Hector Rodriguez

Allosteric inhibition of lysyl oxidase-like-2 impedes the development of a pathologic microenvironment

We have identified a new role for the matrix enzyme lysyl oxidase–like-2 (LOXL2) in the creation and maintenance of the pathologic microenvironment of cancer and fibrotic disease. Our analysis of biopsies from human tumors and fibrotic lung and liver tissues revealed an increase in LOXL2 in disease-associated stroma and limited expression in healthy tissues. Targeting LOXL2 with an inhibitory monoclonal antibody (AB0023) was efficacious in both primary and metastatic xenograft models of cancer, as well as in liver and lung fibrosis models. Inhibition of LOXL2 resulted in a marked reduction in activated fibroblasts, desmoplasia and endothelial cells, decreased production of growth factors and cytokines and decreased transforming growth factor-β (TGF-β) pathway signaling. AB0023 outperformed the small-molecule lysyl oxidase inhibitor β-aminoproprionitrile. The efficacy and safety of LOXL2-specific AB0023 represents a new therapeutic approach with broad applicability in oncologic and fibrotic diseases.

Cardiac Myosin Activation: A Potential Therapeutic Approach for Systolic Heart Failure

A small molecule improves cardiac function by accelerating the transition of myosin into a force-producing state. Decreased cardiac contractility is a central feature of systolic heart failure. Existing drugs increase cardiac contractility indirectly through signaling cascades but are limited by their mechanism-related adverse effects. To avoid these limitations, we previously developed omecamtiv mecarbil, a small-molecule, direct activator of cardiac myosin. Here, we show that it binds to the myosin catalytic domain and operates by an allosteric mechanism to increase the transition rate of myosin into the strongly actin-bound force-generating state. Paradoxically, it inhibits adenosine 5′-triphosphate turnover in the absence of actin, which suggests that it stabilizes an actin-bound conformation of myosin. In animal models, omecamtiv mecarbil increases cardiac function by increasing the duration of ejection without changing the rates of contraction. Cardiac myosin activation may provide a new therapeutic approach for systolic heart failure.

A small-molecule inhibitor of sarcomere contractility suppresses hypertrophic cardiomyopathy in mice.

Powering down yields a healthier heart In hypertrophic cardiomyopathy (HCM), the heart muscle enlarges and becomes progressively less efficient at pumping blood. HCM can be caused by mutations in components of the sarcomere (the hearts contractile unit), most notably myosin. Hypercontractility is among the earliest heart disturbances seen in mice carrying these myosin mutations, implying that the mutations inflict their damage by increasing myosins power production. Green et al. identified a small molecule that binds to myosin and inhibits its activity (see the Perspective by Warshaw). When orally administered to young mice, the molecule prevented the development of several hallmark features of HCM without adversely affecting skeletal muscle. Science, this issue p. 617; see also p. 556 A small molecule that reduces cardiac muscle contraction prevents a certain type of heart disease in mice. [Also see Perspective by Warshaw] Hypertrophic cardiomyopathy (HCM) is an inherited disease of heart muscle that can be caused by mutations in sarcomere proteins. Clinical diagnosis depends on an abnormal thickening of the heart, but the earliest signs of disease are hyperdynamic contraction and impaired relaxation. Whereas some in vitro studies of power generation by mutant and wild-type sarcomere proteins are consistent with mutant sarcomeres exhibiting enhanced contractile power, others are not. We identified a small molecule, MYK-461, that reduces contractility by decreasing the adenosine triphosphatase activity of the cardiac myosin heavy chain. Here we demonstrate that early, chronic administration of MYK-461 suppresses the development of ventricular hypertrophy, cardiomyocyte disarray, and myocardial fibrosis and attenuates hypertrophic and profibrotic gene expression in mice harboring heterozygous human mutations in the myosin heavy chain. These data indicate that hyperdynamic contraction is essential for HCM pathobiology and that inhibitors of sarcomere contraction may be a valuable therapeutic approach for HCM.

Modulation of Lysyl Oxidase-like 2 Enzymatic Activity by an Allosteric Antibody Inhibitor

