Structural basis of the acyl-transfer mechanism of human GPAT1.

Glycerol-3-phosphate acyltransferase (GPAT)1 is a mitochondrial outer membrane protein that catalyzes the first step of de novo glycerolipid biosynthesis. Hepatic expression of GPAT1 is linked to liver fat accumulation and the severity of nonalcoholic fatty liver diseases. Here we present the cryo-EM structures of human GPAT1 in substrate analog-bound and product-bound states. The structures reveal an N-terminal acyltransferase domain that harbors important catalytic motifs and a tightly associated C-terminal domain that is critical for proper protein folding. Unexpectedly, GPAT1 has no transmembrane regions as previously proposed but instead associates with the membrane via an amphipathic surface patch and an N-terminal loop-helix region that contains a mitochondrial-targeting signal. Combined structural, computational and functional studies uncover a hydrophobic pathway within GPAT1 for lipid trafficking. The results presented herein lay a framework for rational inhibitor development for GPAT1.

Generation of a VeroE6 Pgp gene knock out cell line and its use in SARS-CoV-2 antiviral study.

Vero cells are widely used for antiviral tests and virology research for SARS-CoV-2 as well as viruses from various other families. However, Vero cells generally express high levels of multi-drug resistance 1 (MDR1) or Pgp protein, the efflux transporter of foreign substances including many antiviral compounds, affecting the antiviral activity as well as interpretation of data. To address this, a Pgp gene knockout VeroE6 cell line (VeroE6-Pgp-KO) was generated using CRISPR-CAS9 technology. These cells no longer expressed the Pgp protein as indicated by flow cytometry analysis following staining with a Pgp-specific monoclonal antibody. They also showed significantly reduced efflux transporter activity in the calcein acetoxymethyl ester (calcein AM) assay. The VeroE6-Pgp-KO cells and the parental VeroE6 cells were each infected with SARS-CoV-2 to test antiviral activities of remdesivir and nirmatrelvir, two known Pgp substrates, in the presence or absence of a Pgp inhibitor. The compounds showed antiviral activities in VeroE6-Pgp-KO cells similar to that observed in the presence of the Pgp inhibitor. Thus, the newly established VeroE6-Pgp-KO cell line adds a new in vitro virus infection system for SARS-CoV-2 and possibly other viruses to test antiviral therapies without a need to control the Pgp activity. Removal of the Pgp inhibitor for antiviral assays will lead to less data variation and prevent failed assays.

Structural basis for the in vitro efficacy of nirmatrelvir against SARS-CoV-2 variants.

The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (Mpro) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID (nirmatrelvir tablets and ritonavir tablets). However, the efficacy of nirmatrelvir is underdetermined against evolving SARS-CoV-2 variants. Here, we evaluated the in vitro catalytic activity and potency of nirmatrelvir against the Mpro of prevalent variants of concern (VOCs) or variants of interest (VOIs): Alpha (α, B.1.1.7), Beta (ß, B.1.351), Delta (δ, B1.617.2), Gamma (γ, P.1), Lambda (λ, B.1.1.1.37/C37), Omicron (ο, B.1.1.529), as well as the original Washington or wildtype strain. These VOCs/VOIs carry prevalent mutations at varying frequencies in the Mpro specifically for α, ß, γ (K90R), λ (G15S), and ο (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant Mpros demonstrates that they are catalytically comparable to wildtype. We found that nirmatrelvir has similar potency against each mutant Mpro including P132H that is observed in the Omicron variant with a Ki of 0.635 nM as compared to a Ki of 0.933 nM for wildtype. The molecular basis for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the ο, λ, and ß Mpro at 1.63 to 2.09 Å resolution. These in vitro data suggest that PAXLOVID has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 VOC/VOI, including Omicron, from replicating in cells.

Discovery of Small-Molecule CD33 Pre-mRNA Splicing Modulators.

CD33/Siglec 3 is a myeloid lineage cell surface receptor that is known to regulate microglia activity. Multiple genome-wide association studies (GWAS) have identified genetic variants in the CD33 gene that convey protection from late-onset Alzheimer's disease. Furthermore, mechanistic studies into GWAS-linked variants suggest that disease protection is attributed to the alternative splicing of exon 2 of the CD33 pre-mRNA. Using a phenomimetic screen, a series of compounds were found to enhance the exclusion of CD33 exon 2, acting as a chemomimetic of the GWAS-linked gene variants. Additional studies confirmed that meyloid lineage cells treated with several of these compounds have a reduced full-length V-domain containing CD33 protein, while targeted RNA-seq concordantly demonstrated that compound 1 increases exon 2 skipping in cellular mRNA pools. These studies demonstrate how pharmacological interventions can be used to manipulate disease-relevant pre-mRNA splicing and provide a starting point for future efforts to identify small molecules that alter neuroimmune function that is rooted in the human biology of neurodegenerative disease.

