An NMR-Guided Screening Method for Selective Fragment Docking and Synthesis of a Warhead Inhibitor

Abstract

Selective hits for the glutaredoxin ortholog of Brucella melitensis are determined using STD NMR and verified by trNOE and 15 N-HSQC titration. The most promising hit, RK207, was docked into the target molecule using a scoring function to compare simulated poses to experimental data. After elucidating possible poses, the hit was further optimized into the lead compound by extension with an electrophilic acrylamide warhead. We believe that focusing on selectivity in this early stage of drug discovery will limit cross-reactivity that might occur with the human ortholog as the lead compound is optimized. Kinetics studies revealed that lead compound 5 modified with an ester group results in higher reactivity than an acrylamide control; however, after modification this compound shows little selectivity for bacterial protein versus the human ortholog. In contrast, hydrolysis of compound 5 to the acid form results in a decrease in the activity of the compound. Together these results suggest that more optimization is warranted for this simple chemical scaffold, and opens the door for discovery of drugs targeted against glutaredoxin proteins-a heretofore untapped reservoir for antibiotic agents.

Novel chloroacetamido compound CWR-J02 is an anti-inflammatory glutaredoxin-1 inhibitor

“…Accordingly, the area surrounding Thr-50 may represent a potential recognition site on Grx1to be exploited for subsequent design of more selective Grx1 inhibitors. In silico tools could also be used to optimize J02 derivatives, as recently employed to identify acrylamide-containing compounds as potential inhibitors for bacterial Grx1 [ 20 ].…”

“…Accordingly, by optimizing the non-covalent binding of J02 analogs in the proximity of the active site Cys-22, the chloroacetamido moiety of J02 could be replaced by the less reactive fluroacetamido moiety, thereby greatly limiting its general reactivity with less reactive protein thiols. In addition, in silico tools could also be used to optimize derivative structures of J02 for non-covalent interaction with Grx1, as recently employed for optimizing acetonitrile-derived inhibitors for bacterial Grx1 [ 20 ]…”

Identifying Ortholog Selective Fragment Molecules for Bacterial Glutaredoxins by NMR and Affinity Enhancement by Modification with an Acrylamide Warhead

“…RK395 has about four-fold higher affinity for PaGRX over hGRX1, and eleven fold over BrmGRX. The second best hit, RK207, was found to bind with BrmGRX with two and half fold higher affinity than with hGRX1 and about two fold higher than PaGRX [6]. Upon close scrutinization of the rSTD%, K d and L.E.…”

“…We considered this portion of the fragment to be a reasonable candidate for chemical extension with an acrylamide warhead group into the active site. In our previous FBDD investigation of a GRX we used molecular dynamics (MD) simulations, computational CSP calculations, and fragment docking to predict the binding mode of the species selective lead compound, RK207, to BrMGRX [6]. We were reluctant to use a similar strategy with a preliminary NMR-based model of PaGRX but wanted to determine if other tools such as FTMap, an epitope mapping tool that takes advantage of docking of probe structures, to gain a hint at the binding mode of RK395 with PaGRX.…”

“…With the lead compound now serving as a driving group, it can increase residence time of the warhead near the active site. This confers a kinetic advantage over the warhead as a free ligand [6]. The driving group also enables target specificity, eliminating some of the concerns associated with covalent inhibitors [7].…”