A Second-Generation Oral SARS-CoV-2 Main Protease Inhibitor Clinical Candidate for the Treatment of COVID-19.

Despite the record-breaking discovery, development and approval of vaccines and antiviral therapeutics such as Paxlovid, coronavirus disease 2019 (COVID-19) remained the fourth leading cause of death in the world and third highest in the United States in 2022. Here, we report the discovery and characterization of PF-07817883, a second-generation, orally bioavailable, SARS-CoV-2 main protease inhibitor with improved metabolic stability versus nirmatrelvir, the antiviral component of the ritonavir-boosted therapy Paxlovid. We demonstrate the in vitro pan-human coronavirus antiviral activity and off-target selectivity profile of PF-07817883. PF-07817883 also demonstrated oral efficacy in a mouse-adapted SARS-CoV-2 model at plasma concentrations equivalent to nirmatrelvir. The preclinical in vivo pharmacokinetics and metabolism studies in human matrices are suggestive of improved oral pharmacokinetics for PF-07817883 in humans, relative to nirmatrelvir. In vitro inhibition/induction studies against major human drug metabolizing enzymes/transporters suggest a low potential for perpetrator drug-drug interactions upon single-agent use of PF-07817883.

Novel Multiplexed High Throughput Screening of Selective Inhibitors for Drug-Metabolizing Enzymes Using Human Hepatocytes.

Selective chemical inhibitors are critical for reaction phenotyping to identify drug-metabolizing enzymes that are involved in the elimination of drug candidates. Although relatively selective inhibitors are available for the major cytochrome P450 enzymes (CYP), they are quite limited for the less common CYPs and non-CYPs. To address this gap, we developed a multiplexed high throughput screening (HTS) assay using 20 substrate reactions of multiple enzymes to simultaneously monitor the inhibition of enzymes in a 384-well format. Four 384-well assay plates can be run at the same time to maximize throughput. This is the first multiplexed HTS assay for drug-metabolizing enzymes reported. The HTS assay is technologically enabled with state-of-the-art robotic systems and highly sensitive modern LC-MS/MS instrumentation. Virtual screening is utilized to identify inhibitors for HTS based on known inhibitors and enzyme structures. Screening of ~4600 compounds generated many hits for many drug-metabolizing enzymes including the two time-dependent and selective aldehyde oxidase inhibitors, erlotinib and dibenzothiophene. The hit rate is much higher than that for the traditional HTS for biological targets due to the promiscuous nature of the drug-metabolizing enzymes and the biased compound selection process. Future efforts will focus on using this method to identify selective inhibitors for enzymes that do not currently have quality hits and thoroughly characterizing the newly identified selective inhibitors from our screen. We encourage colleagues from other organizations to explore their proprietary libraries using a similar approach to identify better inhibitors that can be used across the industry.

Characterization and Applications of Permeabilized Hepatocytes in Drug Discovery.

Hepatocytes are one of the most physiologically relevant in vitro liver systems for human translation of clearance and drug-drug interactions (DDI). However, the cell membranes of hepatocytes can limit the entry of certain compounds into the cells for metabolism and DDI. Passive permeability through hepatocytes can be different in vitro and in vivo, which complicates the human translation. Permeabilized hepatocytes offer a useful tool to probe mechanistic understanding of permeability-limited metabolism and DDI. Incubation with saponin of 0.01% at 0.5 million cells/mL and 0.05% at 5 million cells/mL for 5 min at 37°C completely permeabilized the plasma membrane of hepatocytes, while leaving the membranes of subcellular organelles intact. Permeabilized hepatocytes maintained similar enzymatic activity as intact unpermeabilized hepatocytes and can be stored at -80°C for at least 7 months. This approach reduces costs by preserving leftover hepatocytes. The relatively low levels of saponin in permeabilized hepatocytes had no significant impact on the enzymatic activity. As the cytosolic contents leak out from permeabilized hepatocytes, cofactors need to be added to enable metabolic reactions. Cytosolic enzymes will no longer be present if the media are removed after cells are permeabilized. Hence permeabilized hepatocytes with and without media removal may potentially enable reaction phenotyping of cytosolic enzymes. Although permeabilized hepatocytes work similarly as human liver microsomes and S9 fractions experimentally requiring addition of cofactors, they behave more like hepatocytes maintaining enzymatic activities for over 4 h. Permeabilized hepatocytes are a great addition to the drug metabolism toolbox to provide mechanistic insights.

