Geraldine Harriman

Inhibition of acetyl-CoA carboxylase suppresses fatty acid synthesis and tumor growth of non-small-cell lung cancer in preclinical models

Continuous de novo fatty acid synthesis is a common feature of cancer that is required to meet the biosynthetic demands of a growing tumor. This process is controlled by the rate-limiting enzyme acetyl-CoA carboxylase (ACC), an attractive but traditionally intractable drug target. Here we provide genetic and pharmacological evidence that in preclinical models ACC is required to maintain the de novo fatty acid synthesis needed for growth and viability of non-small-cell lung cancer (NSCLC) cells. We describe the ability of ND-646—an allosteric inhibitor of the ACC enzymes ACC1 and ACC2 that prevents ACC subunit dimerization—to suppress fatty acid synthesis in vitro and in vivo. Chronic ND-646 treatment of xenograft and genetically engineered mouse models of NSCLC inhibited tumor growth. When administered as a single agent or in combination with the standard-of-care drug carboplatin, ND-646 markedly suppressed lung tumor growth in the Kras;Trp53−/− (also known as KRAS p53) and Kras;Stk11−/− (also known as KRAS Lkb1) mouse models of NSCLC. These findings demonstrate that ACC mediates a metabolic liability of NSCLC and that ACC inhibition by ND-646 is detrimental to NSCLC growth, supporting further examination of the use of ACC inhibitors in oncology.

Acetyl-CoA carboxylase inhibition by ND-630 reduces hepatic steatosis, improves insulin sensitivity, and modulates dyslipidemia in rats

Significance Using structure-based drug design, we have identified a series of potent allosteric protein–protein interaction acetyl-CoA carboxylase inhibitors, exemplified by ND-630, that interact within the acetyl-CoA carboxylase subunit phosphopeptide acceptor and dimerization site to prevent dimerization and inhibit enzymatic activity. ND-630 reduces fatty acid synthesis and stimulates fatty acid oxidation in cultured cells and experimental animals, reduces hepatic steatosis, improves insulin sensitivity, reduces weight gain without affecting food intake, and favorably affects dyslipidemia in diet-induced obese rats and reduces hepatic steatosis, improves glucose-stimulated insulin secretion, and reduces hemoglobin A1c in Zucker diabetic fatty rats. These data suggest that ND-630 may be useful in treating a variety of metabolic disorders, including metabolic syndrome, type 2 diabetes, and fatty liver disease. Simultaneous inhibition of the acetyl-CoA carboxylase (ACC) isozymes ACC1 and ACC2 results in concomitant inhibition of fatty acid synthesis and stimulation of fatty acid oxidation and may favorably affect the morbidity and mortality associated with obesity, diabetes, and fatty liver disease. Using structure-based drug design, we have identified a series of potent allosteric protein–protein interaction inhibitors, exemplified by ND-630, that interact within the ACC phosphopeptide acceptor and dimerization site to prevent dimerization and inhibit the enzymatic activity of both ACC isozymes, reduce fatty acid synthesis and stimulate fatty acid oxidation in cultured cells and in animals, and exhibit favorable drug-like properties. When administered chronically to rats with diet-induced obesity, ND-630 reduces hepatic steatosis, improves insulin sensitivity, reduces weight gain without affecting food intake, and favorably affects dyslipidemia. When administered chronically to Zucker diabetic fatty rats, ND-630 reduces hepatic steatosis, improves glucose-stimulated insulin secretion, and reduces hemoglobin A1c (0.9% reduction). Together, these data suggest that ACC inhibition by representatives of this series may be useful in treating a variety of metabolic disorders, including metabolic syndrome, type 2 diabetes mellitus, and fatty liver disease.

Accelerating drug discovery through tight integration of expert molecular design and predictive scoring

Modeling protein-ligand interactions has been a central goal of computational chemistry for many years. We here review recent progress toward this goal, and highlight the role free energy calculation methods and computational solvent analysis techniques are now having in drug discovery. We further describe recent use of these methodologies to advance two separate drug discovery programs targeting acetyl-CoA carboxylase and tyrosine kinase 2. These examples suggest that tight integration of sophisticated chemistry teams with state-of-the-art computational methods can dramatically improve the efficiency of small molecule drug discovery.

