The COVID-19 pandemic caused by the SARS-CoV-2 requires a fast development of antiviral drugs. SA... more The COVID-19 pandemic caused by the SARS-CoV-2 requires a fast development of antiviral drugs. SARS-CoV-2 viral main protease (Mpro, also called 3C‐like protease, 3CLpro) is a potential target for drug design. Crystal and co-crystal structures of the SARS-CoV-2 Mpro have been solved, enabling the rational design of inhibitory compounds. In this study we analyzed the available SARS-CoV-2 and the highly similar SARS-CoV-1 crystal structures. We identified within the active site of the Mpro, in addition to the inhibitory ligands’ interaction with the catalytic C145, two key H-bond interactions with the conserved H163 and E166 residues. Both H-bond interactions are present in almost all co-crystals and are likely to occur also during the viral polypeptide cleavage process as suggested from docking of the Mpro cleavage recognition sequence. We screened in silico a library of 6900 FDA-approved drugs (ChEMBL) and filtered using these key interactions and selected 29 non-covalent compounds ...
The COVID-19 pandemic caused by the SARS-CoV-2 requires a fast development of antiviral drugs. SA... more The COVID-19 pandemic caused by the SARS-CoV-2 requires a fast development of antiviral drugs. SARS-CoV-2 viral main protease (Mpro, also called 3C-like protease, 3CLpro) is a potential target for drug design. Crystal and co-crystal structures of the SARS-CoV-2 Mpro have been solved, enabling the rational design of inhibitory compounds. In this study we analyzed the available SARS-CoV-2 and the highly similar SARS-CoV-1 crystal structures. We identified within the active site of the Mpro, in addition to the inhibitory ligands’ interaction with the catalytic C145, two key H-bond interactions with the conserved H163 and E166 residues. Both H-bond interactions are present in almost all co-crystals and are likely to occur also during the viral polypeptide cleavage process as suggested from docking of the Mpro cleavage recognition sequence. We screened in silico a library of 6,900 FDA-approved drugs (ChEMBL) and filtered using these key interactions and selected 29 non-covalent compounds...
Highlights d Mapping dynamic RNA structures provides a strategy to identify regulatory regions d ... more Highlights d Mapping dynamic RNA structures provides a strategy to identify regulatory regions d Translation plays a major role in unwinding secondary structures in vivo d Structurally dynamic 3 0 UTRs are enriched in elements that regulate mRNA stability d CDS regions contain dynamic structural elements that regulate translation Authors
In vivo tumor growth inhibition by novel PKM2 cancer metabolism modulators
Journal of Clinical Oncology, 2011
10530 Background: Cancer cells undergo major metabolic changes to enable efficient conversion of ... more 10530 Background: Cancer cells undergo major metabolic changes to enable efficient conversion of glucose needed for cell growth and proliferation. This phenomenon, which takes place even when oxygen is abundant, is known as the Warburg effect. A key mediator of this effect is pyruvate kinase (PK), a rate-limiting enzyme that catalyzes the last step in glycolysis. PK subtype M2 (PKM2), which is over expressed in cancer cells, facilitates the Warburg effect by switching from a highly active tetrameric form to low activity monomeric or dimeric forms. Thus small molecule PKM2 activators that stabilize the tetrameric form are expected to renormalize cancer metabolism, offering a novel therapeutic strategy. METHODS Novel PKM2 activators were discovered using a structure-based approach, and tested in multiple in vitro and in vivo studies. RESULTS Several series of novel, potent, allosteric PKM2 activators with AC50s as low as 20nM were discovered. By stabilizing the active tetrameric form of PKM2 more than 10-fold, these activators lower the levels of lactate in cancer cells, thus countering the Warburg effect. In cellular assays, these compounds significantly reduce the proliferation rate of multiple cancer cell lines, representing 5 different tumor types (colorectal, renal, lung, cervix, liver). So far, the greatest sensitivity was observed in colorectal cancer (CRC) and renal cell carcinoma (RCC) cell lines. Combination with commonly used chemotherapeutic agents show an additive, and in some cases a slight synergistic, effect on cells. In in vivo mouse xenograft models with the CRC cell line HT29, even a modestly active compound, such as DNX-03013 (AC50=0.9uM), demonstrated within a few days of treatment tumor growth inhibition greater than 50% (200 and 400 mg/kg IP QD). The compound was safe in mouse even at high exposure levels (400 mg/kg IP QD), indicating that these efficacious doses are significantly below the MTD. CONCLUSIONS Taken together, this is the first report of potent PKM2 novel small-molecule cancer metabolism modulators that show promising anti-cancer therapeutic potential in vivo. The favorable DMPK profile of these compounds suggests that they can be further developed either as mono-therapy or as combination therapy.