In this report, we assessed the steady-state enzymatic activity of lysyl oxidase-like 2 (LOXL2) against the substrates 1,5-diaminopentane (DAP), spermine, and fibrillar type I collagen. We find that both DAP and spermine are capable of activating LOXL2 to the same extent and have similar Michaelis constants (Km ∼ 1 mm) and catalytic rates (kcat ∼ 0.02 s−1). We also show that LOXL2 is capable of being inhibited by a known suicide inhibitor of lysyl oxidase (LOX), β-aminopropionitrile, which we find is a potent inhibitor of LOXL2 activity. The modality of inhibition of β-aminopropionitrile was also examined and found to be competitive with respect to the substrates DAP and spermine. In addition, we identified an antibody inhibitor (AB0023) of LOXL2 enzymatic function and have found that the inhibition occurs in a non-competitive manner with respect to both spermine and DAP. The binding epitope of AB0023 was mapped to the scavenger receptor cysteine-rich domain four of human LOXL2. AB0023 binds to a region remote from the catalytic domain making AB0023 an allosteric inhibitor of LOXL2. This affords AB0023 several advantages, because it is specific for LOXL2 and inhibits the enzymatic function of LOXL2 in a non-competitive manner thereby allowing inhibition of LOXL2 regardless of substrate concentration. These results suggest that antibody allosteric modulators of enzymatic function represent a novel drug development strategy and, in the context of LOXL2, suggest that inhibitors such as these might be useful therapeutics in oncology, fibrosis, and inflammation.

Activation of fast skeletal muscle troponin as a potential therapeutic approach for treating neuromuscular diseases

Limited neural input results in muscle weakness in neuromuscular disease because of a reduction in the density of muscle innervation, the rate of neuromuscular junction activation or the efficiency of synaptic transmission. We developed a small-molecule fast-skeletal–troponin activator, CK-2017357, as a means to increase muscle strength by amplifying the response of muscle when neural input is otherwise diminished secondary to neuromuscular disease. Binding selectively to the fast-skeletal–troponin complex, CK-2017357 slows the rate of calcium release from troponin C and sensitizes muscle to calcium. As a consequence, the force-calcium relationship of muscle fibers shifts leftwards, as does the force-frequency relationship of a nerve-muscle pair, so that CK-2017357 increases the production of muscle force in situ at sub-maximal nerve stimulation rates. Notably, we show that sensitization of the fast-skeletal–troponin complex to calcium improves muscle force and grip strength immediately after administration of single doses of CK-2017357 in a model of the neuromuscular disease myasthenia gravis. Troponin activation may provide a new therapeutic approach to improve physical activity in diseases where neuromuscular function is compromised.

Discovery of Omecamtiv Mecarbil the First, Selective, Small Molecule Activator of Cardiac Myosin

We report the design, synthesis, and optimization of the first, selective activators of cardiac myosin. Starting with a poorly soluble, nitro-aromatic hit compound (1), potent, selective, and soluble myosin activators were designed culminating in the discovery of omecamtiv mecarbil (24). Compound 24 is currently in clinical trials for the treatment of systolic heart failure.

A small molecule modulator of cardiac myosin acts on multiple stages of the myosin chemomechanical cycle

Mavacamten, formerly known as MYK-461 is a recently discovered novel small-molecule modulator of cardiac myosin that targets the underlying sarcomere hypercontractility of hypertrophic cardiomyopathy, one of the most prevalent heritable cardiovascular disorders. Studies on isolated cells and muscle fibers as well as intact animals have shown that mavacamten inhibits sarcomere force production, thereby reducing cardiac contractility. Initial mechanistic studies have suggested that mavacamten primarily reduces the steady-state ATPase activity by inhibiting the rate of phosphate release of β-cardiac myosin-S1, but the molecular mechanism of action of mavacamten has not been described. Here we used steady-state and presteady-state kinetic analyses to investigate the mechanism of action of mavacamten. Transient kinetic analyses revealed that mavacamten modulates multiple steps of the myosin chemomechanical cycle. In addition to decreasing the rate-limiting step of the cycle (phosphate release), mavacamten reduced the number of myosin-S1 heads that can interact with the actin thin filament during transition from the weakly to the strongly bound state without affecting the intrinsic rate. Mavacamten also decreased the rate of myosin binding to actin in the ADP-bound state and the ADP-release rate from myosin-S1 alone. We, therefore, conclude that mavacamten acts on multiple stages of the myosin chemomechanical cycle. Although the primary mechanism of mavacamten-mediated inhibition of cardiac myosin is the decrease of phosphate release from β-cardiac myosin-S1, a secondary mechanism decreases the number of actin-binding heads transitioning from the weakly to the strongly bound state, which occurs before phosphate release and may provide an additional method to modulate myosin function.

Abstract 5604: Preclinical antitumor activity of novel small molecule glutaminase inhibitors in triple-negative breast cancer.