The Comparison of Machine Learning and Mechanistic In Vitro-In Vivo Extrapolation Models for the Prediction of Human Intrinsic Clearance.

Accurate prediction of human pharmacokinetics (PK) remains one of the key objectives of drug metabolism and PK (DMPK) scientists in drug discovery projects. This is typically performed by using in vitro-in vivo extrapolation (IVIVE) based on mechanistic PK models. In recent years, machine learning (ML), with its ability to harness patterns from previous outcomes to predict future events, has gained increased popularity in application to absorption, distribution, metabolism, and excretion (ADME) sciences. This study compares the performance of various ML and mechanistic models for the prediction of human IV clearance for a large (645) set of diverse compounds with literature human IV PK data, as well as measured relevant in vitro end points. ML models were built using multiple approaches for the descriptors: (1) calculated physical properties and structural descriptors based on chemical structure alone (classical QSAR/QSPR); (2) in vitro measured inputs only with no structure-based descriptors (ML IVIVE); and (3) in silico ML IVIVE using in silico model predictions for the in vitro inputs. For the mechanistic models, well-stirred and parallel-tube liver models were considered with and without the use of empirical scaling factors and with and without renal clearance. The best ML model for the prediction of in vivo human intrinsic clearance (CLint) was an in vitro ML IVIVE model using only six in vitro inputs with an average absolute fold error (AAFE) of 2.5. The best mechanistic model used the parallel-tube liver model, with empirical scaling factors resulting in an AAFE of 2.8. The corresponding mechanistic model with full in silico inputs achieved an AAFE of 3.3. These relative performances of the models were confirmed with the prediction of 16 Pfizer drug candidates that were not part of the original data set. Results show that ML IVIVE models are comparable to or superior to their best mechanistic counterparts. We also show that ML IVIVE models can be used to derive insights into factors for the improvement of mechanistic PK prediction.

In Vitro-In Vivo Extrapolation and Scaling Factors for Clearance of Human and Preclinical Species with Liver Microsomes and Hepatocytes.

In vitro-in vivo extrapolation ((IVIVE) and empirical scaling factors (SF) of human intrinsic clearance (CLint) were developed using one of the largest dataset of 455 compounds with data from human liver microsomes (HLM) and human hepatocytes (HHEP). For extended clearance classification system (ECCS) class 2/4 compounds, linear SFs (SFlin) are approximately 1, suggesting enzyme activities in HLM and HHEP are similar to those in vivo under physiological conditions. For ECCS class 1A/1B compounds, a unified set of SFs was developed for CLint. These SFs contain both SFlin and an exponential SF (SFß) of fraction unbound in plasma (fu,p). The unified SFs for class 1A/1B eliminate the need to identify the transporters involved prior to clearance prediction. The underlying mechanisms of these SFs are not entirely clear at this point, but they serve practical purposes to reduce biases and increase prediction accuracy. Similar SFs have also been developed for preclinical species. For HLM-HHEP disconnect (HLM > HHEP) ECCS class 2/4 compounds that are mainly metabolized by cytochrome P450s/FMO, HLM significantly overpredicted in vivo CLint, while HHEP slightly underpredicted and geometric mean of HLM and HHEP slightly overpredicted in vivo CLint. This observation is different than in rats, where rat liver microsomal CLint correlates well with in vivo CLint for compounds demonstrating permeability-limited metabolism. The good CLint IVIVE developed using HLM and HHEP helps build confidence for prospective predictions of human clearance and supports the continued utilization of these assays to guide structure-activity relationships to improve metabolic stability.

COVID-19 and neurodegeneration: The mitochondrial connection.

There is still a significant lack of knowledge regarding many aspects of the etiopathology and consequences of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. For example, the variety of molecular mechanisms mediating this infection, and the long-term consequences of the disease remain poorly understood. It first seemed like the SARS-CoV-2 infection primarily caused a serious respiratory syndrome. However, over the last years, an increasing number of studies also pointed towards the damaging effects of this infection has on the central nervous system (CNS). In fact, evidence suggests a possible disruption of the blood-brain barrier and deleterious effects on the CNS, especially in patients who already suffer from other pathologies, such as neurodegenerative disorders. The molecular mechanisms behind these effects on the CNS could involve the dysregulation of mitochondrial physiology, a well-known early marker of neurodegeneration and a hallmark of aging. Moreover, mitochondria are involved in the activation of the inflammatory response, which has also been broadly described in the CNS in COVID-19. Here, we critically review the current bibliography regarding the presence of neurodegenerative symptoms in COVID-19 patients, with a special emphasis on the mitochondrial mechanisms of these disorders.