Acetyl‐coenzyme A carboxylase inhibition reduces de novo lipogenesis in overweight male subjects: A randomized, double‐blind, crossover study

NDI‐010976, an allosteric inhibitor of acetyl‐coenzyme A carboxylases (ACC) ACC1 and ACC2, reduces hepatic de novo lipogenesis (DNL) and favorably affects steatosis, inflammation, and fibrosis in animal models of fatty liver disease. This study was a randomized, double‐blind, placebo‐controlled, crossover trial evaluating the pharmacodynamic effects of a single oral dose of NDI‐010976 on hepatic DNL in overweight and/or obese but otherwise healthy adult male subjects. Subjects were randomized to receive either NDI‐010976 (20, 50, or 200 mg) or matching placebo in period 1, followed by the alternate treatment in period 2; and hepatic lipogenesis was stimulated with oral fructose administration. Fractional DNL was quantified by infusing a stable isotope tracer, [1‐13C]acetate, and monitoring 13C incorporation into palmitate of circulating very low‐density lipoprotein triglyceride. Single‐dose administration of NDI‐010976 was well tolerated at doses up to and including 200 mg. Fructose administration over a 10‐hour period stimulated hepatic fractional DNL an average of 30.9 ± 6.7% (mean ± standard deviation) above fasting DNL values in placebo‐treated subjects. Subjects administered single doses of NDI‐010976 at 20, 50, or 200 mg had significant inhibition of DNL compared to placebo (mean inhibition relative to placebo was 70%, 85%, and 104%, respectively). An inverse relationship between fractional DNL and NDI‐010976 exposure was observed with >90% inhibition of fractional DNL associated with plasma concentrations of NDI‐010976 >4 ng/mL. Conclusion: ACC inhibition with a single dose of NDI‐010976 is well tolerated and results in a profound dose‐dependent inhibition of hepatic DNL in overweight adult male subjects. Therefore, NDI‐010976 could contribute considerable value to the treatment algorithm of metabolic disorders characterized by dysregulated fatty acid metabolism, including nonalcoholic steatohepatitis. (Hepatology 2017;66:324–334).

Inhibition of Acetyl-CoA Carboxylase by Phosphorylation or the Inhibitor ND-654 Suppresses Lipogenesis and Hepatocellular Carcinoma

The incidence of hepatocellular carcinoma (HCC) is rapidly increasing due to the prevalence of obesity and non-alcoholic fatty liver disease, but the molecular triggers that initiate disease development are not fully understood. We demonstrate that mice with targeted loss-of-function point mutations within the AMP-activated protein kinase (AMPK) phosphorylation sites on acetyl-CoA carboxylase 1 (ACC1 Ser79Ala) and ACC2 (ACC2 Ser212Ala) have increased liver de novo lipogenesis (DNL) and liver lesions. The same mutation in ACC1 also increases DNL and proliferation in human liver cancer cells. Consistent with these findings, a novel, liver-specific ACC inhibitor (ND-654) that mimics the effects of ACC phosphorylation inhibits hepatic DNL and the development of HCC, improving survival of tumor-bearing rats when used alone and in combination with the multi-kinase inhibitor sorafenib. These studies highlight the importance of DNL and dysregulation of AMPK-mediated ACC phosphorylation in accelerating HCC and the potential of ACC inhibitors for treatment.

Abstract 1048: Modulation of lipid metabolism through inhibition of acetyl-CoA carboxylase with ND-646 leads to potent inhibition of breast cancer cell growth in vitro and in vivo

Metabolic attenuation is a promising approach to cancer therapy and rate-limiting steps in key biosynthetic pathways are particularly attractive targets. Many cancer types are dependent on fatty acid synthesis as a primary source of energy and for providing lipids for expansion of cell and nuclear membranes in rapidly proliferating cells. The rate-limiting enzyme in fatty acid synthesis, acetyl-CoA carboxylase (ACC), has been shown to be highly expressed in human breast cancer. ACC is thought to be critical for the growth and survival of cancer cells, especially within a tumor microenvironment where exogenous fatty acids might be limited. Effective therapeutic options for triple negative breast cancer are limited and identification of robust targeted agents without overt toxicity for this indication are especially needed. Dual inhibition of the ACC isozymes, ACC1 and ACC2, results in concomitant inhibition of fatty acid synthesis and stimulation of fatty acid oxidation. We have identified ND-646, a potent, selective, allosteric inhibitor of ACC with broad tissue distribution that binds to the ACC biotin carboxylase domain and potently inhibits the dimerization and enzymatic activity of both ACC1 (IC50 = 3.5nM) and ACC2 (IC50 = 4.1nM). Profiling the potency of ND-646 in vitro in a panel of breast cancer cell lines including triple negative and BRCA1 mutant cell lines demonstrated potent inhibition of cell proliferation with IC50s Citation Format: Jennifer L. Rocnik, Wenyan Miao, Geraldine Harriman, Jeremy Greenwood, Sathesh Bhat, H. James Harwood, Rosana Kapeller, William F. Westlin. Modulation of lipid metabolism through inhibition of acetyl-CoA carboxylase with ND-646 leads to potent inhibition of breast cancer cell growth in vitro and in vivo . [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1048.

