A series of small-molecule YEATS4 binders have been discovered as part of an ongoing research effort to generate high-quality probe molecules for emerging and/or challenging epigenetic targets. ...Analogues such as 4d and 4e demonstrate excellent potency and selectivity for YEATS4 binding versus YEATS1,2,3 and exhibit good physical properties and in vitro safety profiles. A new X-ray crystal structure confirms direct binding of this chemical series to YEATS4 at the lysine acetylation recognition site of the YEATS domain. Multiple analogues engage YEATS4 with nanomolar potency in a whole-cell nanoluciferase bioluminescent resonance energy transfer assay. Rodent pharmacokinetic studies demonstrate the competency of several analogues as in vivo-capable binders.
Preclinical and clinical studies have identified the ghrelin receptor growth hormone secretagogue receptor (GHSR)1a as a potential target for treating alcohol use disorder. A recent phase 1a clinical ...trial of a GHSR1a antagonist/inverse agonist, PF-5190457, in individuals with heavy alcohol drinking identified a previously undetected major hydroxy metabolite of PF-5190457, namely PF-6870961. Here, we further characterized PF-6870961 by screening for off-target interactions in a high-throughput screen and determined its in vitro pharmacodynamic profile at GHSR1a through binding and concentration-response assays. Moreover, we determined whether the metabolite demonstrated an in vivo effect by assessing effects on food intake in male and female rats. We found that PF-6870961 had no off-target interactions and demonstrated both binding affinity and inverse agonist activity at GHSR1a. In comparison with its parent compound, PF-5190457, the metabolite PF-6870961 had lower binding affinity and potency at inhibiting GHSR1a-induced inositol phosphate accumulation. However, PF-6870961 had increased inhibitory potency at GHSR1a-induced
-arrestin recruitment relative to its parent compound. Intraperitoneal injection of PF-6870961 suppressed food intake under conditions of both food restriction and with ad libitum access to food in male and female rats, demonstrating in vivo activity. The effects of PF-6870961 on food intake were abolished in male and female rats knockout for GHSR, thus demonstrating that its effects on food intake are in fact mediated by the GHSR receptor. Our findings indicate that the newly discovered major hydroxy metabolite of PF-5190457 may contribute to the overall activity of PF-5190457 by demonstrating inhibitory activity at GHSR1a. SIGNIFICANCE STATEMENT: Antagonists or inverse agonists of the growth hormone secretagogue receptor (GHSR)1a have demonstrated substantial potential as therapeutics for alcohol use disorder. We here expand understanding of the pharmacology of one such GHSR1a inverse agonist, PF-5190457, by studying the safety and pharmacodynamics of its major hydroxy metabolite, PF-6870961. Our data demonstrate biased inverse agonism of PF-6870961 at GHSR1a and provide new structure-activity relationship insight into GHSR1a inverse agonism.
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.
Discovery efforts leading to the identification of ervogastat (PF-06865571), a systemically acting diacylglycerol acyltransferase (DGAT2) inhibitor that has advanced into clinical trials for the ...treatment of non-alcoholic steatohepatitis (NASH) with liver fibrosis, are described herein. Ervogastat is a first-in-class DGAT2 inhibitor that addressed potential development risks of the prototype liver-targeted DGAT2 inhibitor PF-06427878. Key design elements that culminated in the discovery of ervogastat are (1) replacement of the metabolically labile motif with a 3,5-disubstituted pyridine system, which addressed potential safety risks arising from a cytochrome P450-mediated O-dearylation of PF-06427878 to a reactive quinone metabolite precursor, and (2) modifications of the amide group to a 3-THF group, guided by metabolite identification studies coupled with property-based drug design.
The optimization for selectivity and central receptor occupancy for a series of spirocyclic azetidine–piperidine inverse agonists of the ghrelin receptor is described. Decreased mAChR muscarinic M2 ...binding was achieved by use of a chiral indane in place of a substituted benzylic group. Compounds with desirable balance of human in vitro clearance and ex vivo central receptor occupancy were discovered by incorporation of heterocycles. Specifically, heteroaryl rings with nitrogen(s) vicinal to the indane linkage provided the most attractive overall properties.
The identification of potent, highly selective orally bioavailable ghrelin receptor inverse agonists from a spiro-azetidino-piperidine series is described. Examples from this series have promising in ...vivo pharmacokinetics and increase glucose-stimulated insulin secretion in human whole and dispersed islets. A physicochemistry-based strategy to increase lipophilic efficiency for ghrelin receptor potency and retain low clearance and satisfactory permeability while reducing off-target pharmacology led to the discovery of 16h. Compound 16h has a superior balance of ghrelin receptor pharmacology and off-target selectivity. On the basis of its promising pharmacological and safety profile, 16h was advanced to human clinical trials.
