Mohammed Khysar Pasha

Enhanced activity of human N-myristoyltransferase by dimethyl sulfoxide and related solvents in the presence of serine/threonine-containing peptide substrates

Human N-myristoyltransferase (hNMT) activity was found to be stimulated several-fold by DMSO and its analogues in the presence of serine-containing peptide substrates. DMSO caused a concentration-dependent 10-fold stimulation of hNMT activity in the presence of a pp60(src)-derived peptide substrate (Gly-Ser-Ser-Lys-Ser-Lys-Pro-Lys-Arg). However, the stimulation of hNMT activity was not observed by DMSO when a cyclic AMP (cAMP)-dependent protein kinase-derived Ser-free peptide substrate (Gly-Asn-Ala-Ala-Ala-Ala-Lys-Lys-Arg-Arg) was used. These findings suggested that the effect of DMSO is on the substrate rather than on the enzyme. When a MARCKS (myristoylated alanine-rich C-kinase substrate)-derived peptide substrate (Gly-Ala-Gln-Phe-Ser-Lys-Thr-Ala-Arg-Arg) and the M2 gene segment of the reovirus type 3 peptide substrate (Gly-Asn-Ala-Ser-Ser-Ile-Lys-Lys-Lys) were used, hNMT activity was increased by approximately 8.5- and 7-fold, respectively. Dimethyl sulfone (20%) increased hNMT activity between 2.5- and 3.5-fold in the presence of pp60(src), MARCKS, and M2 gene segment peptides. Dimethyl formamide (20%) increased the hNMT activity by 8.5-, 8.5-, 5.5- and 3.5-fold when pp60(src), MARCKS, M2, and cAMP-dependent protein kinase-derived peptide substrates were used, respectively. Acetone (20%) also increased the hNMT activity by 20-fold in the presence of the pp60(src) peptide substrate. Dimethyl ammonium chloride (20%) caused about 6.5- and 2.5-fold increases in the hNMT activity in the presence of the pp60(src) and cAMP-dependent protein kinase-derived peptide substrates, respectively. Infrared spectroscopy showed a decreased intensity in the band at 3500-3600cm(-1) when the infrared spectrum of the pp60(src)-derived peptide was determined in the presence of DMSO. These results suggest the involvement of hydrogen bonding between the heteroatoms of the organic molecules and the hydrogen atoms of the free hydroxyl groups of the serine/threonine-containing peptide substrates. Such interactions appear to enhance the activity of hNMT towards its serine-containing substrates.

Cytotoxic 1,4-bis(2-oxo-1-cycloalkylmethylene)benzenes and related compounds.

A series of 1,4-bis(2-oxo-1-cycloalkylmethylene)benzenes 2a-c and 4 and a related acyclic analogue 6a were synthesised and converted to the corresponding Mannich bases 3a-c, 5 and 6b. Evaluation of these compounds against murine P388 and L1210 cells as well as human Molt 4/C8 and CEM T-lymphocytes revealed that the Mannich bases were more cytotoxic than the corresponding unsaturated ketones. 1,4-bis(3-Dimethylaminomethyl-2-oxo-1-cyclohexylmethylene)benzene dihydrochloride (3a) had lower IC(50) values than melphalan against the four cell lines and was 15 times more potent than this drug when examined against a panel of human tumours.

N-myristoyltransferase inhibitor protein is homologous to heat shock cognate protein 70.

Many of viral and eukaryotic proteins are required for signal transduction and regulatory functions which undergo a lipid modification by the enzyme N‐myristoyltransferase (NMT). In this study, we demonstrated that heat shock cognate protein 70 (HSC70) is homologous to NMT inhibitor protein (NIP71), which was discovered in our laboratory, based on MALDI‐TOF mass spectrometric analysis. The purified bovine cytosolic HSC70 and particulate NIP71 produced a dose‐dependent inhibition of human NMT having half maximal inhibitions of 235 and 230 nM, respectively. Further, Western blot analysis revealed that the purified particulate NIP71 and cytosolic HSC70 cross‐reacted with both anti‐NIP71 and anti‐HSC70 antibodies. The results we obtained imply that molecular chaperones could be involved in the regulation of NMT in normal and cancerous cells. Further studies directed to revealing the role of HSC70 in the regulation of NMT may lead to the development of gene based therapies of colon cancer.