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.

An Overview of Cell-Based Assay Platforms for the Solute Carrier Family of Transporters.

The solute carrier (SLC) superfamily represents the biggest family of transporters with important roles in health and disease. Despite being attractive and druggable targets, the majority of SLCs remains understudied. One major hurdle in research on SLCs is the lack of tools, such as cell-based assays to investigate their biological role and for drug discovery. Another challenge is the disperse and anecdotal information on assay strategies that are suitable for SLCs. This review provides a comprehensive overview of state-of-the-art cellular assay technologies for SLC research and discusses relevant SLC characteristics enabling the choice of an optimal assay technology. The Innovative Medicines Initiative consortium RESOLUTE intends to accelerate research on SLCs by providing the scientific community with high-quality reagents, assay technologies and data sets, and to ultimately unlock SLCs for drug discovery.

Acoustic Ejection Mass Spectrometry for High-Throughput Analysis.

We describe a mass spectrometry (MS) analytical platform resulting from the novel integration of acoustic droplet ejection (ADE) technology, an open-port interface (OPI), and electrospray ionization (ESI)-MS that creates a transformative system enabling high-speed sampling and label-free analysis. The ADE technology delivers nanoliter droplets in a touchless manner with high speed, precision, and accuracy. Subsequent sample dilution within the OPI, in concert with the capabilities of modern ESI-MS, eliminates the laborious sample preparation and method development required in current approaches. This platform is applied to a variety of experiments, including high-throughput (HT) pharmacology screening, label-free in situ enzyme kinetics, in vitro absorption, distribution, metabolism, elimination, pharmacokinetic and biomarker analysis, and HT parallel medicinal chemistry.

Photoaffinity Labeling and Quantitative Chemical Proteomics Identify LXRß as the Functional Target of Enhancers of Astrocytic apoE.

Utilizing a phenotypic screen, we identified chemical matter that increased astrocytic apoE secretion in vitro. We designed a clickable photoaffinity probe based on a pyrrolidine lead compound and carried out probe-based quantitative chemical proteomics in human astrocytoma CCF-STTG1 cells to identify liver x receptor ß (LXRß) as the target. Binding of the small molecule ligand stabilized LXRß, as shown by cellular thermal shift assay (CETSA). In addition, we identified a probe-modified peptide by mass spectrometry and proposed a model where the photoaffinity probe is bound in the ligand-binding pocket of LXRß. Taken together, our findings demonstrated that the lead chemical matter bound directly to LXRß, and our results highlight the power of chemical proteomic approaches to identify the target of a phenotypic screening hit. Additionally, the LXR photoaffinity probe and lead compound described herein may serve as valuable tools to further evaluate the LXR pathway.

Discovery of Ketone-Based Covalent Inhibitors of Coronavirus 3CL Proteases for the Potential Therapeutic Treatment of COVID-19.

The novel coronavirus disease COVID-19 that emerged in 2019 is caused by the virus SARS CoV-2 and named for its close genetic similarity to SARS CoV-1 that caused severe acute respiratory syndrome (SARS) in 2002. Both SARS coronavirus genomes encode two overlapping large polyproteins, which are cleaved at specific sites by a 3C-like cysteine protease (3CLpro) in a post-translational processing step that is critical for coronavirus replication. The 3CLpro sequences for CoV-1 and CoV-2 viruses are 100% identical in the catalytic domain that carries out protein cleavage. A research effort that focused on the discovery of reversible and irreversible ketone-based inhibitors of SARS CoV-1 3CLpro employing ligand-protease structures solved by X-ray crystallography led to the identification of 3 and 4. Preclinical experiments reveal 4 (PF-00835231) as a potent inhibitor of CoV-2 3CLpro with suitable pharmaceutical properties to warrant further development as an intravenous treatment for COVID-19.

Hit Triage and Validation in Phenotypic Screening: Considerations and Strategies.

