Diverse Redoxome Reactivity Profiles of Carbon Nucleophiles

Targeted covalent
inhibitors have emerged as a powerful approach
in the drug discovery pipeline. Key to this process is the identification
of signaling pathways (or receptors) specific to (or overexpressed
in) disease cells. In this context, fragment-based ligand discovery
(FBLD) has significantly expanded our view of the ligandable proteome
and affords tool compounds for biological inquiry. To date, such covalent
ligand discovery has almost exclusively employed cysteine-reactive
small-molecule fragments. However, functional cysteine residues in
proteins are often redox-sensitive and can undergo oxidation in cells.
Such reactions are particularly relevant in diseases, like cancer,
which are linked to excessive production of reactive oxygen species.
Once oxidized, the sulfur atom of cysteine is much less reactive toward
electrophilic groups used in the traditional FBLD paradigm. To address
this limitation, we recently developed a novel library of diverse
carbon-based nucleophile fragments that react selectively with cysteine
sulfenic acid formed in proteins via oxidation or hydrolysis reactions.
Here, we report analysis of sulfenic acid-reactive C-nucleophile fragments
screened against a colon cancer cell proteome. Covalent ligands were
identified for >1280 S-sulfenylated cysteines
present
in “druggable” proteins and orphan targets, revealing
disparate reactivity profiles and target preferences. Among the unique
ligand–protein interactions identified was that of a pyrrolidinedione
nucleophile that reacted preferentially with protein tyrosine phosphatases.
Fragment-based covalent ligand discovery with C-nucleophiles affords
an expansive snapshot of the ligandable “redoxome” with
significant implications for covalent inhibitor pharmacology and also
affords new chemical tools to investigate redox-regulation of protein
function.

靶向共价抑制剂（Targeted covalent inhibitors）已成为药物发现管线中极具潜力的研究策略。该流程的核心在于鉴定疾病细胞特异性（或过表达）的信号通路（或受体）。在此背景下，基于片段的配体发现（Fragment-based ligand discovery, FBLD）极大拓展了我们对可配体化蛋白质组的认知，并为生物学探究提供了工具化合物。迄今为止，此类共价配体发现几乎仅采用半胱氨酸反应性小分子片段。然而，蛋白质中的功能性半胱氨酸残基往往具有氧化还原敏感性，可在细胞内发生氧化反应。这类反应在与活性氧过度生成相关的疾病（如癌症）中尤为重要。半胱氨酸被氧化后，其硫原子对传统FBLD范式中所用亲电基团的反应性会显著降低。为解决这一局限，我们近期开发了一类全新的多样化碳基亲核片段库，这类片段可与蛋白质中经氧化或水解反应生成的半胱氨酸次磺酸选择性结合。本文报道了针对结肠癌细胞蛋白质组筛选的次磺酸反应性碳亲核片段的分析研究。研究在“可药性”蛋白及孤儿靶点中鉴定出超过1280个S-亚磺酰化半胱氨酸对应的共价配体，揭示了迥异的反应谱与靶点偏好性。其中发现的独特配体-蛋白相互作用包括一类优先与蛋白酪氨酸磷酸酶结合的吡咯烷二酮亲核试剂。基于碳亲核片段的共价配体发现，可全面展现可配体化“氧化还原组”的全景，这对共价抑制剂药理学研究具有重要意义，同时也为探究蛋白质功能的氧化还原调控机制提供了全新的化学工具。