We next investigated the specificity of compounds and for TS

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 GSK J4 free base 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). Previous studies from our lab looked at the effect of interfering with crossover helix interactions in TS-DHFR. One study utilized peptide mimetics of the crossover helix to inhibit wild-type TS-DHFR noncompetitively, and with an IC of 230 μM. A second study used a virtual screening approach to identify small molecules capable of binding in a non-active site pocket below the crossover helix, adjacent to the TS domain. In the latter example, the compound Flavin Mononucleotide (FMN) was also found to inhibit DHFR activity noncompetitively, though with an improved IC value of 55 μM. Here too, we demonstrate that DHFR activity can be inhibited by disrupting interactions between the crossover helix and DHFR with small molecules aimed at a novel non-active site pocket. Collectively, these efforts help underscore the importance of crossover helix interactions for DHFR catalysis. Additionally, our findings lend support to the use of virtual screening and structure-guided modeling approaches in the discovery of compounds targeting novel binding pockets. Computational modeling proved especially helpful in instructing the design of, compound , which is the first example of a covalent inhibitor designed to target a non-catalytic pocket in TS-DHFR. Targeting non-catalytic cysteine residues is a promising strategy for lead generation and optimization, and is yet to be fully explored in drug discovery efforts aimed at developing -specific inhibitors.