Instead of using conventional enzymatic screening, we opted for a direct cellular phenotypic screen, based upon the wisdom that more drug-like molecules would emerge from such an endeavor. investigated a family of neurotoxins are a family of neurotoxins produced by substitution and the resulting = 471 (A) or 577 (B)). GCSecG was reduced with TCEP cleaving the SeCS bond and then added to6. After 1 h of incubation, acylated peptides (A and B) were observed. Samples without 6 or peptide were used Rabbit polyclonal to PHF13 as controls. Having established TrxR as the protein of relevance for our most potent inhibitors, we next sought to ascertain kinetic parameters of 6 and 23 through Ellmans reagent, DTNB (5, 5-dithio-bis-[2-nitrobenzoic acid]) assay. Here, a series of concentrations were tested for each of the Oglemilast two inhibitors when TrxR and DTNB were added to the reaction milieu, while the whole process was monitored at 412 nm. Initial velocity analysis suggested the inhibitors underwent a two-step mechanism, a noncovalent, reversible binding step that was then followed by covalent killing of Oglemilast the enzymes activity (see the Supporting Information). Further kinetic analysis was conducted by DynaFit4 using a two-step model, (35) granting a Ki of 4.2 0.3 and 51 8 M, and a kinact of (0.78 0.09) 10?3 and (3.2 0.5) 10?3 s?1 for 6 and 23, respectively. From these metrics, what is clear is that 6s acetyl ester has greater affinity to TrxR than 23s t-butyl sulfinic ester. However, 23 displayed a more potent kinact, indicating a greater reactivity of the sulfinic ester, which in turn led to a less selective process between the thiol- and selenol-residues found within TrxR. On the basis of the summation of all analyses, a credible mechanistic scheme that fits the data acquired is proposed in Figure 8. Open in a separate window Figure 8 Cartoon presenting a plausible mechanistic scheme for TrxR inhibition by inhibitors 6 and 3. Note, X = C or S representing 6 or 23, respectively. Having proposed an acyl transfer mechanistic scheme, we make note that a comparable pathway was reported by Badet and co-workers, where they presented the mechanism-based inactivation of glucosamine 6-phosphate synthase via enzymatic hydrolysis of an amide bond, ultimately liberating thioquinone.(20g) Here, inhibition relied on the enzymes innate function to hydrolyze glutamine to glutamate. This report led us to query the specificity of our inhibitor, and as a stern test, 6 was examined with glutathione peroxidase (GPx), which possesses a selenocysteine motif within its active site, akin to TrxR. As anticipated, 6 did not show inhibition against GPx at any of the concentrations examined (Figure S8.1), emphasizing the point that 6 does not engage all Sec-containing enzymes. Although 6 did not engage GPx, promiscuous inhibitor profiling between TrxR and GPx has been previously disclosed, (20f, 33) and has been attributed to active site accessibility between these two enzymes. Thus, it has been established that the selenocysteine motif leveraged within TrxR is C-terminal bound(32) and highly flexible, which provides a rationale for TrxRs broad substrate scope, whereas Sec of GPx is sequestered inside of the enzymes active site.(36) Considering the small architecture of 6, however, it is also conceivable that the initial interaction/binding of 6 to the protein plays a critical role, as shown in our two-step model based on the kinetics observed. Finally, in light of the Oglemilast thiol-reactive nature of 23, vide supra, the stability of these inhibitors in a Oglemilast thiol-rich concentric cellular environment needed to be addressed. The sensitivity of the sulfinic ester moiety toward glutathione (GSH), a universal cellular component, has been reported.(19)Hence, we conducted MS-based stability studies on 6 and 23 in various medium using deuterium-labeled compounds as standards. While both 6 and 23 displayed similar stability in PBS and culture medium (see the Supporting Information), a significant difference was observed in 5 mM GSH solution. Inhibitor 6 exhibited approximately an 11 h half-life; in contrast, 23 was completely depleted within 0.5 h. These results are readily interpreted as neurons are known to possess an.