Discovery of PFI-6, a small-molecule chemical probe for the YEATS domain of MLLT1 and MLLT3.

Epigenetic proteins containing YEATS domains (YD) are an emerging target class in drug discovery. Described herein are the discovery and characterization efforts associated with PFI-6, a new chemical probe for the YD of MLLT1 (ENL/YEATS1) and MLLT3 (AF9/YEATS3). For hit identification, fragment-like mimetics of endogenous YD ligands (crotonylated histone-containing proteins), were synthesized via parallel medicinal chemistry (PMC) and screened for MLLT1 binding. Subsequent SAR studies led to iterative MLLT1/3 binding and selectivity improvements, culminating in the discovery of PFI-6. PFI-6 demonstrates good affinity and selectivity for MLLT1/3 vs. other human YD proteins (YEATS2/4) and engages MLLT3 in cells. Small-molecule X-ray co-crystal structures of two molecules, including PFI-6, bound to the YD of MLLT1/3 are also described. PFI-6 may be a useful tool molecule to better understand the biological effects associated with modulation of MLLT1/3.

Convergent Syntheses of Isomeric Imidazolospiroketones as Templates for Acetyl-CoA Carboxylase (ACC) Inhibitors.

The synthesis of imidazole fused spirocyclic ketones as templates for acetyl-CoA carboxylase (ACC) inhibitors is reported. By completing the spirocyclic ring closure via divergent pathways, the synthesis of these regioisomers from common intermediates was developed. Through an aldehyde homologation/transmetalation strategy, one isomer was formed selectively. The second desired isomer was obtained via an intramolecular aromatic homolytic substitution reaction. Preparation of these isomeric spiroketones provided templates which, upon elaboration, led to key structure-activity relationship (SAR) points for delivery of potent ACC inhibitors.

6-Azaspiro[2.5]octanes as small molecule agonists of the human glucagon-like peptide-1 receptor.

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.

Small molecule inhibitors of PCSK9. SAR investigations of head and amine groups.

Our previous work on the optimization of a new class of small molecule PCSK9 mRNA translation inhibitors focused on empirical optimization of the amide tail region of the lead PF-06446846 (1). This work resulted in compound 3 that showed an improved safety profile. We hypothesized that this improvement was related to diminished binding of 3 to non-translating ribosomes and an apparent improvement in transcript selectivity. Herein, we describe our efforts to further optimize this series of inhibitors through modulation of the heterocyclic head group and the amine fragment. Some of the effort was guided by an emerging cryo electron microscopy structure of the binding mode of 1 in the ribosome. These efforts led to the identification of 15 that was deemed suitable for evaluation in a humanized PCSK9 mouse model and a rat toxicology study. Compound 15 demonstrated a dose dependent reduction of plasma PCSK9 levels. The rat toxicological profile was not improved over that of 1, which precluded 15 from further consideration as a clinical candidate.

Bioorthogonal Tethering Enhances Drug Fragment Affinity for G Protein-Coupled Receptors in Live Cells.

G protein-coupled receptors (GPCRs) modulate diverse cellular signaling pathways and are important drug targets. Despite the availability of high-resolution structures, the discovery of allosteric modulators remains challenging due to the dynamic nature of GPCRs in native membranes. We developed a strategy to covalently tether drug fragments adjacent to allosteric sites in GPCRs to enhance their potency and enable fragment-based drug screening in cell-based systems. We employed genetic code expansion to site-specifically introduce noncanonical amino acids with reactive groups in C-C chemokine receptor 5 (CCR5) near an allosteric binding site for the drug maraviroc. We then used molecular dynamics simulations to design heterobifunctional maraviroc analogues consisting of a drug fragment connected by a flexible linker to a reactive moiety capable of undergoing a bioorthogonal coupling reaction. We synthesized a library of these analogues and employed the bioorthogonal inverse electron demand Diels-Alder reaction to couple the analogues to the engineered CCR5 in live cells, which were then assayed using cell-based signaling assays. Tetherable low-affinity maraviroc fragments displayed an increase in potency for CCR5 engineered with reactive unnatural amino acids that were adjacent to the maraviroc binding site. The strategy we describe to tether novel drug fragments to GPCRs should prove useful to probe allosteric or cryptic binding site functionality in fragment-based GPCR-targeted drug discovery.

A Small-Molecule Oral Agonist of the Human Glucagon-like Peptide-1 Receptor.

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.

