Serendipitous and Systematic Chemoproteomic Discovery of MBLAC2, HINT1, and NME1‑4 Inhibitors from Histone Deacetylase-Targeting Pharmacophores

TL;DR

This study discovers new inhibitors for MBLAC2 and other metalloenzymes, revealing off-target effects and providing tools for future drug development.

Contribution

The paper reports the first selective MBLAC2 inhibitors and identifies the first inhibitors for NME1-4 and HINT1.

Findings

KV-65 and KV-79 are nanomolar MBLAC2 inhibitors with over 60-fold selectivity over HDACs.

KV-30 is the first inhibitor of HINT1, confirmed by cocrystal structure analysis.

Pharmacophores for NME1-4 and HINT1 were identified, enabling future inhibitor design.

Abstract

Metalloenzyme inhibitors
often incorporate a hydroxamic acid moiety
to bind the bivalent metal ion cofactor within the enzyme’s
active site. Recently, inhibitors of Zn2+-dependent histone
deacetylases (HDACs), including clinically advanced drugs, have been
identified as potent inhibitors of the metalloenzyme MBLAC2. However,
selective chemical probes for MBLAC2, which are essential for studying
its inhibitory effects, have not yet been reported. To discover highly
selective MBLAC2 inhibitors, we conducted chemoproteomic target deconvolution
and selectivity profiling of a library of hydroxamic acid-type molecules
and other metal-chelating compounds. This screen revealed MBLAC2 as
a frequent off-target of supposedly selective HDAC inhibitors, including
the HDAC6 inhibitor SW-100. Profiling a focused library of SW-100-related
phenylhydroxamic acids led to identifying two compounds, KV-65 and
KV-79, which exhibit nanomolar binding affinity for MBLAC2 and over
60-fold selectivity compared to HDACs. Interestingly, some phenylhydroxamic
acids were found to bind additional off-targets. We identified KV-30
as the first drug-like inhibitor of the histidine triad nucleotide-binding
protein HINT1 and confirmed its mode of inhibition through a cocrystal
structure analysis. Furthermore, we report the discovery of the first
inhibitors for the undrugged nucleoside diphosphate kinases NME1,
NME2, NME3, and NME4. Overall, this study maps the target and off-target
landscape of 53 metalloenzyme inhibitors, providing the first selective
MBLAC2 inhibitors. Additionally, the discovery of pharmacophores for
NME1-4 and HINT1 establishes a foundation for the future design of
potent and selective inhibitors for these targets.

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