Several hydroxysteroid dehydrogenase 17-beta 13 variants have previously been identified as protective against metabolic dysfunction-associated steatohepatitis (MASH) fibrosis, ballooning and inflammation, and as such this target holds significant therapeutic potential. However, over 5 years later, the function of 17B-HSD13 remains unknown. Structure-aided design enables the development of potent and selective sulfonamide-based 17B-HSD13 inhibitors. In order to probe their inhibitory potency in endogenous expression systems like primary human hepatocytes, inhibitors are transformed into synthetic surrogate substrates with distinct selectivity advantages over substrates previously published. Their application to cells endogenously expressing 17B-HSD13 enables quantitative measures of enzymatic inhibition in primary human hepatocytes which has never been reported to date. Application to multiple cellular systems expressing the protective human variants reveals that the most prevalent IsoD variant maintains NAD-dependent catalytic activity towards some but not all substrates, contradicting reports that the truncation results in loss-of-function.

Activation of the glucagon-like peptide-1 (GLP-1) receptor stimulates insulin release, lowers plasma glucose levels, delays gastric emptying, increases satiety, suppresses food intake, and affords weight loss in humans. These beneficial attributes have made peptide-based agonists valuable tools for the treatment of type 2 diabetes mellitus and obesity. However, efficient, and consistent delivery of peptide agents generally requires subcutaneous injection, which can reduce patient utilization. Traditional orally absorbed small molecules for this target may offer improved patient compliance as well as the opportunity for co-formulation with other oral therapeutics. Herein, we describe an SAR investigation leading to small-molecule GLP-1 receptor agonists that represent a series that parallels the recently reported clinical candidate danuglipron. In the event, identification of a benzyloxypyrimidine lead, using a sensitized high-throughput GLP-1 agonist assay, was followed by optimization of the SAR using substituent modifications analogous to those discovered in the danuglipron series. A new series of 6-azaspiro[2.5]octane molecules was optimized into potent GLP-1 agonists. Information gleaned from cryogenic electron microscope structures was used to rationalize the SAR of the optimized compounds.

Diacylglycerol O-acyltransferase 2 (DGAT2) inhibitors have been shown to lower liver triglyceride content and are being explored clinically as a treatment for non-alcoholic steatohepatitis (NASH). This work details efforts to find an extended-half-life DGAT2 inhibitor. A basic moiety was added to a known inhibitor template, and the basicity and lipophilicity were fine-tuned by the addition of electrophilic fluorines. A weakly basic profile was required to find an appropriate balance of potency, clearance, and permeability. This work culminated in the discovery of PF-07202954 (12), a weakly basic DGAT2 inhibitor that has advanced to clinical studies. This molecule displays a higher volume of distribution and longer half-life in preclinical species, in keeping with its physicochemical profile, and lowers liver triglyceride content in a Western-diet-fed rat model.

Hydroxysteroid 17-beta-dehydrogenase 13 (HSD17B13) is a hepatic lipid droplet-associated enzyme that is upregulated in patients with non-alcoholic fatty liver disease. Recently, there have been several reports that predicted loss of function variants in HSD17B13 protect against the progression of steatosis to non-alcoholic steatohepatitis with fibrosis and hepatocellular carcinoma. Here we report crystal structures of full length HSD17B13 in complex with its NAD+ cofactor, and with lipid/detergent molecules and small molecule inhibitors from two distinct series in the ligand binding pocket. These structures provide insights into a mechanism for lipid droplet-associated proteins anchoring to membranes as well as a basis for HSD17B13 variants disrupting function. Two series of inhibitors interact with the active site residues and the bound cofactor similarly, yet they occupy different paths leading to the active site. These structures provide ideas for structure-based design of inhibitors that may be used in the treatment of liver disease.