Cross-Linked Poly-4-Acrylomorpholine: A Flexible and Reversibly Compressible Aligning Gel for Anisotropic NMR Analysis of Peptides and Small Molecules in Water.

Determination of the solution conformation of both small organic molecules and peptides in water remains a substantial hurdle in using NMR solution conformations to guide drug design due to the lack of easy to use alignment media. Herein we report the design of a flexible compressible chemically cross-linked poly-4-acrylomorpholine gel that can be used for the alignment of both small molecules and cyclic peptides in water. To test the new gel, residual dipolar couplings (RDCs) and J-coupling constants were used in the configurational analysis of strychnine hydrochloride, a molecule that has been studied extensively in organic solvents as well as a small cyclic peptide that is known to form an α-helix in water. The conformational ensembles for each molecule with the best fit to the data are reported. Identification of minor conformers in water that cannot easily be determined by conventional NOE measurements will facilitate the use of RDC experiments in structure-based drug design.

Discovery and evaluation of non-basic small molecule modulators of the atypical chemokine receptor CXCR7.

The atypical chemokine receptor C-X-C chemokine receptor type 7 (CXCR7) is an attractive therapeutic target for a variety of cardiac and immunological diseases. As a strategy to mitigate known risks associated with the development of higher molecular weight, basic compounds, a series of pyrrolidinyl-azolopyrazines were identified as promising small-molecule CXCR7 modulators. Using a highly enabled parallel medicinal chemistry strategy, structure-activity relationship studies geared towards a reduction in lipophilicity and incorporation of saturated heterocycles led to the identification of representative tool compound 20. Notably, compound 20 maintained good potency against CXCR7 with a suitable balance of physicochemical properties to support in vivo pharmacokinetic studies.

PF-07059013: A Noncovalent Modulator of Hemoglobin for Treatment of Sickle Cell Disease.

Sickle cell disease (SCD) is a genetic disorder caused by a single point mutation (ß6 Glu → Val) on the ß-chain of adult hemoglobin (HbA) that results in sickled hemoglobin (HbS). In the deoxygenated state, polymerization of HbS leads to sickling of red blood cells (RBC). Several downstream consequences of polymerization and RBC sickling include vaso-occlusion, hemolytic anemia, and stroke. We report the design of a noncovalent modulator of HbS, clinical candidate PF-07059013 (23). The seminal hit molecule was discovered by virtual screening and confirmed through a series of biochemical and biophysical studies. After a significant optimization effort, we arrived at 23, a compound that specifically binds to Hb with nanomolar affinity and displays strong partitioning into RBCs. In a 2-week multiple dose study using Townes SCD mice, 23 showed a 37.8% (±9.0%) reduction in sickling compared to vehicle treated mice. 23 (PF-07059013) has advanced to phase 1 clinical trials.

Cyclic Peptide Design Guided by Residual Dipolar Couplings, J-Couplings, and Intramolecular Hydrogen Bond Analysis.

Cyclic peptides have long tantalized drug designers with their potential ability to combine the best attributes of antibodies and small molecules. An ideal cyclic peptide drug candidate would be able to recognize a protein surface like an antibody while achieving the oral bioavailability of a small molecule. It has been hypothesized that such cyclic peptides balance permeability and solubility using their solvent-dependent conformational flexibility. Herein we report a conformational deconvolution NMR methodology that combines residual dipolar couplings, J-couplings, and intramolecular hydrogen bond analysis along with conformational analysis using molecular dynamics simulations and density functional theory calculations for studying cyclic peptide conformations in both low-dielectric solvent (chloroform) and high-dielectric solvent (DMSO) to experimentally study the solvent-dependent conformational change hypothesis. Taken together, the combined experimental and computational approaches can illuminate conformational ensembles of cyclic peptides in solution and help identify design opportunities for better permeability.

Discovery of N-(piperidin-3-yl)-N-(pyridin-2-yl)piperidine/piperazine-1-carboxamides as small molecule inhibitors of PCSK9.

A series of N-(piperidin-3-yl)-N-(pyridin-2-yl)piperidine/piperazine-1-carboxamides were identified as small molecule PCSK9 mRNA translation inhibitors. Analogues from this new chemical series, such as 4d and 4g, exhibited improved PCSK9 potency, ADME properties, and in vitro safety profiles when compared to earlier lead structures.

Small Molecule Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibitors: Hit to Lead Optimization of Systemic Agents.

