STX-478, a Mutant-Selective, Allosteric PI3Kα Inhibitor Spares Metabolic Dysfunction and Improves Therapeutic Response in PI3Kα-Mutant Xenografts.

Phosphoinositide 3-kinase α (PIK3CA) is one of the most mutated genes across cancers, especially breast, gynecologic, and head and neck squamous cell carcinoma tumors. Mutations occur throughout the gene, but hotspot mutations in the helical and kinase domains predominate. The therapeutic benefit of isoform-selective PI3Kα inhibition was established with alpelisib, which displays equipotent activity against the wild-type and mutant enzyme. Inhibition of wild-type PI3Kα is associated with severe hyperglycemia and rash, which limits alpelisib use and suggests that selectively targeting mutant PI3Kα could reduce toxicity and improve efficacy. Here we describe STX-478, an allosteric PI3Kα inhibitor that selectively targets prevalent PI3Kα helical- and kinase-domain mutant tumors. STX-478 demonstrated robust efficacy in human tumor xenografts without causing the metabolic dysfunction observed with alpelisib. Combining STX-478 with fulvestrant and/or cyclin-dependent kinase 4/6 inhibitors was well tolerated and provided robust and durable tumor regression in ER+HER2- xenograft tumor models. SIGNIFICANCE: These preclinical data demonstrate that the mutant-selective, allosteric PI3Kα inhibitor STX-478 provides robust efficacy while avoiding the metabolic dysfunction associated with the nonselective inhibitor alpelisib. Our results support the ongoing clinical evaluation of STX-478 in PI3Kα-mutated cancers, which is expected to expand the therapeutic window and mitigate counterregulatory insulin release. See related commentary by Kearney and Vasan, p. 2313. This article is featured in Selected Articles from This Issue, p. 2293.

Next-generation small molecule therapies for heart failure: 2015 and beyond.

Poor prognosis coupled with significant economic burden makes heart failure (HF) one of the largest issues currently facing the world population. Although a significant number of new therapies have emerged over the past 20 years to treat the underlying physiological risk factors, only two new medications specifically for HF have been approved since 2007. This perspective provides an overview of recently approved treatment options for HF and as well as an update on additional small molecule therapies currently in clinical development.

Discovery and Structure-Guided Optimization of Diarylmethanesulfonamide Disrupters of Glucokinase-Glucokinase Regulatory Protein (GK-GKRP) Binding: Strategic Use of a N → S (nN → σ*S-X) Interaction for Conformational Constraint.

The HTS-based discovery and structure-guided optimization of a novel series of GKRP-selective GK-GKRP disrupters are revealed. Diarylmethanesulfonamide hit 6 (hGK-hGKRP IC50 = 1.2 µM) was optimized to lead compound 32 (AMG-0696; hGK-hGKRP IC50 = 0.0038 µM). A stabilizing interaction between a nitrogen atom lone pair and an aromatic sulfur system (nN → σ*S-X) in 32 was exploited to conformationally constrain a biaryl linkage and allow contact with key residues in GKRP. Lead compound 32 was shown to induce GK translocation from the nucleus to the cytoplasm in rats (IHC score = 0; 10 mg/kg po, 6 h) and blood glucose reduction in mice (POC = -45%; 100 mg/kg po, 3 h). X-ray analyses of 32 and several precursors bound to GKRP were also obtained. This novel disrupter of GK-GKRP binding enables further exploration of GKRP as a potential therapeutic target for type II diabetes and highlights the value of exploiting unconventional nonbonded interactions in drug design.

Oxopyrido[2,3-d]pyrimidines as Covalent L858R/T790M Mutant Selective Epidermal Growth Factor Receptor (EGFR) Inhibitors.

In nonsmall cell lung cancer (NSCLC), the threonine(790)-methionine(790) (T790M) point mutation of EGFR kinase is one of the leading causes of acquired resistance to the first generation tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. Herein, we describe the optimization of a series of 7-oxopyrido[2,3-d]pyrimidinyl-derived irreversible inhibitors of EGFR kinase. This led to the discovery of compound 24 which potently inhibits gefitinib-resistant EGFR(L858R,T790M) with 100-fold selectivity over wild-type EGFR. Compound 24 displays strong antiproliferative activity against the H1975 nonsmall cell lung cancer cell line, the first line mutant HCC827 cell line, and promising antitumor activity in an EGFR(L858R,T790M) driven H1975 xenograft model sparing the side effects associated with the inhibition of wild-type EGFR.

