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284 Poly(ADP-ribose) glycohydrolase (PARG) inhibitors increase nuclear poly(ADP-ribose) after methylating DNA damage
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Thursday 20 November 2014
283 POSTER The development of the first selective inhibitors of the UBA5 enzyme to probe for E1 activity in diseased cells S.R. da Silva1 , S.L. Paiva1 , M. Bancerz2 , M. Geletu2 , A.M. Lewis2 , J. Chen3 , Y. Cai3 , H. Li3 , P.T. Gunning1 . 1 University of Toronto, Chemistry, Toronto, Canada; 2 University of Toronto Mississauga, Chemical and Physical Sciences, Mississauga, Canada; 3 Georgia Regents University, Biochemistry and Molecular Biology, Augusta, USA Cancer cells produce and degrade proteins more rapidly than most normal cells, and are in turn more sensitive to changes in protein regulation. Similarly to secretory cells, cancer cells have the propensity to undergo endoplasmic reticulum (ER) stress due to higher protein turnover that, if left uncorrected, can result in apoptosis. To avoid these fates, cells have developed support systems such as the conjugation of the ubiquitin-fold modifier 1 (UFM1) ubiquitin-like protein to other protein targets, which has been implicated in counteracting apoptotic ER stress in pancreatic secretory cells. Our research has recently focused on the development of the first inhibitor of the UFM1 pathway by targeting its E1 enzyme, UBA5, in order to counteract the anti-apoptotic effects of this system and to further study the role of UFM1 conjugation in cancer cells. We have successfully identified a lead inhibitor, 5C-Z, which incorporates an adenosine moiety coupled to a zinc(II) polyazamacrocylic coordination complex. Through enzymatic assays that evaluate the transthiolation ability of UBA5 in the first step of UFM1 activation, our inhibitor exhibits low micromolar activity against UBA5 while demonstrating potent selectivity over other E1 enzymes (>20fold). Kinetic assays reveal that 5C-Z acts non-competitively on UBA5, indicating that the compound could possibly be binding to and inhibiting the “inactive” subunit of the UBA5 homodimer. Furthermore, treatment of lung and leukemia cells that exhibit high levels of UBA5 protein expression leads to a decrease in cell proliferation yet does not induce cell death in diseased or healthy cells, up to 200 mM. This novel strategy of inhibiting UBA5 and UFMylation would make cancer cells that are highly dependent on this system more susceptible to treatment regimens of principle drugs, which could lead to the use of milder drug dosing strategies. Our novel inhibitors produced from this research can be used as a probe to further investigate the role of E1 activating enzymes in cancer progression.
Figure: General formula of the compounds.
284 POSTER Poly(ADP-ribose) glycohydrolase (PARG) inhibitors increase nuclear poly(ADP-ribose) after methylating DNA damage A. Jordan1 , B. Acton1 , E. Fairweather1 , N. Hamilton1 , S. Holt1 , J. Hitchin1 , C. Hutton1 , D. James1 , S. Jones1 , A. McGonagle1 , H. Small1 , K. Smith1 , A. Stowell1 , I. Waddell1 , B. Waszkowycz1 , D. Ogilvie1 . 1 Cancer Research UK Manchester Institute, Drug Discovery, Manchester, United Kingdom Background: DNA single strand breaks (SSBs) are the most common type of damage occurring in cells. Poly(ADP ribose) polymerase (PARP) binds to SSBs and auto-ribosylates itself using NAD+ as a substrate. This creates chains of poly ADP ribose (PAR) which provide a signal for other proteins to repair the lesion. Sequential removal of the PAR chains is accomplished by Poly(ADP-ribose) glycohydrolase (PARG). Failure to complete the DNA repair process, either by inhibition of PARP (e.g. with olaparib) or inhibition of PARG (with shRNA), can lead to cell death. PARG is the only enzyme known to efficiently catalyse the hydrolysis of O-glycosidic linkages of ADP-ribose polymers and exists (unlike PARP) as a single gene. We therefore sought to exploit this vulnerability and as part of a fostering agreement with AstraZeneca have developed novel small molecule inhibitors of PARG activity. Methods: A screening cascade for small molecule inhibition of PARG was developed. Biochemical inhibition of PARG was measured using a bespoke HTRF assay. In addition, selected PARG inhibitors were tested against other enzyme substrates (ARH3, PARP1) to assay for selectivity. Active compounds were then taken forward and tested in cells for PAR chain persistence and for cytotoxicity using a 3-day HeLa assay. Suitable compounds were then evaluated for their physico-chemical properties and their in vivo PK profiles determined.