The optimization of a new class of small molecule PCSK9 mRNA translation inhibitors is described. The potency, physicochemical properties, and off-target pharmacology associated with the hit compound (1) were improved by changes to two regions of the molecule. The last step in the synthesis of the congested amide center was enabled by three different routes. Subtle structural changes yielded significant changes in pharmacology and off-target margins. These efforts led to the identification of 7l and 7n with overall profiles suitable for in vivo evaluation. In a 14-day toxicology study, 7l demonstrated an improved safety profile vs lead 7f. We hypothesize that the improved safety profile is related to diminished binding of 7l to nontranslating ribosomes and an apparent improvement in transcript selectivity due to the lower strength of 7l stalling of off-target proteins.

Discovery of a Novel Small-Molecule Modulator of C-X-C Chemokine Receptor Type 7 as a Treatment for Cardiac Fibrosis.

C-X-C chemokine receptor type 7 (CXCR7) is involved in cardiac and immune pathophysiology. We report the discovery of a novel 1,4-diazepine CXCR7 modulator, demonstrating for the first time the role of pharmacological CXCR7 intervention in cardiac repair. Structure-activity-relationship (SAR) studies demonstrated that a net reduction in lipophilicity (log D) and an incorporation of saturated ring systems yielded compounds with good CXCR7 potencies and improvements in oxidative metabolic stability in human-liver microsomes (HLM). Tethering an ethylene amide further improved the selectivity profile (e.g., for compound 18, CXCR7 Ki = 13 nM, adrenergic α 1a Kb > 10 000 nM, and adrenergic ß 2 Kb > 10 000 nM). The subcutaneous administration of 18 in mice led to a statistically significant increase in circulating concentrations of plasma stromal-cell-derived factor 1α (SDF-1α) of approximately 2-fold. Chronic dosing of compound 18 in a mouse model of isoproterenol-induced cardiac injury further resulted in a statistically significant reduction of cardiac fibrosis.

Liver-Targeted Small-Molecule Inhibitors of Proprotein Convertase Subtilisin/Kexin Type 9 Synthesis.

Targeting of the human ribosome is an unprecedented therapeutic modality with a genome-wide selectivity challenge. A liver-targeted drug candidate is described that inhibits ribosomal synthesis of PCSK9, a lipid regulator considered undruggable by small molecules. Key to the concept was the identification of pharmacologically active zwitterions designed to be retained in the liver. Oral delivery of the poorly permeable zwitterions was achieved by prodrugs susceptible to cleavage by carboxylesterase 1. The synthesis of select tetrazole prodrugs was crucial. A cell-free in vitro translation assay containing human cell lysate and purified target mRNA fused to a reporter was used to identify active zwitterions. In vivo PCSK9 lowering by oral dosing of the candidate prodrug and quantification of the drug fraction delivered to the liver utilizing an oral positron emission tomography 18 F-isotopologue validated our liver-targeting approach.

Discovery of Potent and Orally Bioavailable Macrocyclic Peptide-Peptoid Hybrid CXCR7 Modulators.

The chemokine receptor CXCR7 is an attractive target for a variety of diseases. While several small-molecule modulators of CXCR7 have been reported, peptidic macrocycles may provide advantages in terms of potency, selectivity, and reduced off-target activity. We produced a series of peptidic macrocycles that incorporate an N-linked peptoid functionality where the peptoid group enabled us to explore side-chain diversity well beyond that of natural amino acids. At the same time, theoretical calculations and experimental assays were used to track and reduce the polarity while closely monitoring the physicochemical properties. This strategy led to the discovery of macrocyclic peptide-peptoid hybrids with high CXCR7 binding affinities (Ki < 100 nM) and measurable passive permeability (Papp > 5 × 10-6 cm/s). Moreover, bioactive peptide 25 (Ki = 9 nM) achieved oral bioavailability of 18% in rats, which was commensurate with the observed plasma clearance values upon intravenous administration.

Comparative pharmacokinetic profile of cyclosporine (CsA) with a decapeptide and a linear analogue.

The synthesis and in vivo pharmacokinetic profile of an analogue of cyclosporine is disclosed. An acyclic congener was also profiled in in vitro assays to compare cell permeability. The compounds possess similar calculated and measured molecular descriptors however have different behaviors in an RRCK assay to assess cell permeability.

Nonclassical Size Dependence of Permeation Defines Bounds for Passive Adsorption of Large Drug Molecules.

