Vered Behar

1-(sulfonyl)-5-(arylsulfonyl)indoline as activators of the tumor cell specific M2 isoform of pyruvate kinase

Cancer cells preferentially use glycolysis rather than oxidative phosphorylation for their rapid growth. They consume large amount of glucose to produce lactate even when oxygen is abundant, a phenomenon known as the Warburg effect. This metabolic change originates from a shift in the expression of alternative spliced isoforms of the glycolytic enzyme pyruvate kinase (PK), from PKM1 to PKM2. While PKM1 is constitutively active, PKM2 is switched from an inactive dimer form to an active tetramer form by small molecule activators. The prevalence of PKM2 in cancer cells relative to the prevalence of PKM1 in many normal cells, suggests a therapeutic strategy whereby activation of PKM2 may counter the abnormal cellular metabolism in cancer cells, and consequently decreased cellular proliferation. Herein we describe the discovery and optimization of a series of PKM2 activators derived from the 2-((2,3-dihydrobenzo[b][1,4] dioxin-6-yl)thio)-1-(2-methyl-1-(methylsulfonyl)indolin-5-yl) ethanone scaffold. The synthesis, SAR analysis, enzyme active site docking, enzymatic reaction kinetics, selectivity and pharmaceutical properties are discussed.

Abstract 4065: Modulation of cancer metabolism with novel PKM2 activators exerts anti-tumor activity

Cancer cells adapt altered metabolism to enable efficient conversion of glucose into biomass needed for massive cell growth and proliferation. The metabolic switch takes place even when oxygen is abundant, a phenomenon known as the Warburg effect. A key mediator of this effect is pyruvate kinase type M2 (PKM2), a rate-limiting enzyme, which catalyzes the last step in glycolysis, converting PEP and ADP to pyruvate and ATP. PKM2 switches between a highly active tetrameric form and low activity monomeric or dimeric forms, following binding to its natural allosteric activator FBP (an upstream glycolytic intermediate). Our goal was to identify novel small molecule PKM2 activators that can be used as potential anti-cancer therapeutics. Using our proprietary structure-based screening tools, we identified several novel, potent, allosteric PKM2 activators, with AC50s as low as 50nM and 150% activation levels, similar to the natural activator FBP (AC50=116nM, 150% activation). Kinetics studies showed that these compounds significantly improve the rate and efficiency of the enzymatic reaction to the same level as FBP (a 4-fold decrease in KM, and a 2-fold increase in VMAX). Furthermore, a greater increase in VMAX (3-fold) is observed when combining these compounds with FBP. These small-molecule activators induce the formation of the (active) tetrameric form of PKM2, increasing the PKM2 tetramer:monomer ratio by more than 10-fold, quantitatively similar to the action of FBP. In cellular assays, these compounds show significant reduction in the proliferation rate of cancer cell lines, including colorectal, lung and hepatic carcinomas, in a dose- and time-dependent manner (up to 80% proliferation inhibition). In line with the ability of these novel compounds to re-direct cellular metabolism from aerobic glycolysis to oxidative phosphorylation, we found that inhibition of oxidative phosphorylation with oligomycin extends the anti proliferative effect of our compounds. When used in combination with commonly used chemotherapeutic agents, such as 5FU in colorectal cancer cells, they show an additive effect and yielded close to 100% inhibition of cell growth. Our compounds demonstrate excellent in vitro and in vivo DMPK profiles, including metabolic stability, excellent permeability with no efflux (CaCO-2 PApp=20.5 106/cm·s-1), and in vivo mouse pharmacokinetics with 20% oral bioavailability, good half life PO and volume of distribution. Several animal efficacy studies, as mono-therapy and in combination with chemotherapy or specific kinase inhibitors, are ongoing. Taken together, we present a new class of potent PKM2 activators that modulate the metabolism within cancer cells and show a promising anti cancer therapeutics potential. The favorable DMPK profile of these compounds suggests that they can be further developed either as mono-therapy or in combination with targeted therapeutics or chemo-therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4065. doi:10.1158/1538-7445.AM2011-4065

A Hexokinase 2 Modulator for Field-Directed Treatment of Experimental Actinic Keratoses

Overexpression of hexokinase 2, and its binding to VDAC1 on the outer mitochondrial membrane of cancer cells, is key to their metabolic reprogramming to aerobic glycolysis, which enables them to proliferate. We describe Comp-1, an allosteric small molecule that selectively detaches hexokinase 2 from the mitochondria. Detachment of hexokinase 2 reduces glycolysis and triggers apoptosis in cancer cells, without affecting hexokinase 1-expressing normal cells. The anti-cancer activity of Comp-1 was demonstrated in the UVB-damaged skin model in SKH-1 mice. Topical treatment with Comp-1 led to 70% reduction in lesion number and area. This inxa0vivo efficacy was obtained without local skin reactions or other safety findings. Mechanism-related pharmacodynamic markers, including hexokinase 2 and cleaved caspase 3 levels, are affected by Comp-1 treatment inxa0vivo. Good Laboratory Practice toxicology studies in minipigs for 28 days and 13 weeks established no systemic toxicities and minimal dermal reaction for once-daily application of up to 20% and 15% ointment strengths, respectively. Thus, Comp-1 may address a significant unmet medical need for a non-irritating efficacious topical actinic keratosis treatment.