Cytotoxic mannich bases of 1-arylidene-2-tetralones

Various 1-arylidene-2-tetralones 1 had been shown previously to possess moderate cytotoxic properties unaccompanied by murine toxicity. The objective of the present investigation was to undertake different molecular modifications of representative members of series 1 with a view to discerning those structural features leading to increased potencies. All compounds were evaluated using human Molt 4/C8 and CEM T-lymphocytes as well as murine P388 and L1210 leukemic cells. The Mannich bases 2, 4, 5 and 7 possessed increased potencies compared to the corresponding unsaturated ketones 1 and in general were potent cytotoxics having IC50 values in the 0.2–10 μM range. QSAR using the cytotoxicity data for 2a–e suggested that potency was positively correlated with the size of the substituents in the arylidene aryl ring. Compounds 2a–f were evaluated using a panel of approximately 53 human tumour cell lines and, when all cell lines were considered, were more potent than the reference drug melphalan. In particular, marked antileukemic activity was displayed. Molecular modeling was utilized in order to evaluate whether the shapes of the different compounds contributed to the varying potencies observed. Representative compounds demonstrated minimal or no inhibiting properties towards human N-myristoyltransferase (NMT) and did not bind to calf thymus DNA. This study has revealed a number of unique lead molecules as candidate antineoplastic agents serving as prototypes for future development.

Abstract 3591: Discovery of SB1317: A novel small molecule macrocycle with a unique kinase inhibitory spectrum in phase 1 clinical trials for hematological malignancies

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: During our quest to find novel kinase inhibitor motifs for our protein kinase research projects we discovered a novel series of macrocyclic structures which possess unique but tunable kinase inhibitory profiles. Here we present the chemical structure of SB1317 and the SAR studies leading to the discovery of this novel CDK/FLT3/JAK2 inhibitor, currently in clinical trials for hematological malignancies. Methods/Results: The unusual structure of SB1317 is characterized by a biaryl connection to the hinge-binding 2-anilinopyrimidine and an allylic tertiary amine – homo allylic ether linker forming an 18-membered macrocyclic ring. Combination of the biaryl motif and unsaturation in the linker confers limited conformational freedom and may explain the specific biological activity observed with this series. Following extensive SAR studies a single preferred compound, SB1317, emerged with potent CDK (IC50 against CDKs 1, 2 and 9 = 9, 5 and 3 nM, respectively), FLT3 (IC50 = 19nM) and JAK2 (IC50 = 19nM) potency. SB1317 has low molecular weight and good physicochemical properties conferring good oral exposure and prompting its further evaluation in vitro and in in vivo cancer models. Following promising efficacy data in these studies, SB1317 was nominated as the preferred candidate for progression to the clinic. SB1317 was synthesized via a ring-closing metathesis (RCM) strategy where the macrocycle was formed from coupling the two halves of the molecule to provide a diene which was then closed by RCM using either Grubbs or Zhan catalysts. Crucially the RCM reaction requires acidic conditions to neutralize the basic centre to allow the metathesis to proceed. Conclusions: SB1317 is a low molecular weight compact macrocyclic structure with a unique spectrum of kinase activity suggesting it may have particular utility in the treatment of hematological malignancies and potential for solid tumor therapy as well. SB1317 is currently being developed in the clinic by Tragara Pharmaceuticals (San Diego, CA) under the name TG02. 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 3591. doi:10.1158/1538-7445.AM2011-3591

