Richard K. Harrison

A semicontinuous, high-performance liquid chromatography-based assay for stromelysin

A search for low molecular weight peptide substrates for the metalloendoproteinase, human fibroblast stromelysin, resulted in the discovery that substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) is a substrate for this enzyme and is cleaved exclusively at the Gln6-Phe7 bond. On the basis of this observation, a semicontinuous HPLC-based assay was developed that monitors the production of the hydrolysis product, fragment 7-11 (SP7-11). Steady-state velocities for the production of SP7-11 have been determined as a function of substrate concentration and obey simple, Michaelis-Menten kinetics. For a 1-ml reaction volume, Vmax = (2.4 nmol SP7-11/min)/micrograms protein and Km = 0.38 mM.

Discovery of VTP-27999, an Alkyl Amine Renin Inhibitor with Potential for Clinical Utility.

Structure guided optimization of a series of nonpeptidic alkyl amine renin inhibitors allowed the rational incorporation of additional polar functionality. Replacement of the cyclohexylmethyl group occupying the S1 pocket with a (R)-(tetrahydropyran-3-yl)methyl group and utilization of a different attachment point led to the identification of clinical candidate 9. This compound demonstrated excellent selectivity over related and unrelated off-targets, >15% oral bioavailability in three species, oral efficacy in a double transgenic rat model of hypertension, and good exposure in humans.

Development of Inhibitors of the Aspartyl Protease Renin for the Treatment of Hypertension

Renin is the rate-limiting enzyme in the renin-angiotensin-aldosterone system (RAS) which controls blood pressure and volume. The biological function of renin is to cleave the N-terminus of angiotensinogen releasing the decapeptide, angiotensin I (ANGI). Subsequently, angiotensin I is further processed by the angiotensin converting enzyme (ACE) to produce angiotensin II (ANGII). The RAS cascade is a major target for the clinical management of hypertension. Current clinical treatments include angiotensin converting enzyme inhibitors (ACEi) and ANGII receptor blockers (ARBs). As the rate-limiting enzyme in ANGII production, renin inhibitors have been pursued as an additional class of anti-hypertensives. Clinical studies conducted with renin inhibitors have shown them to be as effective as ACE inhibitors in lowering blood pressure. Most importantly, inhibitors of renin may have a number of potential advantages over ACEi and ARBs. Renin is specific for angiotensinogen and will not carry the ancillary pharmacology associated with ACEi or ARBs. To date, no renin inhibitors have made it to market. The development of these inhibitors has been hindered by poor bioavailability and complex synthesis. However, despite the pharmacokinetic challenges of designing renin inhibitors, the enzyme remains a promising target for the development of novel treatments for hypertension. This review will consist of an overview of renin biology, the pharmacology of renin and RAS and focus in on renin as a target for blood pressure regulation. We also cover the evaluation of renin inhibitors in animal models and clinical studies. Presently a number of new generation inhibitors of renin are in development with at least one in the clinic and these will be discussed. Finally we will discuss what might distinguish renin inhibitors from current therapeutic options and discuss other therapeutic indications renin inhibitors might have.

Structure-based design and synthesis of 1,3-oxazinan-2-one inhibitors of 11β-hydroxysteroid dehydrogenase type 1.

Structure based design led directly to 1,3-oxazinan-2-one 9a with an IC(50) of 42 nM against 11β-HSD1 in vitro. Optimization of 9a for improved in vitro enzymatic and cellular potency afforded 25f with IC(50) values of 0.8 nM for the enzyme and 2.5 nM in adipocytes. In addition, 25f has 94% oral bioavailability in rat and >1000× selectivity over 11β-HSD2. In mice, 25f was distributed to the target tissues, liver, and adipose, and in cynomolgus monkeys a 10 mg/kg oral dose reduced cortisol production by 85% following a cortisone challenge.

Phosphinic acid inhibitors of matrix metalloproteinases

Abstract The matrix metalloproteinase stromelysin-1 (MMP-3) is inhibited more strongly by peptidyl phosphinic acid 7 than by its corresponding phosphonamidate and phosphonate analogs. Extending a benzyl group at P′ 1 to a phenylethyl group in 8 further increases the potency (K i = 1.4 nM). Enhanced potency with an extended substituent into the P 3 region was observed.

Inhibition of stromelysin-1 (MMP-3) by peptidyl phosphinic acids

Abstract A series of phosphinic acid-containing peptide inhibitors of human stromelysin-1 (MMP-3) were prepared. The P1 through P3 subsites were varied in a systematic manner on analogs possessing an invariant P1′-P3′ segment. The in vitro activity of these compounds as inhibitors of stromelysin and collagenase is discussed.

Spirocyclic ureas: Orally bioavailable 11β-HSD1 inhibitors identified by computer-aided drug design

Structure-guided drug design led to the identification of a class of spirocyclic ureas which potently inhibit human 11beta-HSD1 in vitro. Lead compound 10j was shown to be orally bioavailable in three species, distributed into adipose tissue in the mouse, and its (R) isomer 10j2 was efficacious in a primate pharmacodynamic model.

Design and Optimization of Renin Inhibitors: Orally Bioavailable Alkyl Amines

Structure-based drug design led to the identification of a novel class of potent, low MW alkylamine renin inhibitors. Oral administration of lead compound 21l, with MW of 508 and IC(50) of 0.47nM, caused a sustained reduction in mean arterial blood pressure in a double transgenic rat model of hypertension.

Inhibition of matrix metalloproteinases by N-carboxyalkyl dipeptides: Enhanced potency and selectivity with substituted P1′ homophenylalanines

Abstract A series of N-carboxyalkyl dipeptides were synthesized to evaluate their inhibitory activities against human stromelysin-1(MMP-3), collagenase(MMP-1), and gelatinase-A(MMP-2). Structures with a homophenylalanine residue at P 1′ substituted at the para position with small alkyl groups are potent inhibitors of (MMP-3) and (MMP-2) (K i′ s 2–40 nM), but weak inhibitors of (MMP-1).

Optimization of orally bioavailable alkyl amine renin inhibitors.

Structure-guided drug design led to new alkylamine renin inhibitors with improved in vitro and in vivo potency. Lead compound 21a, has an IC(50) of 0.83nM for the inhibition of human renin in plasma (PRA). Oral administration of 21a at 10mg/kg resulted in >20h reduction of blood pressure in a double transgenic rat model of hypertension.