Small Molecule Disruptors of the Glucokinase-Glucokinase Regulatory Protein Interaction: 5. A Novel Aryl Sulfone Series, Optimization Through Conformational Analysis.

The glucokinase-glucokinase regulatory protein (GK-GKRP) complex plays an important role in controlling glucose homeostasis in the liver. We have recently disclosed a series of arylpiperazines as in vitro and in vivo disruptors of the GK-GKRP complex with efficacy in rodent models of type 2 diabetes mellitus (T2DM). Herein, we describe a new class of aryl sulfones as disruptors of the GK-GKRP complex, where the central piperazine scaffold has been replaced by an aromatic group. Conformational analysis and exploration of the structure-activity relationships of this new class of compounds led to the identification of potent GK-GKRP disruptors. Further optimization of this novel series delivered thiazole sulfone 93, which was able to disrupt the GK-GKRP interaction in vitro and in vivo and, by doing so, increases cytoplasmic levels of unbound GK.

Small molecule disruptors of the glucokinase-glucokinase regulatory protein interaction: 4. Exploration of a novel binding pocket.

Structure-activity relationship investigations conducted at the 5-position of the N-pyridine ring of a series of N-arylsulfonyl-N'-2-pyridinyl-piperazines led to the identification of a novel bis-pyridinyl piperazine sulfonamide (51) that was a potent disruptor of the glucokinase-glucokinase regulatory protein (GK-GKRP) interaction. Analysis of the X-ray cocrystal of compound 51 bound to hGKRP revealed that the 3-pyridine ring moiety occupied a previously unexplored binding pocket within the protein. Key features of this new binding mode included forming favorable contacts with the top face of the Ala27-Val28-Pro29 ("shelf region") as well as an edge-to-face interaction with the Tyr24 side chain. Compound 51 was potent in both biochemical and cellular assays (IC50=0.005 µM and EC50=0.205 µM, respectively) and exhibited acceptable pharmacokinetic properties for in vivo evaluation. When administered to db/db mice (100 mg/kg, po), compound 51 demonstrated a robust pharmacodynamic effect and significantly reduced blood glucose levels up to 6 h postdose.

Discovery of Small-Molecule Glucokinase Regulatory Protein Modulators That Restore Glucokinase Activity.

In the nuclei of hepatocytes, glucokinase regulatory protein (GKRP) modulates the activity of glucokinase (GK), a key regulator of glucose homeostasis. Currently, direct activators of GK (GKAs) are in development for the treatment of type 2 diabetes. However, this approach is generally associated with a risk of hypoglycemia. To mitigate such risk, we target the GKRP regulation, which indirectly restores GK activity. Here we describe a screening strategy to look specifically for GKRP modulators, in addition to traditional GKAs. Two high-throughput screening campaigns were performed with our compound libraries using a luminescence assay format, one with GK alone and the other with a GK/GKRP complex in the presence of sorbitol-6-phosphate (S6P). By a subtraction method in the hit triage process of these campaigns, we discovered two close analogs that bind GKRP specifically with sub-µM potency to a site distinct from where fructose-1-phosphate binds. These small molecules are first-in-class allosteric modulators of the GK/GKRP interaction and are fully active even in the presence of S6P. Activation of GK by this particular mechanism, without altering the enzymatic profile, represents a novel pharmacologic modality of intervention in the GK/GKRP pathway.

Nonracemic synthesis of GK-GKRP disruptor AMG-3969.

A nonracemic synthesis of the glucokinase-glucokinase regulatory protein disruptor AMG-3969 (5) is reported. Key features of the synthetic approach are an asymmetric synthesis of the 2-alkynyl piperazine core via a base-promoted isomerization and a revised approach to the synthesis of the aminopyridinesulfonamide with an improved safety profile.

Small molecule disruptors of the glucokinase-glucokinase regulatory protein interaction: 3. Structure-activity relationships within the aryl carbinol region of the N-arylsulfonamido-N'-arylpiperazine series.