Inactivation Kinetics of G-Quadruplex/Hemin Complex and Optimization for More Reliable Catalysis.

Reliable catalysis is critical for the synthesis of various chemicals, molecular sensing and biomedicine. G-quadruplex/Hemin (GQH) complex, a peroxidase-mimicking DNAzyme, has been widely used in various publications. However, a concern exists about the unstable kinetics of GQH-catalyzed peroxidation. This work investigates several factors that result in the inactivation of GQH and the signal degradation during long reaction periods, including pH, buffer component, the selection of substrate and the oxidation damage of cofactor. Using colorimetric and fluorescent assays, GQH was found to be highly unstable under basic conditions with 50 % of GQH activity lost within 2 minutes at high H2 O2 concentrations. Appropriate conditions and substrates are suggested for accurately characterizing GQH-catalyzed reactions, as well as optimization to improve the catalytic reliability, such as the use of polyhistidine and cascade reactions. These results could be useful for GQH-related applications.

Synthesis of Deuterated Heterocycles with Me2NCD(OMe)2 and Evaluation of the Products for Metabolism by Aldehyde Oxidase.

The metabolic oxidation of drug-like small molecules by aldehyde oxidase (AO) has commonly been mitigated through the incorporation of deuterium at the oxidation site. We report that dimethylformamide dimethyl acetal and related compounds undergo rapid CH to CD isotopic exchange upon exposure to methanol-d and similar deuterated alcohols. This isotopic exchange process can be used to synthesize Me2NCD(OMe)2 and has significant implications for the use of Me2NCD(OMe)2 in the synthesis of specifically deuterium-labeled compounds. The application of Me2NCD(OMe)2 to the synthesis of various heterocycles that have been associated with AO metabolism is described, and we report the impact of deuteration on the rate of in vitro AO-mediated metabolism.

Rapid Nucleic Acid Reaction Circuits for Point-of-care Diagnosis of Diseases.

An urgent need exists for a rapid, cost-effective, facile, and reliable nucleic acid assay for mass screening to control and prevent the spread of emerging pandemic diseases. This urgent need is not fully met by current diagnostic tools. In this review, we summarize the current state-of-the-art research in novel nucleic acid amplification and detection that could be applied to point-of-care (POC) diagnosis and mass screening of diseases. The critical technological breakthroughs will be discussed for their advantages and disadvantages. Finally, we will discuss the future challenges of developing nucleic acid-based POC diagnosis.

High and Low Frequency Domino Liver Transplantation Centers Demonstrate Similar Performance Outcomes.

INTRODUCTION: A Domino Liver Transplant (DLT) is a successfully validated surgical option for a subset of patients awaiting liver transplant. Increased utilization of DLTs could increase the donor organ pool. However, DLTs occur primarily at a small number of high volume centers, and are rarely performed at lower volume transplant centers. This study compares DLT recipient performance outcomes between high frequency DLT centers and low frequency DLT centers. METHODS: The UNOS/OPTN STAR database was queried for DLTs performed at transplant centers between 1996-2018. 193 patients were identified and categorized into high (>5 DLTs) or low (≤5 DLTs) frequency centers. Our primary endpoint was allograft survival. Our secondary endpoints were graft status at last follow up and mortality secondary to cardiac, renal, or respiratory failure. RESULTS: Overall median allograft survival between high and low volume DLT centers was similar (48.2 months versus 42.7 months, P >0.314). The one-year (82% versus 76%), three-year (57% versus 56%), and five-year (45% versus 43%) survival percentages were also similar between the high and low volume DLT centers respectively. Overall mortality from cardiac (high 4% versus low 1.7%), renal (high 0.8% versus low 1.7%), or respiratory failure (high 0.8% versus low 1.7%) was similarly low in both groups. CONCLUSION: Low volume and high volume DLT centers are associated with similar outcomes of allograft survival and mortality. DLTs should be utilized more frequently, when the criteria are met, including in centers with limited experience, to expand the donor pool, decrease time on the waitlist, and improve overall survival.

An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19.

The worldwide outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase 1 clinical trial in healthy human participants.

