KRAS mutation occurs in nearly 30% of human cancers, yet the most prevalent and oncogenic KRAS(G12D) variant still lacks inhibitors. Herein, we designed a series of potent inhibitors that can form a salt bridge with KRAS’s Asp12 residue. Our ITC results show that these inhibitors have similar binding affinity with both GDP-bound and GTP-bound KRAS(G12D), and our crystallographic studies reveal the structural basis of inhibitor binding-induced switch-II pocket in KRAS(G12D), experimentally confirming the formation of a salt bridge between the piperazine moiety of the inhibitors and the Asp12 residue of the mutant protein. Among KRAS family proteins and mutants, both ITC and enzymatic assays demonstrate the selectivity of the inhibitors for KRAS(G12D); and the inhibitors disrupt the KRAS–CRAF interaction. We also observed the inhibition of cancer cell proliferation as well as MAPK signaling by a representative inhibitor (TH-Z835). However, since the inhibition was not fully dependent on KRAS mutation status, it is possible that our inhibitors may have off-target effects via targeting non-KRAS small GTPases. Experiments with mouse xenograft models of pancreatic cancer showed that TH-Z835 significantly reduced tumor volume and synergized with an anti-PD-1 antibody. Collectively, our study demonstrates proof-of-concept for a strategy based on salt-bridge and induced-fit pocket formation for KRAS(G12D) targeting, which warrants future medicinal chemistry efforts for optimal efficacy and minimized off-target effects.

Many steroids are important pharmaceutically active compounds, while cytochrome P450 monooxygenases (CYPs) are attractive enzymes for applications in steroidal drug synthesis. However, the catalytic efficiency of existing P450s is not routinely high enough, as well as the molecular basis for selectivity control is unclear, which severely restrict their real applications. Here, a 16β steroid-hydroxylase CYP109B4 from Bacillus sonorensis is identified with excellent selectivity and activity. The crystallization and structural analysis of CYP109B4 reveal potential three "hotspot" residues (V84, V292, and S387) responsible for selectivity control. Then, guided by the sequence−function relationships revealed from the mutability landscape construction on the three residues, focused rational iterative site-specific mutagenesis (FRISM) and limited iterative saturation mutagenesis were performed, which provide variant B4-M7 (L240V/S387F/V84L/V292S/I291T/M290F/F294I) with completely switched regioselectivity from 16β to 15β. The subsequent computational analysis uncovers insights into the substrate binding modes in CYP109B4 and its variants, which further confirms the critical role of the "hotspot" residues for selectivity control. Finally, the generality of conserved-"hotspots"mediated selectivity control is demonstrated by performing scaffold sampling between a panel of CYP109B members. Overall, in addition to the present chemical results, our study provides guidance in rationally designing more excellent P450 biocatalysts for potential practical (industrial) applications.

Two enantiotopic 1D chain compounds, [Cu3(L1)3(H2O)2(H2W12O40)]·4H2O (1a,b; L1 = 2-(4,6-bis(pyridin-2-yl)pyridin-2-yl)pyridine), crystallizing in the chiral space group P212121 were prepared and spontaneously resolved in the absence of any chiral source. Interestingly, compounds 1a,b can be prepared from a [W7O24](6-) aqueous solution, [(n-C4H9)4N]4[W10O32], or Na10[H2W12O42], but when [H2W12O40](6-) aqueous solution was the starting material, the achiral compound [CuL1]2[H4W12O40]·5H2O (2) was obtained. When a terpyridine ligand (L2) having a coordination mode similar to that of L1 was used, the mesomeric dimer [Cu3(L2)3(H2O)(H2W12O40)]2·4H2O (3) was obtained from [W7O24](6-) aqueous solution or Na10[H2W12O42], but from [H2W12O40](6-) aqueous solution only compound [Cu2(L2)2Cl2]2[W10O32] (4) was isolated. It is notable that in compounds 1a,b and 3 the symmetry of the α-[H2W12O40](6-) cluster is broken by asymmetric coordination with metal-organic units in a similar mode. As the asymmetric subunit based on a tridecorated [H2W12O40](6-) cluster can be obtained from several isopolyoxotungstate sources except for [H2W12O40](6-), we speculate that the symmetry breaking of α-[H2W12O40](6-) depends on the transformation of isopolyoxotungstates. Furthermore, during the transformation a possible reaction intermediate as the precursor for 1a,b, compound [Cu3(L1)3(H2O)3(H4W11O38)] (5), has been presented and characterized by density functional theory (DFT) calculations.

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