Host shutoff is a common strategy used by viruses to repress cellular mRNA translation and concom... more Host shutoff is a common strategy used by viruses to repress cellular mRNA translation and concomitantly allow the efficient translation of viral mRNAs. Here we use RNA-sequencing and ribosome profiling to explore the mechanisms that are being utilized by the Influenza A virus (IAV) to induce host shutoff. We show that viral transcripts are not preferentially translated and instead the decline in cellular protein synthesis is mediated by viral takeover on the mRNA pool. Our measurements also uncover strong variability in the levels of cellular transcripts reduction, revealing that short transcripts are less affected by IAV. Interestingly, these mRNAs that are refractory to IAV infection are enriched in cell maintenance processes such as oxidative phosphorylation. Furthermore, we show that the continuous oxidative phosphorylation activity is important for viral propagation. Our results advance our understanding of IAV-induced shutoff, and suggest a mechanism that facilitates the tran...
The invention relates to compounds and methods for modulating components of a kinase signaling ca... more The invention relates to compounds and methods for modulating components of a kinase signaling cascade.
Viruses are by definition fully dependent on the cellular translation machinery, and develop dive... more Viruses are by definition fully dependent on the cellular translation machinery, and develop diverse mechanisms to co-opt this machinery for their own benefit. Unlike many viruses, human cytomegalovirus (HCMV) does suppress the host translation machinery, and the extent to which translation machinery contributes to the overall pattern of viral replication and pathogenesis remains elusive. Here, we combine RNA sequencing and ribosomal profiling analyses to systematically address this question. By simultaneously examining the changes in transcription and translation along HCMV infection, we uncover extensive transcriptional control that dominates the response to infection, but also diverse and dynamic translational regulation for subsets of host genes. We were also able to show that, at late time points in infection, translation of viral mRNAs is higher than that of cellular mRNAs. Lastly, integration of our translation measurements with recent measurements of protein abundance enable...
Abstract 4065: Modulation of cancer metabolism with novel PKM2 activators exerts anti-tumor activity
Cancer Research, 2011
Cancer cells adapt altered metabolism to enable efficient conversion of glucose into biomass need... more 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
Myosin VI, found in organisms from Caenorhabditis elegans to humans, is essential for auditory an... more Myosin VI, found in organisms from Caenorhabditis elegans to humans, is essential for auditory and vestibular function in mammals, since genetic mutations lead to hearing impairment and vestibular dysfunction in both humans and mice. Here, we show that a missense mutation in this molecular motor in an ENU-generated mouse model, Tailchaser, disrupts myosin VI function. Structural changes in the Tailchaser hair bundles include mislocalization of the kinocilia and branching of stereocilia. Transfection of GFP-labeled myosin VI into epithelial cells and delivery of endocytic vesicles to the early endosome revealed that the mutant phenotype displays disrupted motor function. The actin-activated ATPase rates measured for the D179Y mutation are decreased, and indicate loss of coordination of the myosin VI heads or 'gating' in the dimer form. Proper coordination is required for walking processively along, or anchoring to, actin filaments, and is apparently destroyed by the proximity of the mutation to the nucleotide-binding pocket. This loss of myosin VI function may not allow myosin VI to transport its cargoes appropriately at the base and within the stereocilia, or to anchor the membrane of stereocilia to actin filaments via its cargos, both of which lead to structural changes in the stereocilia of myosin VI-impaired hair cells, and ultimately leading to deafness.
Abstract 3219: Changing the metabolism of cancer cells with PKM2 activators - a path to a cancer metabolism drug
Cancer Research, 2012
Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Pyruvate kinase (PK... more Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Pyruvate 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. Citation 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
1-(sulfonyl)-5-(arylsulfonyl)indoline as activators of the tumor cell specific M2 isoform of pyruvate kinase
Bioorganic & Medicinal Chemistry Letters, 2012
Cancer cells preferentially use glycolysis rather than oxidative phosphorylation for their rapid ... more 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.
