Anthony B. Pinkerton

Pathophysiological Role of Vascular Smooth Muscle Alkaline Phosphatase in Medial Artery Calcification

Medial vascular calcification (MVC) is a pathological phenomenon that causes vascular stiffening and can lead to heart failure; it is common to a variety of conditions, including aging, chronic kidney disease, diabetes, obesity, and a variety of rare genetic diseases. These conditions share the common feature of tissue‐nonspecific alkaline phosphatase (TNAP) upregulation in the vasculature. To evaluate the role of TNAP in MVC, we developed a mouse model that overexpresses human TNAP in vascular smooth muscle cells in an X‐linked manner. Hemizygous overexpressor male mice (Tagln‐Cre+/–; HprtALPL/Y or TNAP‐OE) show extensive vascular calcification, high blood pressure, and cardiac hypertrophy, and have a median age of death of 44 days, whereas the cardiovascular phenotype is much less pronounced and life expectancy is longer in heterozygous (Tagln‐Cre+/–; HprtALPL/−) female TNAP‐OE mice. Gene expression analysis showed upregulation of osteoblast and chondrocyte markers and decreased expression of vascular smooth muscle markers in the aortas of TNAP‐OE mice. Through medicinal chemistry efforts, we developed inhibitors of TNAP with drug‐like pharmacokinetic characteristics. TNAP‐OE mice were treated with the prototypical TNAP inhibitor SBI‐425 or vehicle to evaluate the feasibility of TNAP inhibition in vivo. Treatment with this inhibitor significantly reduced aortic calcification and cardiac hypertrophy, and extended lifespan over vehicle‐treated controls, in the absence of secondary effects on the skeleton. This study shows that TNAP in the vasculature contributes to the pathology of MVC and that it is a druggable target.

Advancing Biological Understanding and Therapeutics Discovery with Small-Molecule Probes

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.

Synthesis and SAR of 2-aryl-3-aminomethylquinolines as agonists of the bile acid receptor TGR5

Optimization of a screening hit from uHTS led to the discovery of TGR5 agonist 32, which was shown to have activity in a rodent model for diabetes.

Discovery of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221) as a functional antagonist of the apelin (APJ) receptor

The recently discovered apelin/APJ system has emerged as a critical mediator of cardiovascular homeostasis and is associated with the pathogenesis of cardiovascular disease. A role for apelin/APJ in energy metabolism and gastrointestinal function has also recently emerged. We disclose the discovery and characterization of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221), a potent APJ functional antagonist in cell-based assays that is >37-fold selective over the closely related angiotensin II type 1 (AT1) receptor. ML221 was derived from an HTS of the ~330,600 compound MLSMR collection. This antagonist showed no significant binding activity against 29 other GPCRs, except to the κ-opioid and benzodiazepinone receptors (<50/<70%I at 10 μM). The synthetic methodology, development of structure-activity relationship (SAR), and initial in vitro pharmacologic characterization are also presented.

Recent Progress in the Synthesis and Characterization of Group II Metabotropic Glutamate Receptor Allosteric Modulators

Group II metabotropic glutamate (mGlu) receptors consist of the metabotropic glutamate 2 (mGlu(2)) and metabotropic glutamate 3 (mGlu(3)) receptor subtypes which modulate glutamate transmission by second messenger activation to negatively regulate the activity of adenylyl cyclase. Excessive accumulation of glutamate in the perisynaptic extracellular region triggers mGlu(2) and mGlu(3) receptors to inhibit further release of glutamate. There is growing evidence that the modulation of glutamatergic neurotransmission by small molecule modulators of Group II mGlu receptors has significant potential for the treatment of several neuropsychiatric and neurodegenerative diseases. This review provides an overview of recent progress on the synthesis and pharmacological characterization of positive and negative allosteric modulators of the Group II mGlu receptors.

PPAR-δ is repressed in Huntington's disease, is required for normal neuronal function and can be targeted therapeutically.

Huntingtons disease (HD) is a progressive neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene, which encodes a polyglutamine tract in the HTT protein. We found that peroxisome proliferator-activated receptor delta (PPAR-δ) interacts with HTT and that mutant HTT represses PPAR-δ–mediated transactivation. Increased PPAR-δ transactivation ameliorated mitochondrial dysfunction and improved cell survival of neurons from mouse models of HD. Expression of dominant-negative PPAR-δ in the central nervous system of mice was sufficient to induce motor dysfunction, neurodegeneration, mitochondrial abnormalities and transcriptional alterations that recapitulated HD-like phenotypes. Expression of dominant-negative PPAR-δ specifically in the striatum of medium spiny neurons in mice yielded HD-like motor phenotypes, accompanied by striatal neuron loss. In mouse models of HD, pharmacologic activation of PPAR-δ using the agonist KD3010 improved motor function, reduced neurodegeneration and increased survival. PPAR-δ activation also reduced HTT-induced neurotoxicity in vitro and in medium spiny-like neurons generated from stem cells derived from individuals with HD, indicating that PPAR-δ activation may be beneficial in HD and related disorders.Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by a CAG-polyglutamine repeat expansion in the huntingtin (htt) gene. We found that peroxisome proliferator-activated receptor delta (PPARδ) interacts with htt and that mutant htt represses PPARδ-mediated transactivation. Increased PPARδ transactivation ameliorated mitochondrial dysfunction and improved cell survival of HD neurons. Expression of dominant-negative PPARδ in CNS was sufficient to induce motor dysfunction, neurodegeneration, mitochondrial abnormalities, and transcriptional alterations that recapitulated HD-like phenotypes. Expression of dominant-negative PPARδ specifically in the striatum of medium spiny neurons in mice yielded HD-like motor phenotypes, accompanied by striatal neuron loss. In mouse models of HD, pharmacologic activation of PPAR δ, using the agonist KD3010, improved motor function, reduced neurodegeneration, and increased survival. PPAR δ activation also reduced htt-induced neurotoxicity in vitro and in medium spiny-like neurons generated from human HD stem cells, indicating that PPAR δ activation may be beneficial in individuals with HD and related disorders.

