Turner Lewis D, Nielsen Alexander L, Lin Lucy, Pellett Sabine, Sugane Takashi, Olson Margaret E, Johnson Eric A, Janda Kim D
Departments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology, Worm Institute of Research and Medicine (WIRM), Scripps Research 10550 N Torrey Pines Road La Jolla CA 92037 USA
Center for Biopharmaceuticals & Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen Universitetsparken 2 DK-2100 Copenhagen Denmark.
RSC Med Chem. 2021 May 19;12(6):960-969. doi: 10.1039/d1md00089f. eCollection 2021 Jun 23.
Botulinum neurotoxin A (BoNT/A) is categorized as a Tier 1 bioterrorism agent and persists within muscle neurons for months, causing paralysis. A readily available treatment that abrogates BoNT/A's toxicity and longevity is a necessity in the event of a widespread BoNT/A attack and for clinical treatment of botulism, yet remains an unmet need. Herein, we describe a comprehensive warhead screening campaign of bifunctional hydroxamate-based inhibitors for the irreversible inhibition of the BoNT/A light chain (LC). Using the 2,4-dichlorocinnamic hydroxamic acid (DCHA) metal-binding pharmacophore modified with a pendent warhead, a total of 37 compounds, possessing 13 distinct warhead types, were synthesized and evaluated for time-dependent inhibition against the BoNT/A LC. Iodoacetamides, maleimides, and an epoxide were found to exhibit time-dependent inhibition and their measured as a description of reactivity. The epoxide exhibited superior time-dependent inhibition over the iodoacetamides, despite reacting with glutathione (GSH) 51-fold slower. The proximity-driven covalent bond achieved with the epoxide inhibitor was contingent upon the vital hydroxamate-Zn anchor in placing the warhead in an optimal position for reaction with Cys165. Monofunctional control compounds exemplified the necessity of the bifunctional approach, and Cys165 modification was confirmed through high-resolution mass spectrometry (HRMS) and ablation of time-dependent inhibitory activity against a C165A variant. Compounds were also evaluated against BoNT/A-intoxicated motor neuron cells, and their cell toxicity, serum stability, and selectivity against matrix metalloproteinases (MMPs) were characterized. The bifunctional approach allows the use of less intrinsically reactive electrophiles to intercept Cys165, thus expanding the toolbox of potential warheads for selective irreversible BoNT/A LC inhibition. We envision that this dual-targeted strategy is amenable to other metalloproteases that also possess non-catalytic cysteines proximal to the active-site metal center.
肉毒杆菌神经毒素A（BoNT/A）被列为一级生物恐怖主义制剂，可在肌肉神经元中持续存在数月，导致麻痹。在发生广泛的BoNT/A攻击以及肉毒中毒的临床治疗中，急需一种能够消除BoNT/A毒性和延长其寿命的现成治疗方法，但这一需求仍未得到满足。在此，我们描述了一项针对基于双功能异羟肟酸酯的抑制剂的全面弹头筛选活动，用于不可逆地抑制BoNT/A轻链（LC）。使用带有悬垂弹头修饰的2,4-二氯肉桂酸异羟肟酸（DCHA）金属结合药效基团，合成了总共37种具有13种不同弹头类型的化合物，并评估了它们对BoNT/A LC的时间依赖性抑制作用。发现碘乙酰胺、马来酰亚胺和一种环氧化物表现出时间依赖性抑制作用，并将其 作为反应活性的描述进行测量。尽管环氧化物与谷胱甘肽（GSH）的反应速度慢51倍，但其表现出比碘乙酰胺更好的时间依赖性抑制作用。环氧化物抑制剂实现的邻近驱动共价键取决于至关重要的异羟肟酸锌锚定基团，该基团将弹头置于与Cys165反应的最佳位置。单功能对照化合物证明了双功能方法的必要性，通过高分辨率质谱（HRMS）和对C165A变体的时间依赖性抑制活性的消除证实了Cys165的修饰。还对化合物进行了针对BoNT/A中毒运动神经元细胞的评估，并对其细胞毒性、血清稳定性和对基质金属蛋白酶（MMPs）的选择性进行了表征。双功能方法允许使用内在反应性较低的亲电试剂来拦截Cys165，从而扩展了用于选择性不可逆抑制BoNT/A LC的潜在弹头工具箱。我们设想这种双靶点策略适用于其他在活性位点金属中心附近也具有非催化性半胱氨酸的金属蛋白酶。
