KRAS-G12D mutations are the one of most frequent oncogenic drivers in human cancers. Unfortunately, no therapeutic agent directly targeting KRAS-G12D has been clinically approved yet, with such mutated species remaining undrugged. Notably, cofactor Mg${}^{2+}$ is closely related to the function of small GTPases, but no investigation has been conducted yet on Mg${}^{2+}$ when associated with KRAS. Herein, through microsecond scale molecular dynamics simulations, we found that Mg${}^{2+}$ plays a crucial role in the conformational changes of the KRAS-GDP complex. We located two brand new druggable dynamic pockets exclusive to KRAS-G12D. Using the structural characteristics of these two dynamic pockets, we designed in silico the inhibitor DBD15-21-22, which can specifically and tightly target the KRAS-G12D-GDP-Mg${}^{2+}$ ternary complex. Overall, we provide two brand new druggable pockets located on KRAS-G12D and suitable strategies for its inhibition.

The use of drugs derived from benzothiadiazine, a bicyclic heterocyclic benzene derivative, has become a widespread treatment for diseases such as hypertension, low blood sugar or the human immunodeficiency virus, among others. In this work we have investigated the interactions of benzothiadiazine and four of its derivatives designed in silico with model zwitterionic cell membranes formed by dioleoylphosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphoserine and cholesterol at the liquid–crystal phase inside aqueous potassium chloride solution. We have elucidated the local structure of benzothiadiazine by means of microsecond molecular dynamics simulations of systems including a benzothiadiazine molecule or one of its derivatives. Such derivatives were obtained by the substitution of a single hydrogen site of benzothiadiazine by two different classes of chemical groups, one of them electron-donating groups (methyl and ethyl) and another one by electron-accepting groups (fluorine and trifluoromethyl). Our data have revealed that benzothiadiazine derivatives have a strong affinity to stay at the cell membrane interface although their solvation characteristics can vary significantly—they can be fully solvated by water in short periods of time or continuously attached to specific lipid sites during intervals of 10–70 ns. Furthermore, benzothiadiazines are able to bind lipids and cholesterol chains by means of single and double hydrogen-bonds of characteristic lengths between 1.6 and 2.1 Å.

Using Molecular Dynamics simulations, we propose a new lipid-like compound that can target both oncogenic GDP/GTP-bound KRAS4B-G12D mutant and PDE- δ so that it can foster drug discovery research on the RAS family and beyond.

The NRAS-mutant subset of melanoma is one of the most aggressive and lethal types associated with poor overall survival.

Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous bacterial secondary messenger with diverse functions. A previous Escherichia coli proteome microarray identified that c-di-GMP binds to the 23S rRNA methyltransferases RlmI and RlmE. Here we show that c-di-GMP inhibits RlmI activity in rRNA methylation assays, and that it modulates ribosome assembly in the presence of kanamycin. Molecular dynamics simulation and mutagenesis studies reveal that c-di-GMP binds to RlmI at residues R64, R103, G114, and K201. Structural simulations indicate that c-di-GMP quenches RlmI activity by inducing the closure of the catalytic pocket. We also show that c-di-GMP promotes antibiotic tolerance through RlmI. Binding and methylation assays indicate that the inhibitory effect of c-di-GMP on RlmI is conserved across various pathogenic bacteria. Our data suggest an unexpected role for c-di-GMP in regulating ribosome assembly under stress through the inhibition of rRNA methyltransferases.

KRAS is among the most frequently mutated oncogenes in human cancers, with codon 12 representing a dominant hotspot. Despite decades of study, the atomic-level conformational dynamics of KRAS and its G12 mutations remain insufficiently resolved, posing a central barrier to rational drug discovery. Elucidating these dynamics is critical for revealing hidden druggable pockets and enabling the development of mutation-specific inhibitors. Here, we delineate the impact of KRAS G12 mutations on conformational dynamics and GTP binding by performing microsecond-scale, well-tempered metadynamics simulations. This approach yielded precise free energy landscapes that expose mutation-specific alterations to structural states. By introducing biologically meaningful collective variables, including key angles (ϕ) and distances (d1, d2) defining Switch-I, Switch-II, and the P-loop, we identified distinct local and global minima as well as their transition states. Analysis of these transitions reveals how G12 mutations reshape GTP affinity and drive aberrant KRAS activation. We further demonstrated the convergence of the simulations and discussed the physiological relevance of the calculation results in comparison to previously published experimental and theoretical data. Importantly, our analyses uncovered druggable pockets unique to nonpolar G12 mutants, offering novel entry points for inhibitor design. Collectively, this work provides an atomic-level framework for understanding KRAS G12 mutations, establishes transferable collective variables for broader RAS family studies, and opens new avenues for mutation-specific therapeutic development.