Abstract 3781: Combination therapy with a liver selective acetyl-CoA carboxylase inhibitor ND-654 and sorafenib improves efficacy in the treatment of cirrhotic rats with hepatocellular carcinoma

Background: Hepatocellular carcinoma (HCC) is increasing in incidence worldwide. Current treatment options for HCC are limited, and as such, prognosis is extremely poor with a 5-year survival less than 12%. Sorafenib is the only FDA-approved drug for the treatment of HCC but its effects are marginal as it only extends survival by a few months. Therefore, new treatment options are urgently needed. Metabolic attenuation is a promising approach to cancer therapy, and rate-limiting steps in key biosynthetic pathways are particularly attractive targets. We recently identified ND-654, a hepatoselective (∼3000:1 liver to muscle exposure), allosteric ACC inhibitor that binds to the ACC subunit dimerization site and inhibits the enzymatic activity of both ACC1 (IC50 = 3 nM) and ACC2 (IC50 = 8 nM). Daily oral administration of 10 mg/kg ND-654 reduced tumor incidence by 55% and significantly improved median survival time in a rat model of cirrhosis and HCC. Here, we examine the effects of ND-654 alone and in combination with sorafenib on HCC development in cirrhotic rats. Methods: Male Wistar rats were treated once weekly with 50 mg/kg diethylnitrosamine (DEN) for 18 weeks to induce sequential development of fibrosis, cirrhosis and HCC. After establishment of cirrhosis and when HCCs are first developing (13 weeks), rats were treated daily by oral gavage with either 1) vehicle control, 2) 10 mg/kg ND-654, 3) 10 mg/kg sorafenib, or 4) 10 mg/kg ND-654 and 10 mg/kg sorafenib. After 18 weeks, tumor nodules were counted and liver and tumor tissue was harvested for analysis. Results: Similar to our previous study, simultaneous inhibition of ACC1 and ACC2 significantly reduced HCC incidence by 41% (p Citation Format: Lan Wei, Geraldine Harriman, Sarani Ghoshal, Omeed Moaven, Jeremy Greenwood, Sathesh Bhat, William F. Westlin, H. James Harwood, Rosana Kapeller, Kenneth K. Tanabe, Bryan C. Fuchs. Combination therapy with a liver selective acetyl-CoA carboxylase inhibitor ND-654 and sorafenib improves efficacy in the treatment of cirrhotic rats with hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3781.

Abstract 2679: Acetyl-CoA carboxylase inhibition by ND646 reduces fatty acid synthesis and inhibits cell proliferation in human non-small cell lung cancer cells