Production of active recombinant human aldehyde oxidase (AOX) in the baculovirus expression vector system (BEVS) and deployment in a pre-clinical fraction-of-control AOX compound exposure assay.

Human aldehyde oxidase (AOX) has emerged as a key enzyme activity for consideration in modern drug discovery. The enzyme catalyzes the oxidation of a wide variety of compounds, most notably azaheterocyclics that often form the building blocks of small molecule therapeutics. Failure to consider and assess AOX drug exposure early in the drug development cycle can have catastrophic consequences for novel compounds entering the clinic. AOX is a complex molybdopterin-containing iron-sulfur flavoprotein comprised of two identical 150 kDa subunits that has proven difficult to produce in recombinant form, and a commercial source of the purified human enzyme is currently unavailable. Thus, the potential exposure of novel drug development candidates to human AOX metabolism is usually assessed by using extracts of pooled human liver cytosol as a source of the enzyme. This can complicate the assignment of AOX-specific compound exposure due to its low activity and the presence of contaminating enzymes that may have overlapping substrate specificities. Herein is described a two-step process for the isolation of recombinant human AOX dimers to near homogeneity following production in the baculovirus expression vector system (BEVS). The deployment of this BEVS-produced recombinant human AOX as a substitute for human liver extracts in a fraction-of-control AOX compound-exposure screening assay is described. The ability to generate this key enzyme activity readily in a purified recombinant form provides for a more accurate and convenient approach to the assessment of new compound exposure to bona fide AOX drug metabolism.

Mechanistic insights on clearance and inhibition discordance between liver microsomes and hepatocytes when clearance in liver microsomes is higher than in hepatocytes.

Human liver microsomes (HLM) and human hepatocytes (HHEP) are two common in vitro systems used in metabolic stability and inhibition studies. The comparison between the assays using the two systems can provide mechanistic insights on the interplay of metabolism, passive permeability and transporters. This study investigated the critical factors impacting the unbound intrinsic clearance (CLint,u) and IC50 of CYP3A inhibition between HLM and HHEP. The HLM/HHEP CLint,u ratio and HHEP/HLM IC50 ratio are inversely correlated to passive permeability, but have no correlation with P-gp efflux ratio. Cofactor-supplemented permeabilized HHEP (MetMax™) collapses the IC50 differences between HHEP and HLM. P-gp inhibitor, encequidar, shows minimal impact on CLint,u and IC50 in HHEP. This is the first study that is able to separately investigate the effects of passive permeability and efflux transport. These data collectively show that passive permeability plays a critical role in metabolism and enzyme inhibition in HHEP, while P-gp efflux has a minor role. This may be due to low functional P-gp activity in suspension HHEP under the assay conditions. Low passive permeability may limit metabolism and enzyme inhibition in HHEP, leading to lower CLint,u and higher IC50 in HHEP compared to HLM. When liver microsomes give higher CLint,u than hepatocytes, microsomes are more predictive of in vivo clearance than hepatocytes.

A Physiologically Based in Silico Tool to Assess the Risk of Drug-Related Crystalluria.

Drug precipitation in the nephrons of the kidney can cause drug-induced crystal nephropathy (DICN). To aid mitigation of this risk in early drug discovery, we developed a physiologically based in silico model to predict DICN in rats, dogs, and humans. At a minimum, the likelihood of DICN is determined by the level of systemic exposure to the molecule, the molecule's physicochemical properties and the unique physiology of the kidney. Accordingly, the proposed model accounts for these properties in order to predict drug exposure relative to solubility along the nephron. Key physiological parameters of the kidney were codified in a manner consistent with previous reports. Quantitative structure-activity relationship models and in vitro assays were used to estimate drug-specific physicochemical inputs to the model. The proposed model was calibrated against urinary excretion data for 42 drugs, and the utility for DICN prediction is demonstrated through application to 20 additional drugs.

In Vitro-In Vivo Extrapolation of Key Transporter Activity at the Blood-Brain Barrier.

Understanding the quantitative implications of P-glycoprotein and breast cancer resistance protein efflux is a key hurdle in the design of effective, centrally acting or centrally restricted therapeutics. Previously, a comprehensive physiologically based pharmacokinetic model was developed to describe the in vivo unbound brain-to-plasma concentration ratio as a function of efflux activity measured in vitro. In the present work, the predictive utility of this framework was examined through application to in vitro and in vivo data generated on 133 unique compounds across three preclinical species. Two approaches were examined for the scaling of efflux activity to in vivo, namely relative expression as determined by independent proteomics measurements and relative activity as determined via fitting the in vivo neuropharmacokinetic data. The results with both approaches indicate that in vitro efflux data can be used to accurately predict the degree of brain penetration across species within the context of the proposed physiologically based pharmacokinetic framework.