Poster Session – Drug Design Results: Early PARG inhibitors increased nuclear PAR chain levels in cells after 1 h treatment with the methylating agent methyl methanesulfonate (MMS) but also displayed off-target cytotoxicity in the absence of MMS. Applying computational chemistry enabled us to discover novel series of compounds with sub-micromolar potency with a wide differential to acute cytotoxicity. Compounds were also highly selective against PARP1 and ARH3 in vitro. PAR chain persistence in cells was also maintained after using the more clinically-relevant methylating agent temozolomide (TMZ). PARG inhibitors displayed satisfactory in vitro and in vivo PK profiles. Conclusions: We have developed PARG inhibitors that block the breakdown of PAR chains in cells after exogenous DNA damage by methylating agents. These tool compounds are potent and selective and enable us to both explore in more detail the cellular mode of action and investigate pre-clinical in vivo models for PARG inhibition. 285 POSTER A nanomolar-potency small molecule inhibitor of the STAT5 protein A.A. Cumaraswamy1 , A. Lewis2 , M. Geletu2 , A. Todic2 , D.B. Diaz2 , X.R. Cheng3 , C.E. Brown2 , R. Laister4 , D. Muench5 , K. Kerman3 , H.L. Grimes5 , M.D. Minden4 , P.T. Gunning1 . 1 University of Toronto, Department of Chemistry, Mississauga ON, Canada; 2 University of Toronto, Chemistry, Mississauga ON, Canada; 3 University of Toronto, Department of Physical & Environmental Sciences, Scarborough ON, Canada; 4 Princess Margaret Cancer Institute, Ontario Cancer Institute, Toronto ON, Canada; 5 Cincinnati Children’s Hospital Medical Centre, Division of Experimental Hematology, Cincinnati OH, USA Signal Transducer and Activator of Transcription 5 (STAT5) protein has gained notoriety for its aberrant role in many human cancers. In contrast to normal cells where STAT5 activity is rapid and transient, in cancer cells, including leukemias, STAT5 activity is routinely hyper-activated, conferring resistance to cell death and driving excessive expression of proto-oncogenes. Despite significant evidence showing STAT5’s role in human cancers, there has been little progress in developing direct inhibitors of STAT5 function. Potent and selective small molecule inhibitors of STAT5 will be effective therapeutics for treatment of hematological cancers and overcoming the side effects of current treatments. STAT5 is activated extracellularly by ligand-receptor binding, which results in STAT5 recruitment to intracellular receptor sites via their Src Homology 2 (SH2) domain. STAT5 is then phosphorylated, facilitating STAT5-STAT5 dimerization via reciprocal phosphotyrosine-SH2 interactions. The dimer shuttles to the nucleus and induces target gene transcription. In cancer cells, STAT5 is persistently activated, leading to the aberrant expression of STAT5 target genes that promote cancer cell survival and prevent cell death. To achieve this goal, we have employed a structure-based drug design strategy using computational analysis, medicinal chemistry and synthetic methods amenable to generating a diverse library. Herein, we report the first nanomolar, STAT5-selective inhibitor, AC-3-019, possessing a phosphotyrosyl-mimicking salicylic acid group, which potently and selectively binds to STAT5 over STAT3, inhibits STAT5-SH2 domain complexation events in vitro, silences activated STAT5 in leukemic cells, as well as STAT5’s downstream transcriptional targets, including MYC and MCL1, and as a result, leads to apoptosis. We believe AC-3-019 represents a useful probe for interrogating STAT5 function in cells as well as being a potential candidate for advanced preclinical trials.
