Covalent Inhibitors Allosterically Block the Activation of Rho Family Proteins and Suppress Cancer Cell Invasion.

Zhongya Sun,Hong Ding,Feng Yang,Minjia Tan,Pan Xu,Hongru Tao,Fulin Lian,Jing Huang,Fengcai Zhang,Mingrui Zhu,Wen Zhou,Yongjun Dang,Yuanyuan Zhang,Shijie Chen,Bing Zhou,Rukang Zhang,Bo Liu,Kaixian Chen,Hualiang Jiang,Hao Zhang,Cheng Luo,Chuanpeng Liu,Naixia Zhang,Liping Liao,Wenchao Lu,Yue Li

doi:10.1002/advs.202000098

Abstract

The Rho family GTPases are crucial drivers of tumor growth and metastasis. However, it is difficult to develop GTPases inhibitors due to a lack of well‐characterized binding pockets for compounds. Here, through molecular dynamics simulation of the RhoA protein, a groove around cysteine 107 (Cys107) that is relatively well‐conserved within the Rho family is discovered. Using a combined strategy, the novel inhibitor DC‐Rhoin is discovered, which disrupts interaction of Rho proteins with guanine nucleotide exchange factors (GEFs) and guanine nucleotide dissociation inhibitors (GDIs). Crystallographic studies reveal that the covalent binding of DC‐Rhoin to the Cys107 residue stabilizes and captures a novel allosteric pocket. Moreover, the derivative compound DC‐Rhoin04 inhibits the migration and invasion of cancer cells, through targeting this allosteric pocket of RhoA. The study reveals a novel allosteric regulatory site within the Rho family, which can be exploited for anti‐metastasis drug development, and also provides a novel strategy for inhibitor discovery toward “undruggable” protein targets.

Highlights

It is difficult to develop GTPases inhibitors due to a lack of well-characterized binding pockets for compounds
From our comprehensive analysis of cysteine residue locations in all small GTPases, we observed that cysteine 107 (Cys107) is only present in Rho family proteins, thereby affording the opportunity to use this residue as a means to achieve selectivity for Rho within the Ras superfamily (Figure 1b; Figure S1e, Supporting Information)
We synthesized a series of derivative compounds of DC-Rhoin, the biological effectiveness of these compounds were determined by pull down assay and nucleotide exchange assay, these derivatives inhibited the interaction of RhoA with Leukemia-associated RhoGEF (LARG) or guanine nucleotide dissociation inhibitors (GDIs), as well as the exchange rate of guanosine diphosphate (GDP)/guanosine triphosphate (GTP) catalyzed by LARG with similar or little inferior activity compared with DC-Rhoin (Figure S4, Supporting Information)

Summary

Introduction

It is difficult to develop GTPases inhibitors due to a lack of well-characterized binding pockets for compounds. The divided into several subfamilies on the basis of structures and functions.[4,5] Among them, RhoA, Rac, and Cdc are the best characterized members.[6] The Rho derivative compound DC-Rhoin inhibits the migration and invasion of family proteins repeatedly cycle between cancer cells, through targeting this allosteric pocket of RhoA. Starting with molecular dynamics simulations of the RhoA protein, we discovered a relatively targetable cysteine residue Cys107, as well as a potential compound binding pocket, which is close to a functional phosphorylation site. Our studies revealed that the Rho family GTPases can be regulated by covalent inhibitors via targeting the relatively conserved Cys107 residue, demonstrating that the Cys107-Lock pocket (CLocK) pocket is druggable and provides an ideal starting point for the design of more potent and selective RhoA inhibitors

Results

Discussion

Experimental Section

Conflict of Interest