Continuous de novo lipogenesis is a common feature of tumor cells and is required to meet the bioenergetic and biosynthetic demands of a growing tumor. The expression and activity of the fatty acid synthetic enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase are up-regulated in many types of cancer, where lipogenesis is essential for proliferation and tumor cell survival. These enzymes are therefore attractive targets for anti-neoplastic intervention. Inhibition of ACC results in inhibition of fatty acid synthesis (FASyn) and stimulation of fatty acid oxidation in cultured cells and in animals and has been shown to reduce cancer cell growth in vitro. Our efforts to develop novel ACC inhibitors have focused on the subunit dimerization site of the biotin carboxylase (BC) domain of the enzyme. This site also binds the inhibitory phosphopeptide of ACC phosphorylated by AMPK and the fungal metabolite Soraphen A, both of which suppress dimerization and inhibit enzymatic activity. Using state-of-the-art structure-based drug design and crystal structures of human ACC2 BC domain, we have identified a unique series of allosteric inhibitors with low nanomolar potency, represented by ND646, that bind to the Soraphen binding site, exhibit potent and selective activity in vitro and in vivo and show anti-neoplastic properties in cultured human non-small cell lung cancer (NSCLC) cells. The anti-proliferative action of ND646 in NSCLC cell lines A549, H460, and H358 was enhanced in delipidated media and attenuated by supplementation with palmitic acid, suggesting that the anti-proliferative effect of ND646 is induced by depletion of cellular fatty acids. Current efforts are focused on determining if the cell growth inhibitory actions of ND646 also possess apoptotic properties and on assessing the anti-neoplastic activity of ND646 in preclinical models of lung cancer. During the course of these studies, we also discovered that interaction of ND646 with the ACC phosphopeptide binding site markedly reduced the phosphorylation state of the enzyme in A549 cells, as determined by western blot analysis using anti-phosphoACC (pACC) antibody. Furthermore, in HepG2 cells, the reduction in pACC by the related analog ND630 (EC50 = 66 nM), which closely paralleled inhibition of FASyn (EC50 = 42 nM), resulted in complete loss of pACC at high doses. These observations, that were corroborated in hepatic tissue isolated from both ND646 and ND630 treated mice and in situ in tumors of genetically engineered mouse models of lung cancer dosed with ND646, suggest that assessment of pACC could provide a sensitive biomarker of target engagement for ACC inhibitors interacting in this region of the enzyme. Additional efforts are focused on examining the genetic contexts and optimal therapeutic combinations such that ACC inhibitors may find the greatest clinical utility as anti-neoplastic agents. Citation Format: Robert Svensson, Geraldine Harriman, Jeremy Greenwood, Sathesh Bhat, H.James Harwood, Rosana Kapeller, Reuben Shaw. Acetyl-CoA carboxylase inhibition by ND646 reduces fatty acid synthesis and inhibits cell proliferation in human non-small cell lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2679. doi:10.1158/1538-7445.AM2014-2679

Abstract 1427: Liver selective Acetyl-CoA Carboxylase inhibition by ND-654 decreases hepatocellular carcinoma development in cirrhotic rats

Background: Hepatocellular carcinoma (HCC) is increasing in incidence worldwide. This increase is in part due to the obesity epidemic which is an independent risk factor for both nonalcoholic fatty liver disease (NAFLD) and HCC. In fact, recent evidence suggests that enhanced de novo lipogenesis is a critical mediator for HCC development. Current treatment options for HCC are limited, and as such, prognosis is extremely poor with a 5-year survival less than 12%. Therefore, there is an urgent medical need for new therapeutic strategies. Simultaneous inhibition of the acetyl-CoA carboxylase isozymes, ACC1 and ACC2, results in concomitant inhibition of fatty acid synthesis (FASyn) and stimulation of fatty acid oxidation (FAOxn) in cultured cells and in animals, reduces hepatic steatosis, and impedes tumor cell growth in vitro. Here, we test the hypothesis that simultaneous reduction of hepatic FASyn through inhibition of ACC1 and stimulation of hepatic FAOxn through inhibition ACC2 is a potential therapeutic target for HCC. Results: Using state-of-the-art structure-based drug design and crystal structures of the human ACC2 biotin carboxylase domain, we identified ND-654, a hepatoselective (2700:5:1 liver to plasma to muscle exposure), allosteric inhibitor that binds to the ACC subunit dimerization site, inhibits the enzymatic activity of both ACC1 (IC50 = 3 nM) and ACC2 (IC50 = 8 nM), and inhibits FASyn in HepG2 cells (IC50 = 14 nM) and in rats (ED50 = 0.3 mg/kg). Male Wistar rats were treated weekly with 50 mg/kg diethylnitrosamine (DEN) to induce sequential development of fibrosis, cirrhosis and HCC. After establishment of cirrhosis (13 wks), rats were treated daily by oral gavage with either vehicle or 10 mg/kg ND-654. At the end of the study (18 wks), rats were sacrificed and tumor nodules were counted. Liver tissue and plasma were also analyzed to assess the effects of ND-654 on disease progression. When measured four hours after the final dose, ND-654 showed preferential liver exposure, with steady state levels reaching 3.7±1.8 µM in cirrhotic tissue and 2.2±1.8 µM in tumor tissue. ND-654 did not alter body weight or food consumption, but reduced serum triglyceride levels by 35%. Importantly, ND-654 significantly (p Citation Format: Danielle K. DePeralta, Lan Wei, Geraldine Harriman, Jeremy Greenwood, Sathesh Bhat, William Westlin, H. James Harwood, Rosana Kapeller, Kenneth K. Tanabe, Bryan C. Fuchs. Liver selective Acetyl-CoA Carboxylase inhibition by ND-654 decreases hepatocellular carcinoma development in cirrhotic rats. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1427. doi:10.1158/1538-7445.AM2014-1427