Discovery of a novel, highly potent and orally bioavailable pyrrolidinone indole series of irreversible Myeloperoxidase (MPO) inhibitors.

Myeloperoxidase (MPO) is a heme-containing peroxidase from phagocytic cells, which plays an important role in the innate immune response. The primary anti-microbial function of MPO is achieved by catalyzing the oxidation of halides by hydrogen peroxide (H2O2). Upon activation of phagocytes, MPO activity is detectable in both phagosomes and extracellularly, where it can remain or transcytose into interstitial compartments. Activated MPO leads to oxidative stress and tissue damage in many inflammatory states, including cardiovascular disease. Starting from a low molecular weight (LMW) high throughput screening (HTS) hit, here we report the discovery of a novel pyrrolidinone indole (IN-4) as a highly potent MPO inhibitor. This compound displays similar in vitro potency across peroxidation, plasma and NETosis assays. In a dilution/dialysis study, <5% of the original MPO activity was detected post-incubation of MPO with IN-4, suggesting irreversible enzyme inhibition. A fast MPO inactivation rate (kinact/Ki) and low partition ratio (k3/k4) make IN-4 kinetic properties attractive for an MPO inhibitor. This compound also displays significant selectivity over the closely related thyroid peroxidase (TPO), and is selective for extracellular MPO over intracellular (neutrophil) MPO. Moreover, IN-4 shows good exposure, low clearance and high oral bioavailability in mice, rats and dogs. The high in vitro MPO activity and high oral exposure observed with IN-4 result in a dose-dependent inhibition of MPO activity in three mouse models of inflammation. In conclusion, IN-4 is a novel, potent, mechanism-based and selective MPO inhibitor, which may be used as superior therapeutic agent to treat multiple inflammatory conditions, including cardiovascular disease.

Preclinical Characterization of Vadadustat (AKB-6548), an Oral Small Molecule Hypoxia-Inducible Factor Prolyl-4-Hydroxylase Inhibitor, for the Potential Treatment of Renal Anemia.

Pharmacological inhibition of prolyl-4-hydroxylase domain (PHD) enzymes stabilizes hypoxia-inducible factors (HIFs), transcription factors that activate target genes that, among others, increase erythropoietin (EPO) synthesis, resulting in the production of new red blood cells (RBCs). Herein, we summarize the preclinical characteristics of the small molecule HIF prolyl-4-hydroxylase inhibitor vadadustat (AKB-6548), which is in development for the treatment of anemia in patients with chronic kidney disease (CKD). Vadadustat inhibits the enzyme activity of all three human PHD isozymes, PHD1, PHD2, and PHD3, with similar low nanomolar inhibitory constant values. PHD enzyme inhibition by vadadustat is competitive with endogenous cofactor 2-oxoglutarate and is insensitive to free iron concentration. In the human hepatocellular carcinoma cell line (Hep 3B) and human umbilical vein endothelial cells, PHD inhibition by vadadustat leads to the time- and concentration-dependent stabilization of HIF-1α and HIF-2α In Hep 3B cells, this in turn results in the synthesis and secretion of EPO; vascular endothelial growth factor is not measured at detectable levels. A single oral dose of vadadustat in rats potently increases circulating levels of EPO, and daily oral dosing for 14 days increases RBC indices in healthy rats and in the 5/6 nephrectomy model of CKD. In mice and dogs, once-daily repeat oral dosing increases hemoglobin and hematocrit. Vadadustat has a relatively short half-life in all nonclinical species evaluated and does not accumulate when administered as a single bolus dose (oral or intravenous) or upon repeat oral dosing. The pharmacological profile of vadadustat supports continued development for treatment of renal anemia. SIGNIFICANCE STATEMENT: Vadadustat (AKB-6548) is an orally bioavailable small molecule prolyl-4-hydroxylase inhibitor in development for anemia of chronic kidney disease. It is an equipotent inhibitor of the three human prolyl-4-hydroxylase domain isoforms, which activates erythropoiesis through stabilization of hypoxia-inducible factor (HIF)-1α and HIF-2α, increasing production of erythropoietin, without detectable stimulation of vascular endothelial growth factor.

Discovery of a novel indole pharmacophore for the irreversible inhibition of myeloperoxidase (MPO).

Myeloperoxidase (MPO) activity and subsequent generation of hypochlorous acid has been associated with the killing of host-invading microorganisms (e.g. bacteria, viruses, and fungi). However, during oxidative stress, high MPO activity can damage host tissue and is linked to several chronic inflammatory conditions. Herein, we describe the development of a novel biaryl, indole-pyrazole series of irreversible mechanism-based inhibitors of MPO. Derived from an indole-containing high-throughput screen hit, optimization efforts resulted in potent and selective 6-substituted indoles with good oral bioavailability and in vivo activity.

Discovery of 1-((6-Aminopyridin-3-yl)Methyl)-3-(4-Bromophenyl)Urea as a Potent, Irreversible Myeloperoxidase Inhibitor.

Myeloperoxidase (MPO) is a leukocyte-derived redox enzyme that has been linked to oxidative stress and damage in many inflammatory states, including cardiovascular disease. We have discovered aminopyridines that are potent mechanism-based inhibitors of MPO, with significant selectivity over the closely related thyroid peroxidase. 1-((6-Aminopyridin-3-yl)methyl)-3-(4-bromophenyl)urea (Aminopyridine 2) inhibited MPO in human plasma and blocked MPO-dependent vasomotor dysfunction ex vivo in rat aortic rings. Aminopyridine 2 also showed high oral bioavailability and inhibited MPO activity in vivo in a mouse model of peritonitis. Aminopyridine 2 could effectively be administered as a food admixture, making it an important tool for assessing the relative importance of MPO in preclinical models of chronic inflammatory disease.