Peptide agonists of the glucagon-like peptide-1 receptor (GLP-1R) have revolutionized diabetes therapy, but their use has been limited because they require injection. Herein, we describe the discovery of the orally bioavailable, small-molecule, GLP-1R agonist PF-06882961 (danuglipron). A sensitized high-throughput screen was used to identify 5-fluoropyrimidine-based GLP-1R agonists that were optimized to promote endogenous GLP-1R signaling with nanomolar potency. Incorporation of a carboxylic acid moiety provided considerable GLP-1R potency gains with improved off-target pharmacology and reduced metabolic clearance, ultimately resulting in the identification of danuglipron. Danuglipron increased insulin levels in primates but not rodents, which was explained by receptor mutagensis studies and a cryogenic electron microscope structure that revealed a binding pocket requiring a primate-specific tryptophan 33 residue. Oral administration of danuglipron to healthy humans produced dose-proportional increases in systemic exposure (NCT03309241). This opens an opportunity for oral small-molecule therapies that target the well-validated GLP-1R for metabolic health.

Drug Metabolism Reviews has an impressive track record of providing scientific reviews in the area of xenobiotic biotransformation over 47 years. It has consistently proved to be resourceful to many scientists from pharmaceutical industry, academia, regulatory agencies working in diverse areas including enzymology, pharmacology, pharmacokinetics, and toxicology. Over the last 5 years Drug metabolism Reviews has annually published an industry commentary aimed to highlight novel insights and approaches that have made significant impacts on the field of biotransformation (led by Cyrus Khojasteh). We hope to continue this tradition by providing an overview of advances made in the field of drug transporters during 2020. The field of drug transporters is rapidly evolving as they play an essential role in drug absorption, distribution, clearance, and elimination. In this review, we have selected outstanding drug transporter articles that have significantly contributed to moving forward the field of transporter science with respect to translation and improved understanding of diverse aspects including uptake clearance, clinical biomarkers, induction, proteomics, emerging transporters, and tissue targeting. The theme of this review consists of a synopsis that summarizes each article followed by our commentary. The objective of this work is not to provide a comprehensive review but rather to exemplify novel insights and state-of-the-art highlights of recent research that have advanced our understanding of drug transporters in drug disposition. We are hopeful that this effort will prove useful to the scientific community and as such request feedback, and further extend an invitation to anyone interested in contributing to future reviews.

: Peptide agonists of the glucagon-like peptide-1 receptor (GLP-1R) have revolutionized diabetes therapy, but their use has been limited by the requirement for injection. Here we describe the first effective, orally bioavailable small molecule GLP-1R agonists. A 27 sensitized high-throughput screen identified a series of small molecule GLP-1R agonists that 28 were optimized to promote endogenous GLP-1R signaling with nM potency. These small 29 molecule agonists increased insulin levels in primates but not rodents, which is explained by a 30 cryo-EM structure that revealed a binding pocket requiring primate-specific tryptophan 33. Importantly, oral administration of agonist PF-06882961 to healthy humans produced dose- 32 dependent declines in serum glucose (NCT03309241). This opens the door to a new era of oral 33 small molecule therapies that target the well-validated GLP-1R pathway for metabolic health. standard error of the area CHO,

Preclinical and clinical data suggest that acetyl-CoA carboxylase (ACC) inhibitors have the potential to rebalance disordered lipid metabolism, leading to improvements in nonalcoholic steatohepatitis (NASH). Consistent with these observations, first-in-human clinical trials with our ACC inhibitor PF-05175157 led to robust reduction of de novo lipogenesis (DNL), albeit with concomitant reductions in platelet count, which were attributed to the inhibition of fatty acid synthesis within bone marrow. Herein, we describe the design, synthesis, and evaluation of carboxylic acid-based ACC inhibitors with organic anion transporting polypeptide (OATP) substrate properties, which facilitated selective distribution of the compounds at the therapeutic site of action (liver) relative to the periphery. These efforts led to the discovery of clinical candidate PF-05221304 (12), which selectively inhibits liver DNL in animals, while demonstrating considerable safety margins against platelet reduction in a nonhuman primate model.