Abstract 1725: A bi-functional mechanism of action: Activating the NLRP3 inflammasome and triggering apoptosis in cancer via a HK2-VDAC modulator

Introduction: Cancer cells undergo metabolic reprogramming to enable the efficient conversion of glucose needed for massive cell growth and proliferation, a well-documented phenomenon known as the Warburg effect. A key enzyme in this process is hexokinase 2 (HK2), which catalyzes the first step of glucose metabolism. Unlike HK1, which is ubiquitously expressed in normal cells, high levels of HK2 are found in cancer where it is required for cancer initiation and transformation. HK2 in cancer cells is attached to the outer mitochondrial membrane via the VDAC1 channel. VDAC1/HK2 association blocks pro-apoptotic signals as well as allows a continuous flux of mitochondrial ATP to HK, leading to apoptosis prevention and a high rate of glycolysis. Temporal high HK2 expression, and binding to VDAC, is also found in a variety of activated immune cells to support their changing metabolic needs. It has been recently published that detachment of HK2 from VDAC is one of the first events leading to the NLRP3-inflammasome activation, resulting in IL-1β and IL-18 secretion from macrophages (Cell 166:624 (2016)). Methods: A novel small molecule VDAC/HK2 modulator, VDA-1102, is being developed as a bi-functional drug for the treatment of solid tumors – triggering apoptosis in cancer cells while simultaneously activating the NLRP3-inflammasome in macrophages to induce an anti-tumoral immune response. Results: In vitro studies established that VDA-1102 selectively detaches HK2, but not HK1, from VDAC leading to cancer cell apoptosis, glycolysis inhibition, and prevention of cancer cell proliferation. VDA-1102 treatment of mouse primary bone marrow-derived macrophages and of human macrophage cell line, THP-1 cells, established a dose-dependent NLRP3-inflammasome activation and cytokine secretion. In vivo efficacy studies demonstrated significant tumor growth delay in syngeneic solid tumor models. Analysis of tumor-associated macrophages indicated a treatment-induced change in these macrophage phenotype from M2 to M1. This change was associated with a notable increase in spleen size, an increase ratio of naive T cell in the spleen, and a significant increase in CD8+ and CD4+ tumor-infiltrating T cells. Conclusions: This data supports the notion that VDA-1102 is a bi-functional drug that targets both cancer and the innate immune system. In cancer cells it induces apoptosis, whereas in macrophages it activates the NLRP3-inflammasome machinery and stimulates an anti-tumor immune response. Our findings support further development of VDA-1102 to evaluate its potential as an anti-cancer therapy, either as a monotherapy or in combination with checkpoint inhibitors in high HK2-expressing solid tumors. Citation Format: Vered Behar, Reut Yosef Hamo, Eyal Dor-On, Oren M. Becker. A bi-functional mechanism of action: Activating the NLRP3 inflammasome and triggering apoptosis in cancer via a HK2-VDAC modulator [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1725.

Abstract 3219: Changing the metabolism of cancer cells with PKM2 activators - a path to a cancer metabolism drug

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnPyruvate kinase (PK) M2 which catalyzes the last step in glycolysis, is the alternative spliced isoform expressed in cancer cells, and a key player in exerting the Warburg effect. One of the mechanisms by which PKM2 modulate cancer cell metabolism is by switching between the low activity monomer and the high activity tetramer forms. This process is controlled by the varying concentration of an upstream glycolytic intermediate, FBP. These changes enable the cancer cell to manage its usage of glucose carbon backbones, whether for ATP production or for biomass generation, according to its changing demands. Further, it has been recently shown that the elevated levels of ROS in cancer cells contribute to the decreased activity of PKM2 to support NADPH production to increase cellular anti-oxidation capacity to sustain proliferation. Our goal is to disrupt the metabolic adaptation of cancer cells with small molecule PKM2 modulators. We hypothesized that an activator will redirect the consumption of nutrients, especially of glucose, away from biomass production and ultimately send the cells to die. Using our proprietary structure-based technology, we identified several series of novel allosteric PKM2 activators. Chemical optimization resulted in potent compounds with AC50 as low as 10nM, which were selective against the other PK isoforms. These compounds were proven to stabilize the active tetramer form of PKM2 in cancer cells. Bioenergetic experiments in several cell lines demonstrated that not only do these agents reduce lactate production; they also reduce the oxygen consumption rate. Analysis of cell cycle showed that treatment with PKM2 activators causes the cells to arrest at the G1 phase. In outcome-based assays, these compounds significantly reduced the proliferation rate of various cancer cell lines, and this effect was sensitive to media conditions, such as glucose levels. Taken together, our data supports the hypothesis that activation of PKM2 effectively deprives the cancer cell of building blocks and reduces the detoxification capacity that are required to support growth and proliferation. An in vivo colorectal cancer HT29 cell line mouse xenograft model with a modestly active compound (IC50=0.9uM) demonstrated tumor growth inhibition greater than 50% (100 mg/kg Q2D and 200 and 400 mg/kg IP QD). The compound was very safe in mouse, even at the highest exposure levels (200 and 400 mg/kg IP QD), indicating that these efficacious doses are significantly lower than the MTD. Additional xenograft models are ongoing. Taken together, there is strong support for the effect of potent PKM2 small molecule activators on the cellular metabolism of cancer cells, demonstrating statistically significant anti-cancer effect in an animal model of colorectal cancer. The favorable DMPK profile of these compounds further supports their development as anti-proliferative agents, both as a single agent and in combination therapy.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3219. doi:1538-7445.AM2012-3219