Highlights d Unprecedented allosteric small-molecule binder to PCSK9 was identified using AS/MS d Biased and unbiased hit-to-lead strategy identified binders through divergent SAR d Demonstrated binding of lead compound to PCSK9 in a cellular thermal shift assay d Developed lead compound into targeted degrader achieving 60% reduction of PCSK9 levels

<div>Proprotein convertase substilisin-like/kexin type 9 (PCSK9) is a serine protease involved in a protein-protein interaction with the low-density lipoprotein (LDL) receptor that has both human genetic and clinical validation. Our pursuit of small molecule direct binders for this difficult to drug PPI target utilized affinity selection / mass spectrometry (AS/MS) which identified one confirmed hit compound. An x-ray crystal structure revealed this compound was binding in an unprecedented allosteric pocket located between the catalytic and C-terminal domain. Optimization of this initial hit, using two distinct strategies, led to compounds with high binding affinity to PCSK9. Direct target engagement was demonstrated in cell lysate with a cellular thermal shift assay (CETSA). Finally, ligand-induced protein degradation was shown with a proteasome recruiting tag attached to the high-affinity allosteric ligand for PCSK9.</div>

Hit-to-lead efforts resulted in the discovery of compound 19, a potent CYP11B2 inhibitor that displays high selectivity vs related CYPs, good pharmacokinetic properties in rat and rhesus, and lead-like physical properties. In a rhesus pharmacodynamic model, compound 19 displays robust, dose-dependent aldosterone lowering efficacy, with no apparent effect on cortisol levels. KEYWORDS:Aldosterone synthase, CYP11B2, hit-to-lead, hypertension A ldosterone is a steroid hormone produced in the adrenal zona glomerulosa, and is one of several endogenous ligands that bind the mineralocorticoid receptor (MR). 1,2 Binding of aldosterone to MR yields a complex that can activate gene transcription, ultimately leading to the production of proteins such as ENaC, the epithelial sodium channel responsible for reabsorption of sodium in the kidney. Classically, this sodium reabsorption and its attendant water retention and blood volume increase were thought to drive aldosterone's known ability to increase blood pressure. However, more recent studies indicate that aldosterone raises blood pressure primarily by promoting vasoconstriction, and by acting in the CNS to increase central sympathetic drive. 3 In addition to its effects on blood pressure (BP), aldosterone is also known to produce BPindependent organ damage via inflammatory and pro-fibrotic pathways, and to play a role in insulin resistance. 4 Aldosterone synthase (CYP11B2) is a mitochondrial cytochrome P450 (CYP) enzyme that catalyzes the final three steps of aldosterone biosynthesis. Compounds that inhibit CYP11B2 should thus inhibit the formation of aldosterone, and may be useful as treatments for hypertension, heart failure, and diabetes. Small molecule CYP11B2 inhibitors have been reported in the literature, and one, LCI-699, has recently been shown to lower blood pressure in the clinic, thus providing validation for the use of CYP11B2 inhibitors as treatments for hypertension. 5−10 Steroid-11β-hydroxylase (CYP11B1) is a related enzyme that catalyzes the formation of glucocorticoids such as cortisol, an important regulator of glucose metabolism. Human CYP11B2 and CYP11B1 are >93% homologous, and given the physiological importance of cortisol, selectivity for inhibition of CYP11B2 vs 11B1 is required. LCI-699 displays only ∼4-fold selectivity for inhibition of human CYP11B2 vs 11B1 in cellbased in vitro assays. 7 In the clinic, doses of LCI-699 higher than 0.5 mg elicited suppression of cortisol levels, presumably as a result of CYP11B1 inhibition. Selectivity greater than that exhibited by LCI-699 would thus be desired to derisk the potential for adverse effects resulting from cortisol suppression.Related CYPs 17 and 19, though less homologous to CYP11B2, play important roles in the conversion of steroidal precursors such as pregnenelone and progesterone to end products such as estrone and testosterone. Selectivity with regard to these CYPs as well as the major hepatic CYPs involved in drug metabolism is also required.Our goal is to discover selective CYP1...

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Herein we report the discovery and hit-to-lead optimization of a series of spirocyclic piperidine aldosterone synthase (CYP11B2) inhibitors. Compounds from this series display potent CYP11B2 inhibition, good selectivity versus related CYP enzymes, and lead-like physical and pharmacokinetic properties.KEYWORDS: aldosterone synthase, CYP11B2, hypertension H ypertension is the most prevalent chronic disease state, afflicting an estimated 80 million people in the United States and >1 billion people worldwide.1 Roughly half of that patient population exhibits resistant hypertension, blood pressure that remains high even after treatment with a combination of three or more antihypertensive agents.2 Such uncontrolled hypertension can contribute to organ damage, cardiovascular disease, stroke, heart attack, and heart failure. Current treatment options notwithstanding, there remains a significant need for additional, mechanistically novel, antihypertensive therapies.One novel approach involves the reduction of plasma aldosterone levels.4 Elevated plasma aldosterone levels promote sodium and water retention, vasoconstriction, and increased sympathetic drive, ultimately leading to increased blood pressure. Aldosterone synthase, also known as CYP11B2, catalyzes the final three rate-limiting steps of aldosterone's biosynthesis. Based on this rationale, inhibitors of CYP11B2 should prevent aldosterone biosynthesis, thereby leading to a reduction in plasma aldosterone and consequently blood pressure.The role of aldosterone synthase inhibitors (ASIs) has received much attention in recent years.5−15 Of note, the inhibitor LCI-699 (Figure 1) has been shown to lower plasma aldosterone levels and blood pressure in the clinic, thus providing proof of concept for the use of ASIs to treat hypertension.16,17 LCI-699 is a modestly selective CYP11B2 inhibitor, displaying ∼4-fold selectivity for inhibition of CYP11B2 versus a close structural homologue, CYP11B1, an enzyme that catalyzes the biosynthesis of cortisol. 12 In the clinic, LCI-699 produced an undesired impairment of the stress response, an adverse effect presumed to be caused by inhibition of CYP11B1. Given the physiological importance of cortisol in glucose metabolism and stress response, selectivity for inhibition of CYP11B2 over CYP11B1 is required in a clinically viable candidate.With this in mind the goal was to discover a structurally novel class of potent and selective inhibitors of aldosterone synthase and to assess their potential therapeutic value in models of resistant hypertension. In this regard spiro-fused scaffolds were explored as replacements for the aromatic, planar-fused bicyclic and tricyclic scaffolds that are present in previously described inhibitors.5 Recent literature reports have highlighted the benefits that such spirocyclic scaffolds can