Prospective predictions of human hepatic clearance for anionic/zwitterionic compounds, which are oftentimes subjected to transporter-mediated uptake, are challenging in drug discovery. We evaluated the utility of preclinical species, rats and cynomolgus monkeys [nonhuman primates (NHPs)], to predict the human hepatic clearance using a diverse set of acidic/zwitterionic drugs. Preclinical clearance data were generated following intravenous dosing in rats/NHPs and compared to the human clearance data (n = 18/27). Single-species scaling of NHP clearance with an allometric exponent of 0.50 allowed for good prediction of human clearance (fold error ∼2.1, bias ∼1.0), with ∼86% predictions within 3-fold. In comparison, rats underpredicted the clearance of lipophilic acids, while overprediction was noted for hydrophilic acids. Finally, an in vitro clearance assay based on human hepatocytes, which is routinely used in discovery setting, markedly underpredicted human clearance (bias ∼0.12). Collectively, this study provides insights into the usefulness of the preclinical models in enabling pharmacokinetic optimization for acid/zwitterionic drug candidates.

Optimization of the pharmacokinetic (PK) properties of a series of activators of adenosine monophosphate-activated protein kinase (AMPK) is described. Derivatives of the previously described 5-aryl-indole-3-carboxylic acid clinical candidate (1) were examined with the goal of reducing glucuronidation rate and minimizing renal excretion. Compounds 10 (PF-06679142) and 14 (PF-06685249) exhibited robust activation of AMPK in rat kidneys as well as desirable oral absorption, low plasma clearance, and negligible renal clearance in preclinical species. A correlation of in vivo renal clearance in rats with in vitro uptake by human and rat renal organic anion transporters (human OAT/rat Oat) was identified. Variation of polar functional groups was critical to mitigate active renal clearance mediated by the Oat3 transporter. Modification of either the 6-chloroindole core to a 4,6-difluoroindole or the 5-phenyl substituent to a substituted 5-(3-pyridyl) group provided improved metabolic stability while minimizing propensity for active transport by OAT3.

Studies on indole-3-carboxylic acid derivatives as direct activators of human adenosine monophosphate-activated protein kinase (AMPK) alpha 1 beta 1 gamma 1 isoform have culminated in the identification of PF-06409577 (1), PF-06885249 (2), and PF-06679142 (3) as potential clinical candidates. Compounds 1-3 are primarily cleared in animals and humans via glucuronidation. Herein, we describe the biosynthetic preparation, purification, and structural characterization of the glucuronide conjugates of 1-3. Spectral characterization of the purified glucuronides M1, M2, and M3 indicated that they were acyl glucuronide derivatives. In vitro pharmacological evaluation revealed that all three acyl glucuronides retained selective activation of beta 1-containing AMPK isoforms. Inhibition of de novo lipogenesis with representative parent carboxylic acids and their respective acyl glucuronide conjugates in human hepatocytes demonstrated their propensity to activate cellular AMPK. Cocrystallization of the AMPK alpha 1 beta 1 gamma 1 isoform with 1-3 and M1-M3 provided molecular insights into the structural basis for AMPK activation by the glucuronide conjugates.

Indole acids 1, 2, and 3 are potent 5′-adenosine monophosphate-activated protein kinase (AMPK) activators for the potential treatment of diabetic nephropathy. Compounds 1–3 were scaled to supply material for preclinical studies, and indole 3 was selected for advancement to first-in-human clinical trials and scaled to kilogram quantities. The progression of the synthesis strategy for these AMPK activators is described, as routes were selected for efficient structure–activity relationship generation and then improved for larger scales. The developed sequences employed practical isolations of intermediates and APIs, reproducible cross-coupling, hydrolysis, and other transformations, and enhanced safety and purity profiles and led to the production of 40–50 g of 1 and 2 and 2.4 kg of 3. Multiple polymorphs of 3 were observed, and conditions for the reproducible formation of crystalline material suitable for clinical development were identified.