Postgenomics data are produced in large volumes by life sciences and clinical applications of nov... more Postgenomics data are produced in large volumes by life sciences and clinical applications of novel omics diagnostics and therapeutics for precision medicine. To move from ''data-to-knowledge-to-innovation,'' a crucial missing step in the current era is, however, our limited understanding of biological and clinical contexts associated with data. Prominent among the emerging remedies to this challenge are the gene set enrichment tools. This study reports on GeneAnalyticsÔ (geneanalytics.genecards.org), a comprehensive and easy-to-apply gene set analysis tool for rapid contextualization of expression patterns and functional signatures embedded in the postgenomics Big Data domains, such as Next Generation Sequencing (NGS), RNAseq, and microarray experiments. GeneAnalytics' differentiating features include in-depth evidence-based scoring algorithms, an intuitive user interface and proprietary unified data. GeneAnalytics employs the LifeMap Science's GeneCards suite, including the GeneCardsÒ-the human gene database; the MalaCards-the human diseases database; and the PathCards-the biological pathways database. Expression-based analysis in GeneAnalytics relies on the LifeMap DiscoveryÒ-the embryonic development and stem cells database, which includes manually curated expression data for normal and diseased tissues, enabling advanced matching algorithm for gene-tissue association. This assists in evaluating differentiation protocols and discovering biomarkers for tissues and cells. Results are directly linked to gene, disease, or cell ''cards'' in the GeneCards suite. Future developments aim to enhance the GeneAnalytics algorithm as well as visualizations, employing varied graphical display items. Such attributes make GeneAnalytics a broadly applicable postgenomics data analyses and interpretation tool for translation of data to knowledge-based innovation in various Big Data fields such as precision medicine, ecogenomics, nutrigenomics, pharmacogenomics, vaccinomics, and others yet to emerge on the postgenomics horizon.
The COVID-19 pandemic caused by the SARS-CoV-2 requires a fast development of antiviral drugs. SA... more The COVID-19 pandemic caused by the SARS-CoV-2 requires a fast development of antiviral drugs. SARS-CoV-2 viral main protease (Mpro, also called 3C‐like protease, 3CLpro) is a potential target for drug design. Crystal and co-crystal structures of the SARS-CoV-2 Mpro have been solved, enabling the rational design of inhibitory compounds. In this study we analyzed the available SARS-CoV-2 and the highly similar SARS-CoV-1 crystal structures. We identified within the active site of the Mpro, in addition to the inhibitory ligands’ interaction with the catalytic C145, two key H-bond interactions with the conserved H163 and E166 residues. Both H-bond interactions are present in almost all co-crystals and are likely to occur also during the viral polypeptide cleavage process as suggested from docking of the Mpro cleavage recognition sequence. We screened in silico a library of 6900 FDA-approved drugs (ChEMBL) and filtered using these key interactions and selected 29 non-covalent compounds ...
The COVID-19 pandemic caused by the SARS-CoV-2 requires a fast development of antiviral drugs. SA... more The COVID-19 pandemic caused by the SARS-CoV-2 requires a fast development of antiviral drugs. SARS-CoV-2 viral main protease (Mpro, also called 3C-like protease, 3CLpro) is a potential target for drug design. Crystal and co-crystal structures of the SARS-CoV-2 Mpro have been solved, enabling the rational design of inhibitory compounds. In this study we analyzed the available SARS-CoV-2 and the highly similar SARS-CoV-1 crystal structures. We identified within the active site of the Mpro, in addition to the inhibitory ligands’ interaction with the catalytic C145, two key H-bond interactions with the conserved H163 and E166 residues. Both H-bond interactions are present in almost all co-crystals and are likely to occur also during the viral polypeptide cleavage process as suggested from docking of the Mpro cleavage recognition sequence. We screened in silico a library of 6,900 FDA-approved drugs (ChEMBL) and filtered using these key interactions and selected 29 non-covalent compounds...