Thursday 20 November 2014
283 POSTER The development of the first selective inhibitors of the UBA5 enzyme to probe for E1 activity in diseased cells S.R. da Silva1 , S.L. Paiva1 , M. Bancerz2 , M. Geletu2 , A.M. Lewis2 , J. Chen3 , Y. Cai3 , H. Li3 , P.T. Gunning1 . 1 University of Toronto, Chemistry, Toronto, Canada; 2 University of Toronto Mississauga, Chemical and Physical Sciences, Mississauga, Canada; 3 Georgia Regents University, Biochemistry and Molecular Biology, Augusta, USA Cancer cells produce and degrade proteins more rapidly than most normal cells, and are in turn more sensitive to changes in protein regulation. Similarly to secretory cells, cancer cells have the propensity to undergo endoplasmic reticulum (ER) stress due to higher protein turnover that, if left uncorrected, can result in apoptosis. To avoid these fates, cells have developed support systems such as the conjugation of the ubiquitin-fold modifier 1 (UFM1) ubiquitin-like protein to other protein targets, which has been implicated in counteracting apoptotic ER stress in pancreatic secretory cells. Our research has recently focused on the development of the first inhibitor of the UFM1 pathway by targeting its E1 enzyme, UBA5, in order to counteract the anti-apoptotic effects of this system and to further study the role of UFM1 conjugation in cancer cells. We have successfully identified a lead inhibitor, 5C-Z, which incorporates an adenosine moiety coupled to a zinc(II) polyazamacrocylic coordination complex. Through enzymatic assays that evaluate the transthiolation ability of UBA5 in the first step of UFM1 activation, our inhibitor exhibits low micromolar activity against UBA5 while demonstrating potent selectivity over other E1 enzymes (>20fold). Kinetic assays reveal that 5C-Z acts non-competitively on UBA5, indicating that the compound could possibly be binding to and inhibiting the “inactive” subunit of the UBA5 homodimer. Furthermore, treatment of lung and leukemia cells that exhibit high levels of UBA5 protein expression leads to a decrease in cell proliferation yet does not induce cell death in diseased or healthy cells, up to 200 mM. This novel strategy of inhibiting UBA5 and UFMylation would make cancer cells that are highly dependent on this system more susceptible to treatment regimens of principle drugs, which could lead to the use of milder drug dosing strategies. Our novel inhibitors produced from this research can be used as a probe to further investigate the role of E1 activating enzymes in cancer progression.
Figure: General formula of the compounds.
284 POSTER Poly(ADP-ribose) glycohydrolase (PARG) inhibitors increase nuclear poly(ADP-ribose) after methylating DNA damage A. Jordan1 , B. Acton1 , E. Fairweather1 , N. Hamilton1 , S. Holt1 , J. Hitchin1 , C. Hutton1 , D. James1 , S. Jones1 , A. McGonagle1 , H. Small1 , K. Smith1 , A. Stowell1 , I. Waddell1 , B. Waszkowycz1 , D. Ogilvie1 . 1 Cancer Research UK Manchester Institute, Drug Discovery, Manchester, United Kingdom Background: DNA single strand breaks (SSBs) are the most common type of damage occurring in cells. Poly(ADP ribose) polymerase (PARP) binds to SSBs and auto-ribosylates itself using NAD+ as a substrate. This creates chains of poly ADP ribose (PAR) which provide a signal for other proteins to repair the lesion. Sequential removal of the PAR chains is accomplished by Poly(ADP-ribose) glycohydrolase (PARG). Failure to complete the DNA repair process, either by inhibition of PARP (e.g. with olaparib) or inhibition of PARG (with shRNA), can lead to cell death. PARG is the only enzyme known to efficiently catalyse the hydrolysis of O-glycosidic linkages of ADP-ribose polymers and exists (unlike PARP) as a single gene. We therefore sought to exploit this vulnerability and as part of a fostering agreement with AstraZeneca have developed novel small molecule inhibitors of PARG activity. Methods: A screening cascade for small molecule inhibition of PARG was developed. Biochemical inhibition of PARG was measured using a bespoke HTRF assay. In addition, selected PARG inhibitors were tested against other enzyme substrates (ARH3, PARP1) to assay for selectivity. Active compounds were then taken forward and tested in cells for PAR chain persistence and for cytotoxicity using a 3-day HeLa assay. Suitable compounds were then evaluated for their physico-chemical properties and their in vivo PK profiles determined.
