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CDC42 GTPases (RHOJ, CDC42, and RHOQ) are overexpressed in multiple tumor types and activate pathways critical for tumor growth, angiogenesis, and metastasis. Recently, we reported the discovery of a novel lead compound, ARN22089, which blocks the interaction of CDC42 GTPases with specific downstream effectors. ARN22089 blocks tumor growth in BRAF mutant mouse melanoma models and patient-derived xenografts (PDXs) in vivo. ARN22089 also inhibits tumor angiogenesis in three-dimensional vascularized microtumor models in vitro. Notably, ARN22089 belongs to a novel class of trisubstituted pyrimidines. Based on these results, we describe an extensive structure–activity relationship of ∼30 compounds centered on ARN22089. We discovered and optimized two novel inhibitors (27, ARN25062, and 28, ARN24928), which are optimal back-up/follow-up leads with favorable drug-like properties and in vivo efficacy in PDX tumors. These findings further demonstrate the potential of this class of CDC42/RHOJ inhibitors for cancer treatment, with lead candidates ready for advanced preclinical studies.

Here, we show that alchemical free energy calculations can quantitatively compute the effect of mutations at the protein–protein interface. As a test case, we have used the protein complex formed by the small Rho-GTPase CDC42 and its downstream effector PAK1, a serine/threonine kinase. Notably, the CDC42/PAK1 complex offers a wealth of structural, mutagenesis, and binding affinity data because of its central role in cellular signaling and cancer progression. In this context, we have considered 16 mutations in the CDC42/PAK1 complex and obtained excellent agreement between computed and experimental data on binding affinity. Importantly, we also show that a careful analysis of the side-chain conformations in the mutated amino acids can considerably improve the computed estimates, solving issues related to sampling limitations. Overall, this study demonstrates that alchemical free energy calculations can conveniently be integrated into the design of experimental mutagenesis studies.

The L-type amino acid transporter LAT1, involved in many biological processes including the overexpression of some tumors, is considered a potential pharmacological target. The 1,2,3-Dithiazole scaffold was predicted to inhibit LAT1 by the formation of an intermolecular disulfide bond with the thiolate group of cysteine(s). As a result of the identification of these irreversible covalent inhibitors, we decided to deeply investigate the recognition stage and the covalent interaction, characterizing the chemical structures of the selected ligands. With the aim to provide new insights into the access of the ligands to the binding pocket and to reveal the residues involved in the inhibition, we performed docking, molecular dynamics simulations, and density functional theory-based investigation of three 1,2,3-dithiazoles against LAT1. Our computational analysis further highlighted the crucial role played by water molecules in the inhibition mechanism. The results here presented are consistent with experimental observations and provide insights that can be helpful for the rational design of new-to-come LAT1’s inhibitors.