Organic Anion Transporter 2-Mediated Hepatic Uptake Contributes to the Clearance of High-Permeability-Low-Molecular-Weight Acid and Zwitterion Drugs: Evaluation Using 25 Drugs.

High-permeability-low-molecular-weight acids/zwitterions [i.e., extended clearance classification system class 1A (ECCS 1A) drugs] are considered to be cleared by metabolism with a minimal role of membrane transporters in their hepatic clearance. However, a marked disconnect in the in vitro-in vivo (IVIV) translation of hepatic clearance is often noted for these drugs. Metabolic rates measured using human liver microsomes and primary hepatocytes tend to underpredict. Here, we evaluated the role of organic anion transporter 2 (OAT2)-mediated hepatic uptake in the clearance of ECCS 1A drugs. For a set of 25 ECCS 1A drugs, in vitro transport activity was assessed using transporter-transfected cells and primary human hepatocytes. All but two drugs showed substrate affinity to OAT2, whereas four (bromfenac, entacapone, fluorescein, and nateglinide) also showed OATP1B1 activity in transfected cells. Most of these drugs (21 of 25) showed active uptake by plated human hepatocytes, with rifamycin SV (pan-transporter inhibitor) reducing the uptake by about 25%-95%. Metabolic turnover was estimated for 19 drugs after a few showed no measurable substrate depletion in liver microsomal incubations. IVIV extrapolation using in vitro data was evaluated to project human hepatic clearance of OAT2-alone substrates considering 1) uptake transport only, 2) metabolism only, and 3) transporter-enzyme interplay (extended clearance model). The transporter-enzyme interplay approach achieved improved prediction accuracy (average fold error = 1.9 and bias = 0.93) compared with the other two approaches. In conclusion, this study provides functional evidence for the role of OAT2-mediated hepatic uptake in determining the pharmacokinetics of several clinically important ECCS 1A drugs.

Discovery and in Vitro Optimization of 3-Sulfamoylbenzamides as ROMK Inhibitors.

Inhibitors of the renal outer medullary potassium channel (ROMK) show promise as novel mechanism diuretics, with potentially lower risk of diuretic-induced hypokalemia relative to current thiazide and loop diuretics. Here, we report the identification of a novel series of 3-sulfamoylbenzamide ROMK inhibitors. Starting from HTS hit 4, this series was optimized to provide ROMK inhibitors with good in vitro potencies and well-balanced ADME profiles. In contrast to previously reported small-molecule ROMK inhibitors, members of this series were demonstrated to be highly selective for inhibition of human over rat ROMK and to be insensitive to the N171D pore mutation that abolishes inhibitory activity of previously reported ROMK inhibitors.

Discovery of an in Vivo Tool to Establish Proof-of-Concept for MAP4K4-Based Antidiabetic Treatment.

Recent studies in adipose tissue, pancreas, muscle, and macrophages suggest that MAP4K4, a serine/threonine protein kinase may be a viable target for antidiabetic drugs. As part of the evaluation of MAP4K4 as a novel antidiabetic target, a tool compound, 16 (PF-6260933) and a lead 17 possessing excellent kinome selectivity and suitable properties were delivered to establish proof of concept in vivo. The medicinal chemistry effort that led to the discovery of these lead compounds is described herein together with in vivo pharmacokinetic properties and activity in a model of insulin resistance.

Discovery of an intravenous hepatoselective glucokinase activator for the treatment of inpatient hyperglycemia.

Glucokinase (hexokinase IV) continues to be a compelling target for the treatment of type 2 diabetes given the wealth of supporting human genetics data and numerous reports of robust clinical glucose lowering in patients treated with small molecule allosteric activators. Recent work has demonstrated the ability of hepatoselective activators to deliver glucose lowering efficacy with minimal risk of hypoglycemia. While orally administered agents require a considerable degree of passive permeability to promote suitable exposures, there is no such restriction on intravenously delivered drugs. Therefore, minimization of membrane diffusion in the context of an intravenously agent should ensure optimal hepatic targeting and therapeutic index. This work details the identification a hepatoselective GKA exhibiting the aforementioned properties.