Poster Session – Drug Design Results: Early PARG inhibitors increased nuclear PAR chain levels in cells after 1 h treatment with the methylating agent methyl methanesulfonate (MMS) but also displayed off-target cytotoxicity in the absence of MMS. Applying computational chemistry enabled us to discover novel series of compounds with sub-micromolar potency with a wide differential to acute cytotoxicity. Compounds were also highly selective against PARP1 and ARH3 in vitro. PAR chain persistence in cells was also maintained after using the more clinically-relevant methylating agent temozolomide (TMZ). PARG inhibitors displayed satisfactory in vitro and in vivo PK profiles. Conclusions: We have developed PARG inhibitors that block the breakdown of PAR chains in cells after exogenous DNA damage by methylating agents. These tool compounds are potent and selective and enable us to both explore in more detail the cellular mode of action and investigate pre-clinical in vivo models for PARG inhibition. 285 POSTER A nanomolar-potency small molecule inhibitor of the STAT5 protein A.A. Cumaraswamy1 , A. Lewis2 , M. Geletu2 , A. Todic2 , D.B. Diaz2 , X.R. Cheng3 , C.E. Brown2 , R. Laister4 , D. Muench5 , K. Kerman3 , H.L. Grimes5 , M.D. Minden4 , P.T. Gunning1 . 1 University of Toronto, Department of Chemistry, Mississauga ON, Canada; 2 University of Toronto, Chemistry, Mississauga ON, Canada; 3 University of Toronto, Department of Physical & Environmental Sciences, Scarborough ON, Canada; 4 Princess Margaret Cancer Institute, Ontario Cancer Institute, Toronto ON, Canada; 5 Cincinnati Children’s Hospital Medical Centre, Division of Experimental Hematology, Cincinnati OH, USA Signal Transducer and Activator of Transcription 5 (STAT5) protein has gained notoriety for its aberrant role in many human cancers. In contrast to normal cells where STAT5 activity is rapid and transient, in cancer cells, including leukemias, STAT5 activity is routinely hyper-activated, conferring resistance to cell death and driving excessive expression of proto-oncogenes. Despite significant evidence showing STAT5’s role in human cancers, there has been little progress in developing direct inhibitors of STAT5 function. Potent and selective small molecule inhibitors of STAT5 will be effective therapeutics for treatment of hematological cancers and overcoming the side effects of current treatments. STAT5 is activated extracellularly by ligand-receptor binding, which results in STAT5 recruitment to intracellular receptor sites via their Src Homology 2 (SH2) domain. STAT5 is then phosphorylated, facilitating STAT5-STAT5 dimerization via reciprocal phosphotyrosine-SH2 interactions. The dimer shuttles to the nucleus and induces target gene transcription. In cancer cells, STAT5 is persistently activated, leading to the aberrant expression of STAT5 target genes that promote cancer cell survival and prevent cell death. To achieve this goal, we have employed a structure-based drug design strategy using computational analysis, medicinal chemistry and synthetic methods amenable to generating a diverse library. Herein, we report the first nanomolar, STAT5-selective inhibitor, AC-3-019, possessing a phosphotyrosyl-mimicking salicylic acid group, which potently and selectively binds to STAT5 over STAT3, inhibits STAT5-SH2 domain complexation events in vitro, silences activated STAT5 in leukemic cells, as well as STAT5’s downstream transcriptional targets, including MYC and MCL1, and as a result, leads to apoptosis. We believe AC-3-019 represents a useful probe for interrogating STAT5 function in cells as well as being a potential candidate for advanced preclinical trials.