prodrug playbook to facilitate the delivery of nucleoside and nucleotide therapeutics, small context-dependent amendments to these popular prodrug strategies can drive dramatic improvements in pharmacokinetic (PK) profiles. Herein we offer a brief overview of current prodrug strategies, as well as a case study involving the fine-tuning of lipid prodrugs of acyclic nucleoside phosphonate tenofovir (TFV), an approved nucleotide HIV reverse transcriptase inhibitor (NtRTI) and the cornerstone of combination antiretroviral therapy (cART). Installation of novel lipid terminal motifs significantly reduced fatty acid hepatic ω-oxidation while maintaining potent antiviral activity. This work contributes important insights to the expanding repertoire of lipid prodrug strategies in general, but particularly for the delivery and distribution of acyclic nucleoside phosphonates.

ω-Functionalized Lipid Prodrugs of HIV NtRTI Tenofovir with Enhanced Pharmacokinetic Properties.

Tenofovir (TFV) is the cornerstone nucleotide reverse transcriptase inhibitor (NtRTI) in many combination antiretroviral therapies prescribed to patients living with HIV/AIDS. Due to poor cell permeability and oral bioavailability, TFV is administered as one of two FDA-approved prodrugs, both of which metabolize prematurely in the liver and/or plasma. This premature prodrug processing depletes significant fractions of each oral dose and causes toxicity in kidney, bone, and liver with chronic administration. Although TFV exalidex (TXL), a phospholipid-derived prodrug of TFV, was designed to address this issue, clinical pharmacokinetic studies indicated substantial hepatic extraction, redirecting clinical development of TXL toward HBV. To circumvent this metabolic liability, we synthesized and evaluated ω-functionalized TXL analogues with dramatically improved hepatic stability. This effort led to the identification of compounds 21 and 23, which exhibited substantially longer t1/2 values than TXL in human liver microsomes, potent anti-HIV activity in vitro, and enhanced pharmacokinetic properties in vivo.

Accelerated Discovery of Potent Fusion Inhibitors for Respiratory Syncytial Virus.

A series of five benzimidazole-based compounds were identified using a machine learning algorithm as potential inhibitors of the respiratory syncytial virus (RSV) fusion protein. These compounds were synthesized, and compound 2 in particular exhibited excellent in vitro potency with an EC50 value of 5 nM. This new scaffold was then further refined leading to the identification of compound 44, which exhibited a 10-fold improvement in activity with an EC50 value of 0.5 nM.

Prevalence of Eight Phthalate Monoesters in Water from the Okavango Delta, Northern Botswana.

Phthalate diesters are used in personal care products, plastics, and pesticides, resulting in widespread human and wildlife exposure. Phthalate diesters leach out of these products and ultimately enter biological systems where they are quickly metabolized to phthalate monoesters and glucuronides. As such, phthalate monoesters can serve as indicators of anthropogenic activity in wilderness areas. The Okavango Delta, an inland seasonal wetland covering 5000-12,000 km2 in Botswana, provides fresh water to many species of birds, fish, reptiles, and large mammals. Water samples (N = 46) were taken from across the Okavango water system, extracted, and analyzed for eight different phthalate monoesters using liquid chromatography and isotope dilution mass spectrometry. Seven of eight phthalate monoesters were detected from the low ng/L to low µg/L levels. Phthalate monoesters were found in samples from all five sampling regions. Sources of these contaminants are unknown, but their presence indicates encroachment of human activity on the Okavango Delta.