The promise of phenotypic screening resides in its track record of novel biology and first-in-class therapies. However, challenges stemming from major differences between target-based and phenotypic screening do exist. These challenges prompted us to rethink the critical stage of hit triage and validation on the road to clinical candidates and novel drug targets. Whereas this process is usually straightforward for target screening hits, phenotypic screening hits act through a variety of mostly unknown mechanisms within a large and poorly understood biological space. Our analysis suggests successful hit triage and validation is enabled by three types of biological knowledge-known mechanisms, disease biology, and safety-while structure-based hit triage may be counterproductive.

Human Pluripotent Stem Cell-Derived Kidney Model for Nephrotoxicity Studies.

Current in vitro models for identifying nephrotoxins are poorly predictive. We differentiated human pluripotent stem cells (hPSCs) into three-dimensional, multicellular structures containing proximal tubule cells (PTCs) and podocytes and evaluated them as a platform for predicting nephrotoxicity. The PTCs showed megalin-dependent, cubilin-mediated endocytosis of fluorescently labeled dextran and active gamma-glutamyl transpeptidase enzymes. Transporters from both the ATP-binding cassette (ABC) and the solute carrier (SLC) families were present at physiological levels in the differentiated cells, but important renal transporters such as organic anion transporter 1 (OAT1), OAT3, and organic cation transporter 2 (OCT2) were present only at lower levels. Radioactive uptake studies confirmed the functional activity of organic cation transporter, novel, type 2 (OCTN2), organic anion transporter polypeptide 4C1 (OATP4C1), and OCTs/multidrug and toxin extrusion proteins (MATEs). When treated with 10 pharmacologic agents as a test of the platform, the known nephrotoxic compounds were distinguished from the more benign compounds by an increase in tubular (PTC, kidney injury molecule 1 (KIM-1), and heme oxygenase 1 (HO-1)) and glomerular (nephrin [NPHS1]/Wilms tumor protein [WT1]) markers associated with nephrotoxicity, and we were able to distinguish the type of nephrotoxin by examining the relative levels of these markers. Given the functions demonstrated and with improved expression of key renal transporters, this hPSC-derived in vitro kidney model shows promise as a platform for detection of mechanistically different nephrotoxins.

Design and Synthesis of Clinical Candidate PF-06751979: A Potent, Brain Penetrant, ß-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) Inhibitor Lacking Hypopigmentation.

A major challenge in the development of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors for the treatment of Alzheimer's disease is the alignment of potency, drug-like properties, and selectivity over related aspartyl proteases such as Cathepsin D (CatD) and BACE2. The potential liabilities of inhibiting BACE2 chronically have only recently begun to emerge as BACE2 impacts the processing of the premelanosome protein (PMEL17) and disrupts melanosome morphology resulting in a depigmentation phenotype. Herein, we describe the identification of clinical candidate PF-06751979 (64), which displays excellent brain penetration, potent in vivo efficacy, and broad selectivity over related aspartyl proteases including BACE2. Chronic dosing of 64 for up to 9 months in dog did not reveal any observation of hair coat color (pigmentation) changes and suggests a key differentiator over current BACE1 inhibitors that are nonselective against BACE2 in later stage clinical development.

Pharmacological inhibition of diacylglycerol acyltransferase-1 and insights into postprandial gut peptide secretion.

AIM: To examine the role that enzyme Acyl-CoA:diacylglycerol acyltransferase-1 (DGAT1) plays in postprandial gut peptide secretion and signaling. METHODS: The standard experimental paradigm utilized to evaluate the incretin response was a lipid challenge. Following a lipid challenge, plasma was collected via cardiac puncture at each time point from a cohort of 5-8 mice per group from baseline at time zero to 10 h. Incretin hormones [glucagon like peptide-1 (GLP-1), peptide tyrosine-tyrosine (PYY) and glucose dependent insulinotropic polypeptide (GIP)] were then quantitated. The impact of pharmacological inhibition of DGAT1 on the incretin effect was evaluated in WT mice. Additionally, a comparison of loss of DGAT1 function either by genetic ablation or pharmacological inhibition. To further elucidate the pathways and mechanisms involved in the incretin response to DGAT1 inhibition, other interventions [inhibitors of dipeptidyl peptidase-IV (sitagliptin), pancreatic lipase (Orlistat), GPR119 knockout mice] were evaluated. RESULTS: DGAT1 deficient mice and wildtype C57/BL6J mice were lipid challenged and levels of both active and total GLP-1 in the plasma were increased. This response was further augmented with DGAT1 inhibitor PF-04620110 treated wildtype mice. Furthermore, PF-04620110 was able to dose responsively increase GLP-1 and PYY, but blunt GIP at all doses of PF-04620110 during lipid challenge. Combination treatment of PF-04620110 and Sitagliptin in wildtype mice during a lipid challenge synergistically enhanced postprandial levels of active GLP-1. In contrast, in a combination study with Orlistat, the ability of PF-04620110 to elicit an enhanced incretin response was abrogated. To further explore this observation, GPR119 knockout mice were evaluated. In response to a lipid challenge, GPR119 knockout mice exhibited no increase in active or total GLP-1 and PYY. However, PF-04620110 was able to increase total GLP-1 and PYY in GPR119 knockout mice as compared to vehicle treated wildtype mice. CONCLUSION: Collectively, these data provide some insight into the mechanism by which inhibition of DGAT1 enhances intestinal hormone release.