We have recently reported a novel approach to increase cytosolic glucokinase (GK) levels through the binding of a small molecule to its endogenous inhibitor, glucokinase regulatory protein (GKRP). These initial investigations culminated in the identification of 2-(4-((2S)-4-((6-amino-3-pyridinyl)sulfonyl)-2-(1-propyn-1-yl)-1-piperazinyl)phenyl)-1,1,1,3,3,3-hexafluoro-2-propanol (1, AMG-3969), a compound that effectively enhanced GK translocation and reduced blood glucose levels in diabetic animals. Herein we report the results of our expanded SAR investigations that focused on modifications to the aryl carbinol group of this series. Guided by the X-ray cocrystal structure of compound 1 bound to hGKRP, we identified several potent GK-GKRP disruptors bearing a diverse set of functionalities in the aryl carbinol region. Among them, sulfoximine and pyridinyl derivatives 24 and 29 possessed excellent potency as well as favorable PK properties. When dosed orally in db/db mice, both compounds significantly lowered fed blood glucose levels (up to 58%).

Small molecule disruptors of the glucokinase-glucokinase regulatory protein interaction: 1. Discovery of a novel tool compound for in vivo proof-of-concept.

Small molecule activators of glucokinase have shown robust efficacy in both preclinical models and humans. However, overactivation of glucokinase (GK) can cause excessive glucose turnover, leading to hypoglycemia. To circumvent this adverse side effect, we chose to modulate GK activity by targeting the endogenous inhibitor of GK, glucokinase regulatory protein (GKRP). Disrupting the GK-GKRP complex results in an increase in the amount of unbound cytosolic GK without altering the inherent kinetics of the enzyme. Herein we report the identification of compounds that efficiently disrupt the GK-GKRP interaction via a previously unknown binding pocket. Using a structure-based approach, the potency of the initial hit was improved to provide 25 (AMG-1694). When dosed in ZDF rats, 25 showed both a robust pharmacodynamic effect as well as a statistically significant reduction in glucose. Additionally, hypoglycemia was not observed in either the hyperglycemic or normal rats.

Small molecule disruptors of the glucokinase-glucokinase regulatory protein interaction: 2. Leveraging structure-based drug design to identify analogues with improved pharmacokinetic profiles.

In the previous report , we described the discovery and optimization of novel small molecule disruptors of the GK-GKRP interaction culminating in the identification of 1 (AMG-1694). Although this analogue possessed excellent in vitro potency and was a useful tool compound in initial proof-of-concept experiments, high metabolic turnover limited its advancement. Guided by a combination of metabolite identification and structure-based design, we have successfully discovered a potent and metabolically stable GK-GKRP disruptor (27, AMG-3969). When administered to db/db mice, this compound demonstrated a robust pharmacodynamic response (GK translocation) as well as statistically significant dose-dependent reductions in fed blood glucose levels.

Metabolism-guided discovery of a potent and orally bioavailable urea-based calcimimetic for the treatment of secondary hyperparathyroidism.

A series of urea based calcimimetics was optimized for potency and oral bioavailability. Crucial to this process was overcoming the poor pharmacokinetic properties of lead thiazole 1. Metabolism-guided modifications, characterized by the use of metabolite identification (ID) and measurement of time dependent inhibition (TDI) of CYP3A4, were essential to finding a compound suitable for oral dosing. Calcimimetic 18 exhibited excellent in vivo potency in a 5/6 nephrectomized rat model and cross-species pharmacokinetics.

Antidiabetic effects of glucokinase regulatory protein small-molecule disruptors.

Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic ß-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.

Design and synthesis of novel amide AKT1 inhibitors with selectivity over CDK2.

Through the analysis of X-ray crystallographic information and previous SAR studies, a novel series of protein kinase B (PKB/AKT) inhibitors was developed. The compounds showed nanomolar inhibition of AKT1 and were selective against cyclin-dependent kinase 2 (CDK2).

From fragment screening to in vivo efficacy: optimization of a series of 2-aminoquinolines as potent inhibitors of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1).

