Designing isothiocyanates that target macrophage migration inhibitory factor
Isothiocyanates (ITCs) are a class of compounds found in edible cruciferous vegetables. They have been extensively studied both in vitro and in vivo for their preventive and therapeutic effects in number of diseases, including inflammation and cancer. Increasing epidemiological evidence demonstrates that increased consumption of cruciferous vegetables leads to improved outcomes in many cancers and inflammatory diseases. These benefits may be attributable to ITCs. Evidence suggests that ITCs exert their biological effects through distinct but interconnected signalling pathways. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with well-characterised roles in inflammation, and innate and adaptive human immune responses. MIF is overexpressed in various cancers, including colorectal cancer, pancreatic cancer, leukemia, prostate cancer and breast cancer as well as inflammatory conditions such as acute pancreatitis, septic shock, rheumatoid arthritis and atherosclerosis. MIF exerts its biological activity by binding to the cell surface receptor CD74 as well as intracellular signalling proteins. MIF also possesses keto-enol tautomerase activity against dopachrome, dopachrome methyl ester, phenylpyruvate and p-hydroxyphenylpyruvate with an N-terminal proline as the key catalytic amino acid. Whilst no physiological substrates of MIF have yet to be identified, inhibition of tautomerase activity has been associated with a loss of biological function of MIF. Recent studies have identified small molecule MIF inhibitors for treating inflammatory diseases and other pathologies. In 2009, our research group identified ITCs as irreversible inhibitors of MIF’s tautomerase activity via covalent attachment to the N-terminal catalytic proline. Benzyl and phenethyl ITC derivatives that orient in “upward” binding mode have become attractive lead compounds for development of MIF inhibitors, especially with a sulfonamide substituent. In this thesis, a library of ITCs featuring benzyl, phenethyl and phenyl propyl isothiocyanates with sulfonamide and carboxylate substituents were designed, synthesised and purified to determine whether the acidic nature of these substituents or hydrogen bonding characteristics are essential for inhibiting MIF. The derivatives were expected to bind in “upward” orientation. Ten ITCs synthesised from commercially available starting materials were tested for their ability to inhibit recombinant human MIF (rhMIF) and MIF activity in Jurkat T-lymphoma cells. rhMIF was expressed in Escherichia coli and purified using three protocols in an attempt to obtain a homogenous form with post-translational cleavage of N-terminal methionine. All the three protocols produced heterogeneous form of protein with some of the protein retaining the N-terminal methionine. Among the three, protein obtained from a single preparation (protocol 1) was used to assess the synthesised ITCs as it contained the least amount of inactive protein. ITCs with sulfonamide substituents LPR07, LPR16, LPR17, LPR25 and LPR26 showed good MIF tautomerase inhibition with submicromolar IC50 values in both enzyme and cell-based assays. ITCs with carboxylate substituents LPR06 and LPR08 showed reduced MIF tautomerase inhibition. Neither showed strong inhibition in a cell-based assay. The phenethyl (LPR25) and phenyl propyl (LPR26) derivatives of racemic sulfonyl methylpiperidine ITC (LPR17) were synthesised and assessed. No correlation was observed between activity with differing linker length (from one to three carbons). Both enantiomers of LPR17 were synthesised to identify the more active stereoisomer. The R enantiomer (R-LPR29) was shown to be more potent than the S enantiomer (S-LPR32) in both rhMIF and Jurkat cell MIF tautomerase assays. LPR16 and LPR17 showed good inhibitory activity in both assays. The binding affinity, Ki, rate of inactivation, kinact and specific reactivity, kinact/ Ki of PEITC, LPR16 and LPR17 was studied using a two-step covalent inhibition mechanism under special experimental conditions and analysed using DynaFit software. LPR17 showed low reactivity with high binding affinity and LPR16 showed high reactivity with less binding affinity. However, a linear correlation between the specific reactivity and IC50 values of LPR16 and LPR17 was not observed. The docking studies of LPR16, LPR02, LPR07, R-LPR29, S-LPR32, LPR25 and LPR26 showed cis and trans thiourea bond formation with “upward” binding orientation as expected and the potential for sulfonamide hydrogen bonding and π stacking interactions with the adjacent Tyr-37 favoured potent MIF tautomerase inhibitory activity. The hydrogen bonding characteristics of the sulfonamide ITC derivatives are required for MIF inhibition but not the acidic nature of either sulfonamide or carboxylate substituents. The lead molecules LPR16 and LPR17 were examined for their cytotoxicity against CT26 colorectal cancer cells. LPR16 and LPR17 showed dose-dependent cytotoxic potential towards CT26 colorectal cancer cell viability. LPR16 and LPR17 were also evaluated by the National Cancer Institute (NCI) 60 human tumour cell lines screen (NCI60) at 10 µM. The single-dose response data of LPR16 and LPR17 showed good growth inhibition of many cancer cell lines, especially gastrointestinal cancer cell lines. At 10 µM, LPR16 and LPR17 reduced the growth (below 60% growth percent) of colon (COLO 205, HCT-116, HCT-15, HT29, KM12 and SW-620) and renal cancer cells (ACHN, CAKI-1 and RXF 393). LPR16 was selected for further screening and evaluated at five doses (10 nM to 100 µM). LPR16 showed strong growth inhibition and cytotoxicity against colon cancer, renal cancer, leukemia, melanoma, ovarian and breast cancer cell lines. In summary, these results show that MIF’s tautomerase activity was inhibited by synthesised ITCs with the majority more potent than other reported ITCs and other MIF covalent inhibitors. There is a considerable scope for further development of ITCs as potent MIF tautomerase inhibitors for different diseases including gastrointestinal cancers or inflammatory diseases.
Advisor: Tyndall, Joel; Vernall, Andrea; Wilbanks, Sigurd
Degree Name: Doctor of Philosophy
Degree Discipline: School of Pharmacy
Publisher: University of Otago
Keywords: Isothiocyanates; Isothiocyanates; ITCs; Macrophage migration inhibitory factor; MIF; Tautomerase activity; Covalent; DynaFit; NCI60
Research Type: Thesis