This review outlines the role of electrostatics in computational molecular biophysics and its implication in altering wild-type characteristics of biological macromolecules, and thus the contribution of electrostatics to disease mechanisms. The work is not intended to review existing computational approaches or to propose further developments. Instead, it summarizes the outcomes of relevant studies and provides a generalized classification of major mechanisms that involve electrostatic effects in both wild-type and mutant biological macromolecules. It emphasizes the complex role of electrostatics in molecular biophysics, such that the long range of electrostatic interactions causes them to dominate all other forces at distances larger than several Angstroms, while at the same time, the alteration of short-range wild-type electrostatic pairwise interactions can have pronounced effects as well. Because of this dual nature of electrostatic interactions, being dominant at long-range and being very specific at short-range, their implications for wild-type structure and function are quite pronounced. Therefore, any disruption of the complex electrostatic network of interactions may abolish wild-type functionality and could be the dominant factor contributing to pathogenicity. However, we also outline that due to the plasticity of biological macromolecules, the effect of amino acid mutation may be reduced, and thus a charge deletion or insertion may not necessarily be deleterious.

Calpain-1 and calpain-2 are heterodimeric proteases consisting of a common small regulatory subunit CAPNS1 and a large catalytic subunit, CAPN1 or CAPN2, respectively. These calpains have emerged as potential therapeutic targets in cancer and other diseases through their roles in cell signaling pathways affecting sensitivity to chemotherapeutic and targeted drugs, and in promoting metastasis. While inhibition of calpains has the potential to provide therapeutic benefit to cancer patients, there are currently no clinically approved active site directed drugs that specifically and effectively inhibit them. However, the structures of calpain-1 and calpain-2 make them susceptible to allosteric inhibition aimed at interfering with heterodimerization of the catalytic and regulatory subunits, which is necessary for stability and proteolytic activity. Split-Nanoluciferase biosensors were generated to quantify the protein-protein interactions (PPIs) between the calcium-binding penta-EF hand (PEF) domains of CAPN1 or CAPN2 and CAPNS1. These biosensors were used to quantify the heterodimer dissociation constants (K_D) of calpain-1 and calpain-2, estimated at 185 nM and 509 nM, respectively, in the presence of 5 mM Ca²⁺; and 362 nM and 1651 nM, respectively, in the presence of Mg²⁺. The half-maximal Ca²⁺ concentrations supporting these PPIs for calpain-1 and calpain-2 were 59.9 μM and 940.8 μM, respectively. Molecular modeling, based on the crystal structure of calpain-2, was used to predict 20 residues of the PEF domains that contribute to heterodimerization. Individual point mutation of CAPNS1 at Q263 reduced the catalytic activity of calpain-2 to 51.0 ± 6.4 % in live cells.

Most human diseases have genetic components, frequently single nucleotide variants (SNVs), which alter the wild type characteristics of macromolecules and their interactions. A straightforward approach for correcting such SNVs-related alterations is to seek small molecules, potential drugs, that can eliminate disease-causing effects. Certain disorders are caused by altered protein-protein interactions, for example, Snyder-Robinson syndrome, the therapy for which focuses on the development of small molecules that restore the wild type homodimerization of spermine synthase. Other disorders originate from altered protein-nucleic acid interactions, as in the case of cancer; in these cases, the elimination of disease-causing effects requires small molecules that eliminate the effect of mutation and restore wild type p53-DNA affinity. Overall, especially for complex diseases, pathogenic mutations frequently alter macromolecular interactions. This effect can be direct, i.e., the alteration of wild type affinity and specificity, or indirect via alterations in the concentration of the binding partners. Here, we outline progress made in methods and strategies to computationally identify small molecules capable of altering macromolecular interactions in a desired manner, reducing or increasing the binding affinity, and eliminating the disease-causing effect. When applicable, we provide examples of the outlined general strategy. Successful cases are presented at the end of the work.

Monogenic disorders present a compelling target for drug discovery due to their clear, single-gene etiology. Yet, effective therapies remain scarce, largely because diverse mutation types scattered across a protein obstruct targeted drug design. In this study, we leverage MOGEDO, a curated database of monogenic disorders, to test whether different pathogenic genotypes within a single protein converge on a common molecular mechanism. By clustering MOGEDO proteins into eight functional families and analyzing representative proteins carrying both benign and pathogenic mutations, we integrated molecular dynamics, network analysis, and side-chain entropy metrics. Our findings reveal that distinct pathogenic mutations disrupt the structural and dynamic properties of the wild-type and benign protein through common pathways. By demonstrating that diverse disease-causing mutations manifest a unified pathogenic phenotype, this work provides a framework for designing broad-spectrum therapeutics that target common dysfunctional mechanisms rather than individual mutations.

The creatine transporter (CRT/SLC6A8) plays a key role in cellular energy homeostasis, yet the molecular mechanism underlying creatine transport remains poorly understood. Here, we reconstruct the complete transport cycle of human CRT using a hybrid simulation strategy that combines constant‐force steered molecular dynamics (cf‐sMD) with targeted molecular dynamics (tMD). This approach captures continuous progression through the outward‐open, outward‐occluded, inward‐occluded, and inward‐open states and reveals a water‐assisted, sequential intracellular release of Na2, creatine, and Na1. Hydration analysis shows that progressive water penetration into the binding pocket weakens protein‐substrate and protein‐ion interactions and destabilizes the bound state before release. Residue‐level contact analysis identifies residues that interact with creatine along the transport pathway, while dynamic network analysis reveals a TM1–TM6 communication backbone that mediates long‐range coupling during transport. Together, these results provide a molecular framework for creatine transport and establish an approach for investigating transport mechanisms across the broader solute carrier family.

Monogenic disorders present a compelling target for drug discovery due to their clear, single-gene etiology. Yet, effective therapies remain scarce, largely because diverse mutation types scattered across a protein obstruct targeted drug design. In this study, we leverage MOGEDO, a curated database of monogenic disorders, to test whether different pathogenic genotypes within a single protein converge on a common molecular mechanism. By clustering MOGEDO proteins into eight functional families and analyzing representative proteins carrying both benign and pathogenic mutations, we integrated molecular dynamics, network analysis, and side-chain entropy metrics. Our findings reveal that distinct pathogenic mutations disrupt the structural and dynamic properties of the wild-type and benign protein through common pathways. By demonstrating that diverse disease-causing mutations manifest a unified pathogenic phenotype, this work provides a framework for designing broad-spectrum therapeutics that target common putative intervention sites rather than individual mutations.