Using fragment-based screening of a focused fragment library, 2-aminoquinoline 1 was identified as an initial hit for BACE1. Further SAR development was supported by X-ray structures of BACE1 cocrystallized with various ligands and molecular modeling studies to expedite the discovery of potent compounds. These strategies enabled us to integrate the C-3 side chain on 2-aminoquinoline 1 extending deep into the P2' binding pocket of BACE1 and enhancing the ligand's potency. We were able to improve the BACE1 potency to subnanomolar range, over 10(6)-fold more potent than the initial hit (900 µM). Further elaboration of the physical properties of the lead compounds to those more consistent with good blood-brain barrier permeability led to inhibitors with greatly improved cellular activity and permeability. Compound 59 showed an IC(50) value of 11 nM on BACE1 and cellular activity of 80 nM. This compound was advanced into rat pharmacokinetic and pharmacodynamic studies and demonstrated significant reduction of Aß levels in cerebrospinal fluid (CSF).

Discovery of a calcimimetic with differential effects on parathyroid hormone and calcitonin secretion.

Calcimimetics are positive allosteric modulators to the calcium-sensing receptor (CaSR). Activation of the CaSR inhibits the secretion of parathyroid hormone (PTH), stimulates the secretion of calcitonin, and decreases serum calcium (Ca(2+)). Cinacalcet, a second-generation calcimimetic, is used therapeutically to control PTH in patients with chronic kidney disease who are on dialysis with secondary hyperparathyroidism. A calcimimetic that displays increased separation of PTH versus Ca(2+) lowering in patients would potentially allow the use of calcimimetics to treat patients in earlier stages of renal disease because hypocalcemia can develop in this population. Toward this end, we developed a third-generation calcimimetic, determined the molecular pharmacological properties of it using an operation model of allosteric modulation/agonism, and measured the compound effects on PTH, serum ionized Ca(2+), and calcitonin levels in 5/6 nephrectomized rats. We found the new molecule effectively reduced PTH levels without promoting calcitonin secretion or hypocalcemia. Furthermore, our third-generation molecule was less efficacious at promoting calcitonin secretion from human thyroid carcinoma cells compared with 3-(2-chlorophenyl)-N-((1R)-1-(3-methoxyphenyl)ethyl)-1-propanamine (R-568), a first-generation calcimimetic. These data provide evidence that calcimimetics with increased potency can be used to lower PTH without production of significant hypocalcemia because the threshold for inhibition of PTH secretion is much lower than the threshold for calcitonin secretion.

Synthesis and Evaluation of the Metabolites of AMG 221, a Clinical Candidate for the Treatment of Type 2 Diabetes.

All eight of the major active metabolites of (S)-2-((1S,2S,4R)-bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one (AMG 221, compound 1), an inhibitor of 11ß-hydroxysteroid dehydrogenase type 1 that has entered the clinic for the treatment of type 2 diabetes, were synthetically prepared and confirmed by comparison with samples generated in liver microsomes. After further profiling, we determined that metabolite 2 was equipotent to 1 on human 11ß-HSD1 and had lower in vivo clearance and higher bioavailability in rat and mouse. Compound 2 was advanced into a pharmacodynamic model in mouse where it inhibited adipose 11ß-HSD1 activity.

The discovery of an orally efficacious positive allosteric modulator of the calcium sensing receptor containing a dibenzylamine core.

The discovery of a series of novel and orally efficacious type II calcimimetics, developed from the lead compound 1, is described herein. Compound 22 suppressed plasma PTH levels relative to vehicle when dosed orally in a rat pharmacodynamic model.

Discovery of a potent, orally active 11beta-hydroxysteroid dehydrogenase type 1 inhibitor for clinical study: identification of (S)-2-((1S,2S,4R)-bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one (AMG 221).

Thiazolones with an exo-norbornylamine at the 2-position and an isopropyl group on the 5-position are potent 11beta-HSD1 inhibitors. However, the C-5 center was prone to epimerization in vitro and in vivo, forming a less potent diastereomer. A methyl group was added to the C-5 position to eliminate epimerization, leading to the discovery of (S)-2-((1S,2S,4R)-bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one (AMG 221). This compound decreased fed blood glucose and insulin levels and reduced body weight in diet-induced obesity mice.