Pharmacological inhibition to examine the role of DGAT1 in dietary lipid absorption in rodents and humans.

Alterations in fat metabolism, in particular elevated plasma concentrations of free fatty acids and triglycerides (TG), have been implicated in the pathogenesis of Type 2 diabetes, obesity, and cardiovascular disease. Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), a member of the large family of membrane-bound O-acyltransferases, catalyzes the final step in triacylglycerol formation. In the intestine, DGAT1 is one of the acyltransferases responsible for the reesterficiation of dietary TG. Following a single dose of a selective pharmacological inhibitor of DGAT1, PF-04620110, a dose-dependent inhibition of TG and vitamin A absorption postprandially was demonstrated in rodents and human subjects. In C57/BL6J mice, acute DGAT1 inhibition alters the temporal and spatial pattern of dietary lipid absorption. To understand the impact of DGAT1 inhibition on enterocyte lipid metabolism, lipomic profiling was performed in rat intestine and plasma as well as human plasma. DGAT1 inhibition causes an enrichment of polyunsaturated fatty acids within the TG class of lipids. This pharmacological intervention gives us insight as to the role of DGAT1 in human dietary lipid absorption.

Insights into the novel hydrolytic mechanism of a diethyl 2-phenyl-2-(2-arylacetoxy)methyl malonate ester-based microsomal triglyceride transfer protein (MTP) inhibitor.

Inhibition of intestinal and hepatic microsomal triglyceride transfer protein (MTP) is a potential strategy for the treatment of dyslipidemia and related metabolic disorders. Inhibition of hepatic MTP, however, results in elevated liver transaminases and increased hepatic fat deposition consistent with hepatic steatosis. Diethyl 2-((2-(3-(dimethylcarbamoyl)-4-(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-ylcarboxamido)phenyl)acetoxy)methyl)-2-phenylmalonate (JTT-130) is an intestine-specific inhibitor of MTP and does not cause increases in transaminases in short-term clinical trials in patients with dyslipidemia. Selective inhibition of intestinal MTP is achieved via rapid hydrolysis of its ester linkage by liver-specific carboxylesterase(s), resulting in the formation of an inactive carboxylic acid metabolite 1. In the course of discovery efforts around tissue-specific inhibitors of MTP, the mechanism of JTT-130 hydrolysis was examined in detail. Lack of ¹8O incorporation in 1 following the incubation of JTT-130 in human liver microsomes in the presence of H2¹8O suggested that hydrolysis did not occur via a simple cleavage of the ester linkage. The characterization of atropic acid (2-phenylacrylic acid) as a metabolite was consistent with a hydrolytic pathway involving initial hydrolysis of one of the pendant malonate ethyl ester groups followed by decarboxylative fragmentation to 1 and the concomitant liberation of the potentially electrophilic acrylate species. Glutathione conjugates of atropic acid and its ethyl ester were also observed in microsomal incubations of JTT-130 that were supplemented with the thiol nucleophile. Additional support for the hydrolysis mechanism was obtained from analogous studies on diethyl 2-(2-(2-(3-(dimethylcarbamoyl)-4-(4'-trifluoromethyl)-[1,1'-biphenyl]-2-ylcarboxamido)phenyl)acetoxy)ethyl)-2-phenylmalonate (3), which cannot participate in hydrolysis via the fragmentation pathway because of the additional methylene group. Unlike the case with JTT-130, ¹8O was readily incorporated into 1 during the enzymatic hydrolysis of 3, suggestive of a mechanism involving direct hydrolytic cleavage of the ester group in 3. Finally, 3-(ethylamino)-2-(ethylcarbamoyl)-3-oxo-2-phenylpropyl 2-(3-(dimethylcarbamoyl)-4-(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-ylcarboxamido)phenyl)acetate (4), which possessed an N,N-diethyl-2-phenylmalonamide substituent (in lieu of the diethyl-2-phenylmalonate motif in JTT-130) proved to be resistant to the hydrolytic cleavage/decarboxylative fragmentation pathway that yielded 1, a phenomenon that further confirmed our hypothesis. From a toxicological standpoint, it is noteworthy to point out that the liberation of the electrophilic acrylic acid species as a byproduct of JTT-130 hydrolysis is similar to the bioactivation mechanism established for felbamate, an anticonvulsant agent associated with idiosyncratic aplastic anemia and hepatotoxicity.

