Al-Horani Rami A, Afosah Daniel K, Kar Srabani, Aliter Kholoud F, Mottamal Madhusoodanan
Division of Basic Pharmaceutical Sciences, College of Pharmacy, Xavier University of Louisiana, New Orleans, LA, USA.
Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, VA, USA.
RPS Pharm Pharmacol Rep. 2023 Jan 6;2(1):rqad001. doi: 10.1093/rpsppr/rqad001. eCollection 2023 Jan.
Cathepsin G (CatG) is a cationic serine protease with wide substrate specificity. CatG is reported to play a role in several inflammatory pathologies. Thus, we aimed at identifying a potent and allosteric inhibitor of CatG to be used as a platform in further drug development opportunities.
Chromogenic substrate hydrolysis assays were used to evaluate the inhibition potency and selectivity of SPGG towards CatG. Salt-dependent studies, Michaelis-Menten kinetics and SDS-PAGE were exploited to decipher the mechanism of CatG inhibition by SPGG. Molecular modelling was also used to identify a plausible binding site.
SPGG displayed an inhibition potency of 57 nM against CatG, which was substantially selective over other proteases. SPGG protected fibronectin and laminin against CatG-mediated degradation. SPGG reduced V of CatG hydrolysis of a chromogenic substrate without affecting K, suggesting an allosteric mechanism. Resolution of energy contributions indicated that non-ionic interactions contribute ~91% of binding energy, suggesting a substantial possibility of specific recognition. Molecular modelling indicated that SPGG plausibly binds to an anion-binding sequence of SRRVRRNRN.
We present the discovery of SPGG as the first small molecule, potent, allosteric glycosaminoglycan mimetic inhibitor of CatG. SPGG is expected to open a major route to clinically relevant allosteric CatG anti-inflammatory agents.
组织蛋白酶G（CatG）是一种具有广泛底物特异性的阳离子丝氨酸蛋白酶。据报道，CatG在多种炎症性疾病中发挥作用。因此，我们旨在鉴定一种有效的CatG变构抑制剂，作为进一步药物开发的平台。
采用发色底物水解试验评估SPGG对CatG的抑制效力和选择性。利用盐依赖性研究、米氏动力学和SDS-PAGE来阐明SPGG对CatG的抑制机制。还使用分子建模来确定一个可能的结合位点。
SPGG对CatG的抑制效力为57 nM，对其他蛋白酶具有显著的选择性。SPGG保护纤连蛋白和层粘连蛋白免受CatG介导的降解。SPGG降低了发色底物CatG水解的Vmax而不影响Km，提示存在变构机制。能量贡献解析表明，非离子相互作用贡献了约91%的结合能，表明存在特异性识别的很大可能性。分子建模表明，SPGG可能与SRRVRRNRN的阴离子结合序列结合。
我们报道了SPGG作为首个小分子、强效、变构糖胺聚糖模拟物CatG抑制剂的发现。预计SPGG将为临床上相关的变构CatG抗炎药物开辟一条主要途径。
