Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening.

Covalent probes can display unmatched potency, selectivity, and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered nonselective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against 10 cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. In contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.

Mechanism of interaction of novel uncharged, centrally active reactivators with OP-hAChE conjugates.

A library of more than 200 novel uncharged oxime reactivators was used to select and refine lead reactivators of human acetylcholinesterase (hAChE) covalently conjugated with sarin, cyclosarin, VX, paraoxon and tabun. N-substituted 2-hydroxyiminoacetamido alkylamines were identified as best reactivators and reactivation kinetics of the lead oximes, RS41A and RS194B, were analyzed in detail. Compared to reference pyridinium reactivators, 2PAM and MMB4, molecular recognition of RS41A reflected in its Kox constant was compromised by an order of magnitude on average for different OP-hAChE conjugates, without significant differences in the first order maximal phosphorylation rate constant k(2). Systematic structural modifications of the RS41A lead resulted in several-fold improvement with reactivator, RS194B. Kinetic analysis indicated K(ox) reduction for RS194B as the main kinetic constant leading to efficient reactivation. Subtle structural modifications of RS194B were used to identify essential determinants for efficient reactivation. Computational molecular modeling of RS41A and RS194B interactions with VX inhibited hAChE, bound reversibly in Michaelis type complex and covalently in the pentacoordinate reaction intermediate suggests that the faster reactivation reaction is a consequence of a tighter RS194B interactions with hAChE peripheral site (PAS) residues, in particular with D74, resulting in lower interaction energies for formation of both the binding and reactivation states. Desirable in vitro reactivation properties of RS194B, when coupled with its in vivo pharmacokinetics and disposition in the body, reveal the potential of this oxime design as promising centrally and peripherally active antidotes for OP toxicity.

Refinement of structural leads for centrally acting oxime reactivators of phosphylated cholinesterases.

We present a systematic structural optimization of uncharged but ionizable N-substituted 2-hydroxyiminoacetamido alkylamine reactivators of phosphylated human acetylcholinesterase (hAChE) intended to catalyze the hydrolysis of organophosphate (OP)-inhibited hAChE in the CNS. Starting with the initial lead oxime RS41A identified in our earlier study and extending to the azepine analog RS194B, reactivation rates for OP-hAChE conjugates formed by sarin, cyclosarin, VX, paraoxon, and tabun are enhanced severalfold in vitro. To analyze the mechanism of intrinsic reactivation of the OP-AChE conjugate and penetration of the blood-brain barrier, the pH dependence of the oxime and amine ionizing groups of the compounds and their nucleophilic potential were examined by UV-visible spectroscopy, (1)H NMR, and oximolysis rates for acetylthiocholine and phosphoester hydrolysis. Oximolysis rates were compared in solution and on AChE conjugates and analyzed in terms of the ionization states for reactivation of the OP-conjugated AChE. In addition, toxicity and pharmacokinetic studies in mice show significantly improved CNS penetration and retention for RS194B when compared with RS41A. The enhanced intrinsic reactivity against the OP-AChE target combined with favorable pharmacokinetic properties resulted in great improvement of antidotal properties of RS194B compared with RS41A and the standard peripherally active oxime, 2-pyridinealdoxime methiodide. Improvement was particularly noticeable when pretreatment of mice with RS194B before OP exposure was combined with RS194B reactivation therapy after the OP insult.