Macrocyclic peptides are considered large enough to inhibit "undruggable" targets, but the design of passively cell-permeable molecules in this space remains a challenge due to the poorly understood role of molecular size on passive membrane permeability. Using split-pool combinatorial synthesis, we constructed a library of cyclic, per-N-methlyated peptides spanning a wide range of calculated lipohilicities (0 < AlogP < 8) and molecular weights (∼800 Da < MW < ∼1200 Da). Analysis by the parallel artificial membrane permeability assay revealed a steep drop-off in apparent passive permeability with increasing size in stark disagreement with current permeation models. This observation, corroborated by a set of natural products, helps define criteria for achieving permeability in larger molecular size regimes and suggests an operational cutoff, beyond which passive permeability is constrained by a sharply increasing penalty on membrane permeation.

Discovery of a Selective Covalent Inhibitor of Lysophospholipase-like 1 (LYPLAL1) as a Tool to Evaluate the Role of this Serine Hydrolase in Metabolism.

Lysophospholipase-like 1 (LYPLAL1) is an uncharacterized metabolic serine hydrolase. Human genome-wide association studies link variants of the gene encoding this enzyme to fat distribution, waist-to-hip ratio, and nonalcoholic fatty liver disease. We describe the discovery of potent and selective covalent small-molecule inhibitors of LYPLAL1 and their use to investigate its role in hepatic metabolism. In hepatocytes, selective inhibition of LYPLAL1 increased glucose production supporting the inference that LYPLAL1 is a significant actor in hepatic metabolism. The results provide an example of how a selective chemical tool can contribute to evaluating a hypothetical target for therapeutic intervention, even in the absence of complete biochemical characterization.

Evaluation of Cu-64 and Ga-68 Radiolabeled Glucagon-Like Peptide-1 Receptor Agonists as PET Tracers for Pancreatic ß cell Imaging.

PURPOSE: Copper-64 (Cu-64) and Galium-68 (Ga-68) radiolabeled DO3A and NODA conjugates of exendin-4 were used for preclinical imaging of pancreatic ß cells via targeting of glucagon-like peptide-1 receptor (GLP-1R). PROCEDURES: DO3A-VS- and NODA-VS-tagged Cys(40)exendin-4 (DO3A-VS-Cys(40)-exendin-4 and NODA-VS-Cys(40)-exendin-4, respectively) were labeled with Cu-64 and Ga-68 using standard techniques. Biodistribution and dynamic positron emission tomography (PET) were carried out in normal Sprague-Dawley (SD) rats. Ex vivo autoradiography imaging was conducted with freshly frozen pancreatic thin sections. RESULTS: DO3A-VS- and NODA-VS-Cys(40)-exendin-4 analogues were labeled with Cu-64 and Ga-68 to a specific activity of 518.7 ± 3.7 Ci/mmol (19.19 ± 0.14 TBq/mmol) and radiochemical yield above 98 %. Biodistribution data demonstrated pancreatic uptake of 0.11 ± 0.02 %ID/g for [(64)Cu]DO3A-VS-, 0.14 ± 0.02 %ID/g for [(64)Cu]NODA-VS-, 0.11 ± 0.03 for [(68)Ga]DO3A-VS-, and 0.26 ± 0.03 for [(68)Ga]NODA-VS-Cys(40)-exendin-4. Excess exendin-4 and exendin-(9-39)-amide displaced all four Cu-64 and Ga-68 labeled exendin-4 derivatives in blocking studies. CONCLUSIONS: [(64)Cu]/[(68)Ga]DO3A-VS-Cys(40)- and [(64)Cu]/[(68)Ga]NODA-VS-Cys(40)-exendin-4 can be used as PET imaging agents specific for GLP-1R expressed on ß cells. Here, we report the first evidence of pancreatic uptake visualized with exendin-4 derivative in a rat animal model via in vivo dynamic PET imaging.

Peptide to Peptoid Substitutions Increase Cell Permeability in Cyclic Hexapeptides.

The effect of peptide-to-peptoid substitutions on the passive membrane permeability of an N-methylated cyclic hexapeptide is examined. In general, substitutions maintained permeability but increased conformational heterogeneity. Diversification with nonproteinogenic side chains increased permeability up to 3-fold. Additionally, the conformational impact of peptoid substitutions within a ß-turn are explored. Based on these results, the strategic incorporation of peptoid residues into cyclic peptides can maintain or improve cell permeability, while increasing access to diverse side-chain functionality.