Epithelial N-methyl-D-aspartate (NMDA) receptors mediate renal vasodilation by affecting kidney autoregulation.

Background: N-methyl-D-aspartate receptor (NMDAR) are amino acid receptors that are well studied in brain physiology; however, their role in kidney is poorly understood. Nonetheless, NMDAR inhibitors can increase serum K+ and reduce GFR, which suggests they have an important physiological role in the kidney. We hypothesized that NMDARs in the distal nephron induce afferent-arteriole vasodilation through the vasodilator mechanism connecting-tubule-glomerular feedback (CNTGF) that involves ENaC activation. Methods and results: Using a tubule-specific transcriptome database combined with molecular biology and microscopy techniques, we showed kidney expression of NMDAR subunits along the nephron and specifically in ENaC-positive cells. This receptor is expressed in both male and female mice, with higher abundance in females (p=0.02). Microperfusing NMDAR agonists into the connecting tubule induced afferent-arteriole vasodilation (EC50 10.7 vs. 24.5 mM; p<0.001) that was blunted or eliminated with the use of NMDAR blocker MK-801 or with the ENaC inhibitor Benzamil, indicating a dependence on CNTGF of the NMDAR-induced vasodilation. In vivo, we confirmed this CNTGF-associated vasodilation using kidney micropuncture (Stop-flow pressure 37.9±2.6 vs. 28.6±1.9 mmHg, NMDAR agonist vs vehicle; p<0.01). We explored NMDAR and ENaC channel interaction by using mpkCCD cells and split-open connecting tubules. We observed increased amiloride-sensitive current following NMDAR activation that was prevented by MK-801 (1.14 vs. 0.4 µAmp; p=0.03). In split-open tubules, NMDAR activation increased ENaC activity (Npo Vehicle vs. NMDA; p=0.04). Conclusion: NMDARs are expressed along the nephron, including ENaC-positive cells, with higher expression in females. Epithelial NMDAR mediates renal vasodilation through the connecting-tubule-glomerular feedback, by increasing ENaC activity.

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.

Neurotherapeutic Potential of Water-Soluble pH-Responsive Prodrugs of EIDD-036 in Traumatic Brain Injury.

The C-20 oxime of progesterone, EIDD-036 (2), demonstrates neuroprotection and improved outcomes in animal models of traumatic brain injury (TBI). However, 2 suffers from poor solubility, which renders it unsuitable for rapid administration. Previous prodrugs of 2 aimed at improving solubility by incorporating enzymatically labile amino acid and phosphate ester promoieties. These approaches were effective but led to limitations with in vivo administration. Herein, we disclose a pH-responsive water-soluble prodrug strategy to improve exposure to 2 through enzyme-independent activation. Compound 13l was identified as a lead that exhibits water-solubility, stability in acidic solutions, and rapid conversion to 2 at physiological pH. Administration of 13l to rats resulted in a twofold increase in exposure to 2 compared to the previous generation phosphate prodrug, EIDD-1723 (6). In a rat model of TBI, treatment with 13l resulted in a significant decrease in cerebral edema when administered postinjury.

Novel GluN2B-Selective NMDA Receptor Negative Allosteric Modulator Possesses Intrinsic Analgesic Properties and Enhances Analgesia of Morphine in a Rodent Tail Flick Pain Model.

Many cases of accidental death associated with drug overdose are due to chronic opioid use, tolerance, and addiction. Analgesic tolerance is characterized by a decreased response to the analgesic effects of opioids, requiring increasingly higher doses to maintain the desired level of pain relief. Overactivation of GluN2B-containing N-methyl-d-Aspartate receptors is thought to play a key role in mechanisms underlying cellular adaptation that takes place in the development of analgesic tolerance. Herein, we describe a novel GluN2B-selective negative allosteric modulator, EU93-108, that shows high potency and brain penetrance. We describe the structural basis for binding at atomic resolution. This compound possesses intrinsic analgesic properties in the rodent tail immersion test. EU93-108 has an acute and significant anodyne effect, whereby morphine when combined with EU93-108 produces a higher tail flick latency compared to that of morphine alone. These data suggest that engagement of GluN2B as a target has utility in the treatment of pain, and EU93-108 could serve as an appropriate tool compound to interrogate this hypothesis. Future structure-activity relationship work around this scaffold could give rise to compounds that can be co-administered with opioids to diminish the onset of tolerance due to chronic opioid use, thereby modifying their utility.