Abstract 5436: The histone deacetylase inhibitor SB939 acts synergistically with Sorafenib in an orthotopic model of hepatucellular carcinoma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: SB939 is an hydroxamic acid based class I, II and IV HDAC inhibitor, with very favourable physiochemical, pharmaceutical and pharmacokinetic properties, resulting in an excellent bioavailability and marked accumulation in tumor tissue. In clinical phase I studies with SB939 in solid tumors, drug exposure was demonstrated to be higher and side effects milder/fewer compared to first generation HDAC inhibitors such as SAHA. Only 6.7% Grade3/4 fatigue (2/30 patients) or 6.9% (2/29 patients) were observed in two independent Phase I clinical studies making SB939 ideally suited for combination therapies. The aim of the present studies was to explore the potential of SB939 for the treatment of hepatocellular carcinoma (HCC). Methods: The effects of SB939 as single agent and combination therapy on the proliferation of HCC cell lines were explored in vitro studies. Single agent and combination therapy together with Sorafenib (Nexavar), a multitargeted VEGFR-b-Raf kinase inhibitor, which is now standard therapy for HCC, was explored in an orthotopic xenograft model of HCC, using Hep3B cells in female SCID mice. Mice were dosed daily with either 10 or 45 mg/kg Sorafenib per oral (p.o.) or with 125 mg/kg SB939 p.o. every other day, alone or in combination with Sorafenib. Sorafenib was dosed first and SB939 dosed 6 h thereafter. Plasma and tumor concentrations of SB939 and Sorafenib were measured using HPLC/MS, analyzed with the MassLynx software and PK parameters were calculated by a non-compartmental method using WinNonlin 4.0 software. Pharmacodynamic (PD) parameters such as HDAC inhibition, inhibition of the Raf-ras MAPK pathway, as well as anti-angiogenic effects of the treatment were analyzed by Western blotting and immunohistochemistry of tumor tissue. Results: HCC cell lines did not show high sensitivity to the anti-proliferative effects of either Sorafanib or SB939 (IC50 values for SB939 ranging from 0.9 to 1.23 μM). In contrast, in the in vivo model of HCC, Sorafenib at a dose of 10 mg/kg had no significant effect on tumor growth (tumor growth inhibition [TGI] was −12.6%), but a dose of 45 mg/kg was very effective (82% TGI). SB939 at a maximum tolerated dose and schedule was well tolerated and moderately effective in arresting tumor grow (40% TGI). When combined with the ineffective lower dose of Sorafenib, the anti-tumor efficacy of SB939 was significantly enhanced (58% TGI), with an in vivo combinatorial index of 0.4, indicating synergy. Conclusion: Although the Hep3B cell line is quite resistant to SB939 in vitro, SB939 is moderately effective as single agent therapy. Anti-tumor efficacy of SB939 was synergistically enhanced in combination with a low and ineffective dose of the VEGFR-Raf tyrosine kinase inhibitor Sorafenib, and the combination was well tolerated, providing a rationale for a phase II trial in combination with Sorafenib in HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5436.

Role of High Molecular Weight Calmodulin Binding Protein in Cardiac Muscle

High molecular weight calmodulin binding protein (HMWCaMBP) was originally discovered and purified in our laboratory and identified as a homologue of the calpains inhibitor, calpastatin. Calpains are Ca2+-dependent cysteine proteases that regulate various enzymes, transcription factors and structural proteins through limited proteolysis. Decreased expression of HMWCaMBP in ischemia suggests that it may be susceptible to proteolysis by calpains during ischemia and reperfusion. HMWCaMBP may protect its substrates from calpains in the normal myocardium, however during an early phase of ischemia and reperfusion with increased Ca2+ influx calpain activity exceeds HMWCaMBP activity leading to its proteolysis and of other substrates resulting in cellular injury. This study will lead to a better understanding of the pathology of disease states and the development of novel therapeutics. The exact role of HMWCaMBP in ischemia and reperfusion is not clear because most of the studies have been carried out in vitro. Therefore, it is essential that further research should be directed to in vivo studies. The nucleic acid and amino acid sequence information of HMWCaMBP will help us to do further characterization of this protein.