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

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.

Progress in the therapeutic inhibition of Cdc42 signalling

“…A distinctly different method of inhibiting Cdc42–GEF interactions exploits a cysteine located spatially close to but not directly within the interface of the complex, to inhibit GEF-catalyzed nucleotide exchange. A set of covalent, irreversible inhibitor molecules have recently been developed targeting Cys107 of RhoA [ 43 ]. The tightest binder, DC-Rhoin04, inhibited nucleotide exchange on RhoA with a IC 50 of ∼3 µM [ 43 ].…”

“…A set of covalent, irreversible inhibitor molecules have recently been developed targeting Cys107 of RhoA [ 43 ]. The tightest binder, DC-Rhoin04, inhibited nucleotide exchange on RhoA with a IC 50 of ∼3 µM [ 43 ]. The screened molecules all contain electron-deficient alkenes capable of reacting with the nucleophilic cysteine of interest.…”

Drug design targeting active posttranslational modification protein isoforms

“…As a proof of concept study, 54 we have developed a covalent and selective inhibitor of RhoA using PTMI‐DD. As a branch of the Ras superfamily, the Rho family proteins also repeatedly cycle between the active guanosine triphosphate (GTP)‐bound state and the inactive guanosine diphosphate (GDP)‐bound state.…”

“…Some of them have shown early treatment benefits in clinical studies, such as the first KRAS G12C irreversible inhibitor, AMG510, 246 in multiple solid tumors and MAK683 of EED targeting allosteric inhibitor of PRC complex. Most recently discovered inhibitors such as a potent reversible and allosteric DNMT1 inhibitor GSK3482364, 374 an irreversible H3K36 methyltransrase NSD1 inhibitor BT5, 375 and the irreversible RhoA inhibitor DC‐Rohin, 54 providing attractive chemical starting points to further investigate those novel mechanism driven compounds into more promising therapeutics as precision medicines.…”

“…Under the guidance of this strategy, the intersection of chemical space and biological space will be further extended. Using PTMI‐DD, we have identified a covalent inhibitor of Ras homolog family member A (RhoA) protein, which is believed to be undruggable target despite its crucial roles in biological functions and drug design 54 . More importantly, due to the specific roles of PTM isoforms in regulating biological functions, small molecules targeting such isoforms will potentially have better selectivity profiles owning to not interfering with normal functions of the wild‐type counterpart.…”