Highlights d Mapping dynamic RNA structures provides a strategy to identify regulatory regions d ... more Highlights d Mapping dynamic RNA structures provides a strategy to identify regulatory regions d Translation plays a major role in unwinding secondary structures in vivo d Structurally dynamic 3 0 UTRs are enriched in elements that regulate mRNA stability d CDS regions contain dynamic structural elements that regulate translation Authors
In vivo tumor growth inhibition by novel PKM2 cancer metabolism modulators
Journal of Clinical Oncology, 2011
10530 Background: Cancer cells undergo major metabolic changes to enable efficient conversion of ... more 10530 Background: Cancer cells undergo major metabolic changes to enable efficient conversion of glucose needed for cell growth and proliferation. This phenomenon, which takes place even when oxygen is abundant, is known as the Warburg effect. A key mediator of this effect is pyruvate kinase (PK), a rate-limiting enzyme that catalyzes the last step in glycolysis. PK subtype M2 (PKM2), which is over expressed in cancer cells, facilitates the Warburg effect by switching from a highly active tetrameric form to low activity monomeric or dimeric forms. Thus small molecule PKM2 activators that stabilize the tetrameric form are expected to renormalize cancer metabolism, offering a novel therapeutic strategy. METHODS Novel PKM2 activators were discovered using a structure-based approach, and tested in multiple in vitro and in vivo studies. RESULTS Several series of novel, potent, allosteric PKM2 activators with AC50s as low as 20nM were discovered. By stabilizing the active tetrameric form of PKM2 more than 10-fold, these activators lower the levels of lactate in cancer cells, thus countering the Warburg effect. In cellular assays, these compounds significantly reduce the proliferation rate of multiple cancer cell lines, representing 5 different tumor types (colorectal, renal, lung, cervix, liver). So far, the greatest sensitivity was observed in colorectal cancer (CRC) and renal cell carcinoma (RCC) cell lines. Combination with commonly used chemotherapeutic agents show an additive, and in some cases a slight synergistic, effect on cells. In in vivo mouse xenograft models with the CRC cell line HT29, even a modestly active compound, such as DNX-03013 (AC50=0.9uM), demonstrated within a few days of treatment tumor growth inhibition greater than 50% (200 and 400 mg/kg IP QD). The compound was safe in mouse even at high exposure levels (400 mg/kg IP QD), indicating that these efficacious doses are significantly below the MTD. CONCLUSIONS Taken together, this is the first report of potent PKM2 novel small-molecule cancer metabolism modulators that show promising anti-cancer therapeutic potential in vivo. The favorable DMPK profile of these compounds suggests that they can be further developed either as mono-therapy or as combination therapy.
Host shutoff is a common strategy used by viruses to repress cellular mRNA translation and concom... more Host shutoff is a common strategy used by viruses to repress cellular mRNA translation and concomitantly allow the efficient translation of viral mRNAs. Here we use RNA-sequencing and ribosome profiling to explore the mechanisms that are being utilized by the Influenza A virus (IAV) to induce host shutoff. We show that viral transcripts are not preferentially translated and instead the decline in cellular protein synthesis is mediated by viral takeover on the mRNA pool. Our measurements also uncover strong variability in the levels of cellular transcripts reduction, revealing that short transcripts are less affected by IAV. Interestingly, these mRNAs that are refractory to IAV infection are enriched in cell maintenance processes such as oxidative phosphorylation. Furthermore, we show that the continuous oxidative phosphorylation activity is important for viral propagation. Our results advance our understanding of IAV-induced shutoff, and suggest a mechanism that facilitates the tran...
The invention relates to compounds and methods for modulating components of a kinase signaling ca... more The invention relates to compounds and methods for modulating components of a kinase signaling cascade.
Viruses are by definition fully dependent on the cellular translation machinery, and develop dive... more Viruses are by definition fully dependent on the cellular translation machinery, and develop diverse mechanisms to co-opt this machinery for their own benefit. Unlike many viruses, human cytomegalovirus (HCMV) does suppress the host translation machinery, and the extent to which translation machinery contributes to the overall pattern of viral replication and pathogenesis remains elusive. Here, we combine RNA sequencing and ribosomal profiling analyses to systematically address this question. By simultaneously examining the changes in transcription and translation along HCMV infection, we uncover extensive transcriptional control that dominates the response to infection, but also diverse and dynamic translational regulation for subsets of host genes. We were also able to show that, at late time points in infection, translation of viral mRNAs is higher than that of cellular mRNAs. Lastly, integration of our translation measurements with recent measurements of protein abundance enable...