Synthesis and Evaluation of Novel Tetrahydronaphthyridine CXCR4 Antagonists with Improved Drug-like Profiles.

Our first-generation CXCR4 antagonist TIQ15 was rationally modified to improve drug-like properties. Introducing a nitrogen atom into the aromatic portion of the tetrahydroisoquinoline ring led to several heterocyclic variants including the 5,6,7,8-tetrahydro-1,6-naphthyridine series, greatly reducing the inhibition of the CYP 2D6 enzyme. Compound 12a demonstrated the best overall properties after profiling a series of isomeric tetrahydronaphthyridine analogues in a battery of biochemical assays including CXCR4 antagonism, CYP 2D6 inhibition, metabolic stability, and permeability. The butyl amine side chain of 12a was substituted with various lipophilic groups to improve the permeability. These efforts culminated in the discovery of compound 30 as a potent CXCR4 antagonist (IC50 = 24 nM) with diminished CYP 2D6 activity, improved PAMPA permeability (309 nm/s), potent inhibition of human immunodeficiency virus entry (IC50 = 7 nM), a cleaner off-target in vitro safety profile, lower human ether a-go-go-related gene channel activity, and higher oral bioavailability in mice (% FPO = 27) compared to AMD11070 and TIQ15.

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.

Amino-Heterocycle Tetrahydroisoquinoline CXCR4 Antagonists with Improved ADME Profiles via Late-Stage Buchwald Couplings.

This work surveys a variety of diamino-heterocycles as an isosteric replacement for the piperazine substructure of our previously disclosed piperarinyl-tetrahydroisoquinoline containing CXCR4 antagonists. A late-stage Buchwald coupling route was developed for rapid access to final compounds from commercial building blocks. Among 13 analogs in this study, compound 31 embodying an aza-piperazine linkage was found to have the best overall profile with potent CXCR4 inhibitory activity and favorable in vitro absorption, distribution, metabolism, and excretion (ADME) properties. An analysis of the calculated physiochemical parameters (ROF, cLogD) and the experimental ADME attributes of the analogs lead to the selection of 31 for pharmacokinetic studies in mice. Compared with the clinical compound AMD11070, compound 31 has no CYP450 3A4 or 2D6 inhibition, higher metabolic stability and PAMPA permeability, greatly improved physiochemical parameters, and superior oral bioavailability (%F = 24). A binding rationale for 31 within CXCR4 was elucidated from docking and molecular simulation studies.

A Glutamate N-Methyl-d-Aspartate (NMDA) Receptor Subunit 2B-Selective Inhibitor of NMDA Receptor Function with Enhanced Potency at Acidic pH and Oral Bioavailability for Clinical Use.

We describe a clinical candidate molecule from a new series of glutamate N-methyl-d-aspartate receptor subunit 2B-selective inhibitors that shows enhanced inhibition at extracellular acidic pH values relative to physiologic pH. This property should render these compounds more effective inhibitors of N-methyl-d-aspartate receptors at synapses responding to a high frequency of action potentials, since glutamate-containing vesicles are acidic within their lumen. In addition, acidification of penumbral regions around ischemic tissue should also enhance selective drug action for improved neuroprotection. The aryl piperazine we describe here shows strong neuroprotective actions with minimal side effects in preclinical studies. The clinical candidate molecule NP10679 has high oral bioavailability with good brain penetration and is suitable for both intravenous and oral dosing for therapeutic use in humans. SIGNIFICANCE STATEMENT: This study identifies a new series of glutamate N-methyl-d-aspartate (NMDA) receptor subunit 2B-selective negative allosteric modulators with properties appropriate for clinical advancement. The compounds are more potent at acidic pH, associated with ischemic tissue, and this property should increase the therapeutic safety of this class by improving efficacy in affected tissue while sparing NMDA receptor block in healthy brain.

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.