Capzimin is a potent and specific inhibitor of proteasome isopeptidase Rpn11

The proteasome is a vital cellular machine that maintains protein homeostasis, which is of particular importance in multiple myeloma and possibly other cancers. Targeting of proteasome 20S peptidase activity with bortezomib and carfilzomib has been widely used to treat myeloma. However, not all patients respond to these compounds, and those who do eventually suffer relapse. Therefore, there is an urgent and unmet need to develop new drugs that target proteostasis through different mechanisms. We identified quinoline-8-thiol (8TQ) as a first-in-class inhibitor of the proteasome 19S subunit Rpn11. A derivative of 8TQ, capzimin, shows >5-fold selectivity for Rpn11 over the related JAMM proteases and >2 logs selectivity over several other metalloenzymes. Capzimin stabilized proteasome substrates, induced an unfolded protein response, and blocked proliferation of cancer cells, including those resistant to bortezomib. Proteomic analysis revealed that capzimin stabilized a subset of polyubiquitinated substrates. Identification of capzimin offers an alternative path to develop proteasome inhibitors for cancer therapy.

HNF4α antagonists discovered by a high-throughput screen for modulators of the human insulin promoter.

Hepatocyte nuclear factor (HNF)4α is a central regulator of gene expression in cell types that play a critical role in metabolic homeostasis, including hepatocytes, enterocytes, and pancreatic β cells. Although fatty acids were found to occupy the HNF4α ligand-binding pocket and were proposed to act as ligands, there is controversy about both the nature of HNF4α ligands as well as the physiological role of the binding. Here, we report the discovery of potent synthetic HNF4α antagonists through a high-throughput screen for effectors of the human insulin promoter. These molecules bound to HNF4α with high affinity and modulated the expression of known HNF4α target genes. Notably, they were found to be selectively cytotoxic to cancer cell lines in vitro and in vivo, although in vivo potency was limited by suboptimal pharmacokinetic properties. The discovery of bioactive modulators for HNF4α raises the possibility that diseases involving HNF4α, such as diabetes and cancer, might be amenable to pharmacologic intervention by modulation of HNF4α activity.

Identification and Characterization of Novel Human Glucose-6-Phosphate Dehydrogenase Inhibitors

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway, converting glucose-6-phosphate to 6-phosphoglucono-δ-lactone with parallel reduction of NADP+. Several human diseases, including cancer, are associated with increased G6PD activity. To date, only a few G6PD inhibitors have been available. However, adverse side effects and high IC50 values hamper their use as therapeutics and basic research probes. In this study, we developed a high-throughput screening assay to identify novel human G6PD (hG6PD) inhibitors. Screening the LOPAC (Sigma-Aldrich; 1280 compounds), Spectrum (Microsource Discovery System; 1969 compounds), and DIVERSet (ChemBridge; 49 971 compounds) small-molecule compound collections revealed 139 compounds that presented ≥50% hG6PD inhibition. Hit compounds were further included in a secondary and orthogonal assay in order to identify false-positives and to determine IC50 values. The most potent hG6PD inhibitors presented IC50 values of <4 µM. Compared with the known hG6PD inhibitors dehydroepiandrosterone and 6-aminonicotinamide, the inhibitors identified in this study were 100- to 1000-fold more potent and showed different mechanisms of enzyme inhibition. One of the newly identified hG6PD inhibitors reduced viability of the mammary carcinoma cell line MCF10-AT1 (IC50 ~25 µM) more strongly than that of normal MCF10-A cells (IC50 >50 µM).

Discovery of a Plasmodium falciparum Glucose-6-phosphate Dehydrogenase 6-phosphogluconolactonase Inhibitor (R,Z)-N-((1-Ethylpyrrolidin-2-yl)methyl)-2-(2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide (ML276) That Reduces Parasite Growth in Vitro

A high-throughput screen of the NIHs MLSMR collection of ∼340000 compounds was undertaken to identify compounds that inhibit Plasmodium falciparum glucose-6-phosphate dehydrogenase (PfG6PD). PfG6PD is important for proliferating and propagating P. falciparum and differs structurally and mechanistically from the human orthologue. The reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PD) is the first, rate-limiting step in the pentose phosphate pathway (PPP), a key metabolic pathway sustaining anabolic needs in reductive equivalents and synthetic materials in fast-growing cells. In P. falciparum , the bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (PfGluPho) catalyzes the first two steps of the PPP. Because P. falciparum and infected host red blood cells rely on accelerated glucose flux, they depend on the G6PD activity of PfGluPho. The lead compound identified from this effort, (R,Z)-N-((1-ethylpyrrolidin-2-yl)methyl)-2-(2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide, 11 (ML276), is a submicromolar inhibitor of PfG6PD (IC(50) = 889 nM). It is completely selective for the enzymes human isoform, displays micromolar potency (IC(50) = 2.6 μM) against P. falciparum in culture, and has good drug-like properties, including high solubility and moderate microsomal stability. Studies testing the potential advantage of inhibiting PfG6PD in vivo are in progress.