Many tumor cells are dependent on glutamine (Gln) and Gln-derived metabolites to meet bioenergetic and biosynthetic demands. A key cellular reaction in the utilization of Gln is its deamidation by the enzyme glutaminase to yield glutamate (Glu). We have developed a series of potent and selective small molecule glutaminase inhibitors for evaluation as novel cancer therapeutics. These inhibitors exhibit 5-20 nM potency against the broadly-expressed form of glutaminase (GLS) with minimal activity against the liver form of the enzyme (GLS2). Expression analysis (see below) has identified triple-negative breast cancer (TNBC) as a potential clinical target population for GLS inhibitors. TNBC is a poor prognosis breast cancer subtype lacking estrogen receptor (ER), progesterone receptor (PR) and the growth factor receptor Her2. TNBC is insensitive to approved targeted therapies (ER antagonists and anti-Her2 agents) and is currently treated with conventional cytotoxic drugs. Therefore, TNBC represents a critical unmet medical need for which new targeted therapeutics are urgently needed. Analysis of a primary breast tumor mRNA expression dataset (The Cancer Genome Atlas; n=756) revealed that low ER, PR, and Her2 expression is associated with high GLS expression and low expression of both GLS2 and glutamine synthetase, an enzyme that opposes the action of glutaminase by synthesizing Gln from Glu. This expression pattern, together with published work on the Gln dependence of breast tumor cell lines [Kung et al. (2011) PLOS Genet 7:e1002229] suggests that TNBC may be particularly dependent on GLS. To test this hypothesis, we evaluated the anti-tumor activity of our GLS inhibitors on a panel of breast tumor-derived cell lines (n>25) that included a mixture of TNBC and ER-positive subtypes. The TNBC subtype displayed the greatest sensitivity to GLS inhibitor treatment (IC50s ranging from 5-100 nM) and this sensitivity correlated with a dependence on extracellular Gln for cell growth. In the TNBC cell line MDA-MB-231 the antiproliferative effect of the GLS inhibitor was associated with a dose-dependent accumulation of Gln and depletion of Glu. Additionally, GLS inhibition showed additive in vitro activity in combination with paclitaxel, a standard-of-care treatment in TNBC. In a mouse orthotopic tumor xenograft model with MDA-MB-231 cells implanted in the mammary fat pad, oral delivery of a GLS inhibitor caused an accumulation of tumor Gln, a reduction in tumor Glu, and enhanced the anti-tumor efficacy of the paclitaxel. Experiments aimed at expanding this observation with other TNBC xenograft models are in progress. Overall, these results demonstrate that a selective inhibitor of GLS, either as a single agent or in combination with standard-of-care chemotherapeutics, may be effective as a targeted therapeutic in TNBC. Citation Format: Susan Demo, Tania Chernov-Rogan, Matthew Gross, Julie Janes, Raja Kawas, Evan Lewis, Francesco Parlati, Hector Rodriguez, Mirna Rodriguez, Jinfu Yang, Frances Zhao, Adam Richardson, Mark K. Bennett. Preclinical antitumor activity of novel small molecule glutaminase inhibitors in triple-negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5604. doi:10.1158/1538-7445.AM2013-5604

Abstract 1877: Anti-tumor activity of novel, potent, selective and orally-bioavailable glutaminase inhibitors.

Glutamine is required for the growth of a broad range of tumor cells. An important step in the metabolism of glutamine is its conversion to glutamate, which is catalyzed by the mitochondrial enzyme glutaminase. GLS, the form of the enzyme expressed in most cells, is up-regulated in a sub-set of glutamine-requiring tumor cells. Suppression of GLS by genetic knockdown or with small molecule inhibitors slows the growth of these tumor cells. To further evaluate GLS as an oncology therapeutic target, we have developed a series of novel and potent GLS inhibitors that are orally bioavailable, permitting both in vitro and in vivo analysis of GLS inhibition. In a reconstituted system with purified enzyme, the inhibitor CB-498 inhibited GLS with an IC50 of CB-839 is an orally bioavailable analog of CB-498 with comparable biochemical and cellular potency but with improved solubility and pharmacokinetic properties. In an H2122 lung adenocarcinoma xenograft model, CB-839 showed single-agent anti-tumor activity. This anti-tumor activity was associated with increased tumor glutamine levels and decreased levels of glutamate and aspartate. Importantly, these glutamate and aspartate decreases were not seen in kidney, liver, or plasma, indicating a tumor specific response to GLS inhibition. Oral administration of CB-839 resulted in significant plasma exposure across multiple species and has been well tolerated in in vivo studies in rodents on either daily or twice-daily dosing schedules. These results motivate a further investigation of these potent and selective GLS inhibitors for clinical utility in oncology. Citation Format: Francesco Parlati, Tania Chernov-Rogan, Susan Demo, Matthew Gross, Julie Janes, Raja Kawas, Evan Lewis, Hector Rodriguez, Mirna Rodriguez, Jinfu Yang, Frances Zhao, Adam Richardson, Mark K. Bennett. Anti-tumor activity of novel, potent, selective and orally-bioavailable glutaminase inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1877. doi:10.1158/1538-7445.AM2013-1877