Modeling the mechanism of action of a DGAT1 inhibitor using a causal reasoning platform.

Triglyceride accumulation is associated with obesity and type 2 diabetes. Genetic disruption of diacylglycerol acyltransferase 1 (DGAT1), which catalyzes the final reaction of triglyceride synthesis, confers dramatic resistance to high-fat diet induced obesity. Hence, DGAT1 is considered a potential therapeutic target for treating obesity and related metabolic disorders. However, the molecular events shaping the mechanism of action of DGAT1 pharmacological inhibition have not been fully explored yet. Here, we investigate the metabolic molecular mechanisms induced in response to pharmacological inhibition of DGAT1 using a recently developed computational systems biology approach, the Causal Reasoning Engine (CRE). The CRE algorithm utilizes microarray transcriptomic data and causal statements derived from the biomedical literature to infer upstream molecular events driving these transcriptional changes. The inferred upstream events (also called hypotheses) are aggregated into biological models using a set of analytical tools that allow for evaluation and integration of the hypotheses in context of their supporting evidence. In comparison to gene ontology enrichment analysis which pointed to high-level changes in metabolic processes, the CRE results provide detailed molecular hypotheses to explain the measured transcriptional changes. CRE analysis of gene expression changes in high fat habituated rats treated with a potent and selective DGAT1 inhibitor demonstrate that the majority of transcriptomic changes support a metabolic network indicative of reversal of high fat diet effects that includes a number of molecular hypotheses such as PPARG, HNF4A and SREBPs. Finally, the CRE-generated molecular hypotheses from DGAT1 inhibitor treated rats were found to capture the major molecular characteristics of DGAT1 deficient mice, supporting a phenotype of decreased lipid and increased insulin sensitivity.

Discovery of a clinical candidate from the structurally unique dioxa-bicyclo[3.2.1]octane class of sodium-dependent glucose cotransporter 2 inhibitors.

Compound 4 (PF-04971729) belongs to a new class of potent and selective sodium-dependent glucose cotransporter 2 inhibitors incorporating a unique dioxa-bicyclo[3.2.1]octane (bridged ketal) ring system. In this paper we present the design, synthesis, preclinical evaluation, and human dose predictions related to 4. This compound demonstrated robust urinary glucose excretion in rats and an excellent preclinical safety profile. It is currently in phase 2 clinical trials and is being evaluated for the treatment of type 2 diabetes.

N-benzylimidazole carboxamides as potent, orally active stearoylCoA desaturase-1 inhibitors.

A potent, small molecule inhibitor with a favorable pharmacokinetic profile to allow for sustained SCD inhibition in vivo was identified. Starting from a low MW acyl guanidine (5a), identified with a RapidFire High-Throughput Mass Spectrometry (RF-MS) assay, iterative library design was used to rapidly probe the amide and tail regions of the molecule. Singleton synthesis was used to probe core changes. Biological evaluation of a SCD inhibitor (5b) included in vitro potency at SCD-1 and in vivo modulation of the plasma desaturation index (DI) in rats on a low essential fatty acid (LEFA) diet. In addition to dose-dependent decrease in DI, effects on rodent ocular tissue were noted. Therefore, in rat, these SCD inhibitors only recapitulate a portion of phenotype exhibited by the SCD-1 knockout mouse.

C-Aryl glycoside inhibitors of SGLT2: Exploration of sugar modifications including C-5 spirocyclization.

Modifications to the sugar portion of C-aryl glycoside sodium glucose transporter 2 (SGLT2) inhibitors were explored, including systematic deletion and modification of each of the glycoside hydroxyl groups. Based on results showing activity to be quite tolerant of structural change at the C-5 position, a series of novel C-5 spiro analogues was prepared. Some of these analogues exhibit low nanomolar potency versus SGLT2 and promote urinary glucose excretion (UGE) in rats. However, due to sub-optimal pharmacokinetic parameters (in particular half-life), predicted human doses did not meet criteria for further advancement.