Glucagon-like peptide (GLP-1) is an endogenous hormone that induces insulin secretion from pancreatic islets and modified forms are used to treat diabetes mellitus type 2. Understanding how GLP-1 interacts with its receptor (GLP-1R) can potentially lead to more effective drugs. Modeling and NMR studies of the N-terminus of GLP-1 suggest a β-turn between residues Glu9-Phe12 and a kinked alpha helix between Val16-Gly37. N-terminal turn constraints attenuated binding affinity and activity (compounds 1–8). Lys-Asp (i, i+4) crosslinks in the middle and at the C-terminus increased alpha helicity and cAMP stimulation without much effect on binding affinity or beta-arrestin 2 recruitment (compounds 9–18). Strategic positioning of helix-inducing constraints and amino acid substitutions (Tyr16, Ala22) increased peptide helicity and produced ten-fold higher cAMP potency (compounds 19–28) over GLP-1(7–37)-NH2. The most potent cAMP activator (compound 23) was also the most potent inducer of insulin secretion.

Diabetic nephropathy remains an area of high unmet medical need, with current therapies that slow down, but do not prevent, the progression of disease. A reduced phosphorylation state of adenosine monophosphate-activated protein kinase (AMPK) has been correlated with diminished kidney function in both humans and animal models of renal disease. Here, we describe the identification of novel, potent, small molecule activators of AMPK that selectively activate AMPK heterotrimers containing the β1 subunit. After confirming that human and rodent kidney predominately express AMPK β1, we explore the effects of pharmacological activation of AMPK in the ZSF1 rat model of diabetic nephropathy. Chronic administration of these direct activators elevates the phosphorylation of AMPK in the kidney, without impacting blood glucose levels, and reduces the progression of proteinuria to a greater degree than the current standard of care, angiotensin-converting enzyme inhibitor ramipril. Further analyses of urine biomarkers and kidney tissue gene expression reveal AMPK activation leads to the modulation of multiple pathways implicated in kidney injury, including cellular hypertrophy, fibrosis, and oxidative stress. These results support the need for further investigation into the potential beneficial effects of AMPK activation in kidney disease.

Glucagon-like peptide-1 (GLP-1) signaling through the glucagon-like peptide 1 receptor (GLP-1R) is a key regulator of normal glucose metabolism, and exogenous GLP-1R agonist therapy is a promising avenue for the treatment of type 2 diabetes mellitus. To date, the development of therapeutic GLP-1R agonists has focused on producing drugs with an extended serum half-life. This has been achieved by engineering synthetic analogs of GLP-1 or the more stable exogenous GLP-1R agonist exendin-4 (Ex-4). These synthetic peptide hormones share the overall structure of GLP-1 and Ex-4, with a C-terminal helical segment and a flexible N-terminal tail. Although numerous studies have investigated the molecular determinants underpinning GLP-1 and Ex-4 binding and signaling through the GLP-1R, these have primarily focused on the length and composition of the N-terminal tail or on how to modulate the helicity of the full-length peptides. Here, we investigate the effect of C-terminal truncation in GLP-1 and Ex-4 on the cAMP pathway. To ensure helical C-terminal regions in the truncated peptides, we produced a series of chimeric peptides combining the N-terminal portion of GLP-1 or Ex-4 and the C-terminal segment of the helix-promoting peptide α-conotoxin pl14a. The helicity and structures of the chimeric peptides were confirmed using circular dichroism and NMR, respectively. We found no direct correlation between the fractional helicity and potency in signaling via the cAMP pathway. Rather, the most important feature for efficient receptor binding and signaling was the C-terminal helical segment (residues 22-27) directing the binding of Phe(22) into a hydrophobic pocket on the GLP-1R.

Adenosine monophosphate-activated protein kinase (AMPK) is a protein kinase involved in maintaining energy homeostasis within cells. On the basis of human genetic association data, AMPK activators were pursued for the treatment of diabetic nephropathy. Identification of an indazole amide high throughput screening (HTS) hit followed by truncation to its minimal pharmacophore provided an indazole acid lead compound. Optimization of the core and aryl appendage improved oral absorption and culminated in the identification of indole acid, PF-06409577 (7). Compound 7 was advanced to first-in-human trials for the treatment of diabetic nephropathy.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.