Abstract 4065: Modulation of cancer metabolism with novel PKM2 activators exerts anti-tumor activity
Cancer Research, 2011
Cancer cells adapt altered metabolism to enable efficient conversion of glucose into biomass need... more 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
Myosin VI, found in organisms from Caenorhabditis elegans to humans, is essential for auditory an... more Myosin VI, found in organisms from Caenorhabditis elegans to humans, is essential for auditory and vestibular function in mammals, since genetic mutations lead to hearing impairment and vestibular dysfunction in both humans and mice. Here, we show that a missense mutation in this molecular motor in an ENU-generated mouse model, Tailchaser, disrupts myosin VI function. Structural changes in the Tailchaser hair bundles include mislocalization of the kinocilia and branching of stereocilia. Transfection of GFP-labeled myosin VI into epithelial cells and delivery of endocytic vesicles to the early endosome revealed that the mutant phenotype displays disrupted motor function. The actin-activated ATPase rates measured for the D179Y mutation are decreased, and indicate loss of coordination of the myosin VI heads or 'gating' in the dimer form. Proper coordination is required for walking processively along, or anchoring to, actin filaments, and is apparently destroyed by the proximity of the mutation to the nucleotide-binding pocket. This loss of myosin VI function may not allow myosin VI to transport its cargoes appropriately at the base and within the stereocilia, or to anchor the membrane of stereocilia to actin filaments via its cargos, both of which lead to structural changes in the stereocilia of myosin VI-impaired hair cells, and ultimately leading to deafness.
Abstract 3219: Changing the metabolism of cancer cells with PKM2 activators - a path to a cancer metabolism drug
Cancer Research, 2012
Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Pyruvate kinase (PK... more Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Pyruvate 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. Citation 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
1-(sulfonyl)-5-(arylsulfonyl)indoline as activators of the tumor cell specific M2 isoform of pyruvate kinase
Bioorganic & Medicinal Chemistry Letters, 2012
Cancer cells preferentially use glycolysis rather than oxidative phosphorylation for their rapid ... more 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.
Postgenomics data are produced in large volumes by life sciences and clinical applications of nov... more Postgenomics data are produced in large volumes by life sciences and clinical applications of novel omics diagnostics and therapeutics for precision medicine. To move from ''data-to-knowledge-to-innovation,'' a crucial missing step in the current era is, however, our limited understanding of biological and clinical contexts associated with data. Prominent among the emerging remedies to this challenge are the gene set enrichment tools. This study reports on GeneAnalyticsÔ (geneanalytics.genecards.org), a comprehensive and easy-to-apply gene set analysis tool for rapid contextualization of expression patterns and functional signatures embedded in the postgenomics Big Data domains, such as Next Generation Sequencing (NGS), RNAseq, and microarray experiments. GeneAnalytics' differentiating features include in-depth evidence-based scoring algorithms, an intuitive user interface and proprietary unified data. GeneAnalytics employs the LifeMap Science's GeneCards suite, including the GeneCardsÒ-the human gene database; the MalaCards-the human diseases database; and the PathCards-the biological pathways database. Expression-based analysis in GeneAnalytics relies on the LifeMap DiscoveryÒ-the embryonic development and stem cells database, which includes manually curated expression data for normal and diseased tissues, enabling advanced matching algorithm for gene-tissue association. This assists in evaluating differentiation protocols and discovering biomarkers for tissues and cells. Results are directly linked to gene, disease, or cell ''cards'' in the GeneCards suite. Future developments aim to enhance the GeneAnalytics algorithm as well as visualizations, employing varied graphical display items. Such attributes make GeneAnalytics a broadly applicable postgenomics data analyses and interpretation tool for translation of data to knowledge-based innovation in various Big Data fields such as precision medicine, ecogenomics, nutrigenomics, pharmacogenomics, vaccinomics, and others yet to emerge on the postgenomics horizon.
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Papers by Alina Shitrit