The discovery of spirocyclic piperidine-azetidine inverse agonists of the ghrelin receptor is described. The characterization and redressing of the issues associated with these compounds is detailed. ...An efficient three-step synthesis and a binding assay were relied upon as the primary means of rapidly improving potency and ADMET properties for this class of inverse agonist compounds. Compound 10n bearing distributed polarity in the form of an imidazo-thiazole acetamide and a phenyl triazole is a unit lower in logP and has significantly improved binding affinity compared to the hit molecule 10a, providing support for further optimization of this series of compounds.
An inhibitor of human liver glycogen phosphorylase a (HLGPa) has been identified and characterized in vitro and in vivo. This substance, R-(R*,S*)-5-chloro-N-3- ...(dimethylamino)-2-hydroxy-3-oxo-1-(phenylmethyl)propyl-1H-indole-2-carboxamide (CP-91149), inhibited HLGPa with an IC50of 0.13 μ M in the presence of 7.5 mM glucose. CP-91149 resembles caffeine, a known allosteric phosphorylase inhibitor, in that it is 5- to 10-fold less potent in the absence of glucose. Further analysis, however, suggests that CP-91149 and caffeine are kinetically distinct. Functionally, CP-91149 inhibited glucagon-stimulated glycogenolysis in isolated rat hepatocytes (P < 0.05 at 10-100 μ M) and in primary human hepatocytes (2.1 μ M IC50). In vivo, oral administration of CP-91149 to diabetic ob/ob mice at 25-50 mg/kg resulted in rapid (3 h) glucose lowering by 100-120 mg/dl (P < 0.001) without producing hypoglycemia. Further, CP-91149 treatment did not lower glucose levels in normoglycemic, nondiabetic mice. In ob/ob mice pretreated with14C-glucose to label liver glycogen, CP-91149 administration reduced14C-glycogen breakdown, confirming that glucose lowering resulted from inhibition of glycogenolysis in vivo. These findings support the use of CP-91149 in investigating glycogenolytic versus gluconeogenic flux in hepatic glucose production, and they demonstrate that glycogenolysis inhibitors may be useful in the treatment of type 2 diabetes.
The chemokines CCL3 and CCL5, as well as their shared receptor CCR1, are believed to play a role in the pathogenesis of several inflammatory diseases including rheumatoid arthritis, multiple ...sclerosis, and transplant rejection. In this study we describe the pharmacological properties of a novel small molecular weight CCR1 antagonist, CP-481,715 (quinoxaline-2-carboxylic acid 4(R)-carbamoyl-1(S)-(3-fluorobenzyl)-2(S),7-dihydroxy-7-methyloctylamide). Radiolabeled binding studies indicate that CP-481,715 binds to human CCR1 with a Kd of 9.2 nm and displaces 125I-labeled CCL3 from CCR1-transfected cells with an IC50 of 74 nm. CP-481,715 lacks intrinsic agonist activity but fully blocks the ability of CCL3 and CCL5 to stimulate receptor signaling (guanosine 5′-O-(thiotriphosphate) incorporation; IC50 = 210 nm), calcium mobilization (IC50 = 71 nm), monocyte chemotaxis (IC50 = 55 nm), and matrix metalloproteinase 9 release (IC50 = 54 nm). CP-481,715 retains activity in human whole blood, inhibiting CCL3-induced CD11b up-regulation and actin polymerization (IC50 = 165 and 57 nm, respectively) on monocytes. Furthermore, it behaves as a competitive and reversible antagonist. CP-481,715 is >100-fold selective for CCR1 as compared with a panel of G-protein-coupled receptors including related chemokine receptors. Evidence for its potential use in human disease is suggested by its ability to inhibit 90% of the monocyte chemotactic activity present in 11/15 rheumatoid arthritis synovial fluid samples. These data illustrate that CP-481,715 is a potent and selective antagonist for CCR1 with therapeutic potential for rheumatoid arthritis and other inflammatory diseases.
Several polar heteroaromatic acetic acids and their piperidine amides were synthesized and evaluated as ghrelin or type 1a growth hormone secretagogue receptor (GHS-R1a) inverse agonists. Efforts to ...improve pharmacokinetic and safety profile was achieved by modulating physicochemical properties and, more specifically, emphasizing increased polarity of our chemical series. ortho-Carboxamide containing compounds provided optimal physicochemical, pharmacologic, and safety profile. pH-dependent chemical stability was also assessed with our series.
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