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  • In order to derive a better understanding of the


    In order to derive a better understanding of the results from our SAR study, we utilized Glide to model the binding of the SAR compounds with the non-active site pocket of TS-DHFR shown in C. The majority of models position the phenolic moiety of these compounds within the non-active site pocket, while exposing a chlorine substituent to solvent. In some models, the chlorine substituent is positioned near residue Cys44, located at the C-terminal end of the DHFR B helix. A representative model for the binding of the SAR compounds with the non-active site pocket of TS-DHFR is shown in C. Because of the close proximity of this cysteine residue to the non-active site pocket, compounds and were designed to covalently target Cys44 with a sulfhydryl group (B and 5C, ). Initially, 500 µM of caffeic acid australia and demonstrated modest inhibitory activity, about 14% and 21%, respectively, using the original assay conditions in which each compound was incubated for 10 min with DHFR. Because the formation of a disulfide bond between the covalent compounds and Cys44 may take longer than 10 min to occur, we incubated the potential covalent inhibitors with TS-DHFR for 1 h, 16 h, 48 h and 72 h. We observed that both covalent inhibitors demonstrated time-dependent inhibition, with almost complete inhibition at 72 h for and 48 h for (). DMSO controls incubated simultaneously with compound-containing samples did not suffer a loss of enzymatic activity, indicating that compound and are responsible for the observed inhibition. We prepared a Cys44Ser mutant TS-DHFR to further evaluate whether compound and bind within the non-active site pocket and form a covalent interaction with residue Cys44 of the protein. Both compounds were pre-incubated with Cys44Ser mutant TS-DHFR for 16 h prior to initiation of the reaction. Compound displayed a 2-fold decrease in inhibition between wild-type and Cys44Ser DHFR, about 68% and 38%, respectively (). This significant decrease in inhibition would suggest that does indeed bind within our non-active site pocket, forming a disulfide bond with residue Cys44. We did not, however, observe a discernable change in DHFR inhibition for compound (). As a negative control, we also incubated compound , the parent compound of which does not contain a sulfhydryl group, with both wild-type and Cys44Ser mutant TS-DHFR. We observed no significant change in inhibition in the control experiment, indicating that, unlike , the activity of is mediated by disulfide bond formation. When 10 mM DTT was added to the reaction mixture before incubation of enzyme and covalent compounds, inhibition was abolished, while inhibited DHFR by ∼31% with no change in inhibition for (). This suggest that DTT in the preincubation solution sequesters compound and prevents it from interacting with the enzyme. Additionally, this data suggests that does not inhibit DHFR by interacting with the active site or an alternative cysteine residue in the DHFR domain, or through aggregation. While DTT does negate some of inhibition of DHFR, this data would suggest that may be binding in more than one location. There is no such effect observed for the control compound , as expected given the lack of a sulfhydryl group. We next investigated the specificity of compounds and for TS-DHFR over human DHFR, which does not have a cysteine residue corresponding to Cys44 of TS-DHFR (). Both compounds were incubated with human DHFR for 16 h to evaluate any inhibitory effects. Compound , was found to be highly selective for DHFR, having negligible effect on human DHFR. This is in dramatic contrast to , which inhibited human DHFR by about 95% (). The results for compound and suggest that while both compounds demonstrate time-dependent inhibition of DHFR, appears to bind the non-active site pocket, and demonstrates high specificity for DHFR. The decrease in inhibition observed for between wild-type and Cys44Ser TS-DHFR, suggests that the formation of a disulfide bond is required for to bind with the non-active site pocket. Furthermore, our preincubation of with enzyme and 10 mM DTT data suggests that, does not indiscriminately inhibit DHFR by binding to the active site or randomly interacting with the enzyme. On the other hand, the time-dependent inhibition demonstrated by suggests that this compound may be targeting a different cysteine residue. DHFR contains two additional cysteine residues in addition to Cys44, Cys113 and Cys164 () Cys113 resides in the active site pocket, while Cys164 is located near the interface between the TS and DHFR domains. Perhaps the time-dependent inhibition is due to targeting one of these residues. Since this inhibition is only reversed by 31% for DHFR when was preincubated with enzyme and 10 mM DTT, this suggests an alternate inhibition pathway may also be operative. The observed time dependence inhibition of may occur through another mechanism, possibly due to the para-bromo phenol ring of the compound being unstable and susceptible towards oxidation. This could in turn produce a quinonoid type electrophilic species. Similar time dependent inhibition results are observed for the human DHFR and (data not shown).