The accurate parameterization of drug-like molecules is a fundamental prerequisite for molecular dynamics simulations in structure-based drug design. While the AM1-BCC charge model has served as the de facto "standard" for GAFF2 parameterization for two decades, the recently introduced ABCG2 model offers substantially improved accuracy in predicting hydration free energies and other key physicochemical properties. However, accessing ABCG2 parameters traditionally requires downloading and installing the full AmberTools suite-a multi-gigabyte software package with complex dependencies-presenting a significant barrier for many practitioners, particularly experimental collaborators and researchers new to the field. Here we present a major upgrade to the PrimaDORAC web interface that makes ABCG2 parameterization readily accessible to the entire drug design community. Through a minimalistic integration of essential AmberTools components directly into the web application framework, users can now obtain GAFF2 parameters with ABCG2 charges for any drug-like molecule in seconds, without any software installation. The interface accepts a single SMILES string or structure file and returns a complete archive containing GROMACS-compatible topology files and a ready-to-use PDB structure. By removing all technical barriers to accessing state-of-the-art ABCG2 parameters, the upgraded PrimaDORAC interface is aimed at empowering medicinal chemists, pharmacologists, and computational researchers alike to conduct more accurate MD simulations with minimal effort. The service is freely available at www1.chim.unifi.it/orac.

An upgraded GAFF2 force field has been used to simulate two fluorinated alcohols, TFE and HFIP, in aqueous solutions at several concentrations. The same force field has also been employed to simulate a 26-residue amphiphilic peptide in several cosolvent/water mixtures to verify and clarify its efficacy in stabilizing the secondary structure. The calculated thermodynamic and structural properties are in agreement with experimental findings. The force field allows a correct description of the secondary structure and affords an accurate characterization of the spatial organization of cosolvent molecules around the peptide.

Single-topology (ST) free energy perturbation (FEP) methods, while accurate for congeneric series, require chemical similarity between ligands, bespoke perturbation networks, and offer no direct route to absolute binding free energies (ABFEs). Here we introduce DT-NE-Alchemy, a dual-topology nonequilibrium (NE) framework that eliminates these constraints through a universal Lennard-Jones (LJ) blob reference state. The LJ blob─a minimal, rigid molecule (as small as a single LJ particle) designed to fit the binding pocket─serves as a common anchor for alchemical transformations. Relative binding free energies between any two ligands are obtained by two-edge transformations Li → blob → Lj, yielding all n(n - 1)/2 pairwise values from only n blob-based calculations. Crucially, ABFEs follow directly as ΔGblob+ΔGblob→Li, converting the long-sought "holy grail" of computational drug design into a practical reality. Applied to 16 MCL-1 ligands spanning indole, benzothiophene, and benzofuran scaffolds, the method achieves Pearson correlations up to 0.88, Kendall τ of ≃0.70, and mean unsigned errors of 1.2-1.5 kcal/mol for 120 relative pairs. The methodology delivers credible, tunable confidence intervals directly from BAR analysis, controllable via switching time τ and trajectory count N without costly replicate simulations. All computational tasks are embarrassingly parallel by design, perfectly aligned with leadership-class HPC systems. DT-NE-Alchemy is automatable and poised to effectively complement ST/FEP for the exploration of chemically diverse libraries and scaffold identification, with the latter remaining the gold standard for lead optimization within strictly congeneric series.

Cyclodextrins are amphiphilic macromolecular containers that are particularly valuable in applications ranging from biomedicine to environmental science. Despite years of development of computational techniques for cyclodextrin host‐guest coordination, accurate modelling of these supramolecular systems remains challenging. In this work, an integrative computational technique is presented to solve this problem. The protocol integrates a force‐field recalibration procedure, equilibrium enhanced sampling, nonequilibrium Hamiltonian switching, a work convolution algorithm, a series of finite‐size corrections, and energy decomposition analysis. A large‐scale survey of 222 CD host–guest systems is performed to demonstrate that the protocol enables the fast calculation of cyclodextrin host‐guest binding strength without compromise in accuracy, an unbiased capture of cyclodextrin dynamics in the free CD, and the multi‐modal behavior of the coordination patterns, and further the identification of the physicochemical driving force stabilizing host‐guest complexes. Especially, the protocol consistently provides accurate results for various systems while conventional transferable force fields systematically fail for larger and more flexible 𝛾‐CD. It thus opens new opportunities for high‐throughput screening and rational design across diverse macrocyclic host families.

We present PDBrestore, a free web interface for repairing protein PDB chains extracted from either a local PDB file or a PDB file downloaded from the Protein Data Bank. PDBrestore performs several key tasks: It adds hydrogen atoms, completes missing atoms in side chains, fills gaps in the sequence, derives the itp parameter file for a ligand according to the GAFF2 force field for GROMACS applications, and generates a reasonably pre-equilibrated solvated simulation box. The interface is designed to streamline the cumbersome preparatory work required to set up an initial protein-ligand coordinates PDB file for use in drug design projects, such as free energy perturbation or thermodynamic integration calculations of ligand binding affinities. Additionally, PDBrestore is available as a command-line application within the open-source ORAC distribution, which can be freely downloaded from the website: www1.chim.unifi.it/orac.

We present a dual biosensing strategy integrating Quartz Crystal Microbalance (QCM) and Surface-Enhanced Raman Spectroscopy (SERS) for the quantitative and molecular-specific detection of FKBP12. Silver nanodendritic arrays were electrodeposited onto QCM sensors, optimized for SERS enhancement using Rhodamine 6G, and functionalized with a custom-designed receptor to selectively capture FKBP12. QCM measurements revealed a two-step Langmuir adsorption behavior, enabling sensitive mass quantification with a low limit of detection. Concurrently, in situ SERS analysis on the same sensor provided vibrational fingerprints of FKBP12, resolved through comparative studies of the free protein, surface-bound receptor, and surface-bound receptor–protein complex. Ethanol-induced denaturation confirmed protein-specific peaks, while shifts in receptor vibrational modes—linked to FKBP12 binding—demonstrated dynamic molecular interactions. A ratiometric parameter, derived from key peak intensities, served as a robust, concentration-dependent signature of complex formation. This platform bridges quantitative (QCM) and structural (SERS) biosensing, offering real-time mass tracking and conformational insights. The nanodendritic substrate’s dual functionality, combined with the receptor’s selectivity, advances label-free protein detection for applications in drug diagnostics, with potential adaptability to other target analytes.

We developed and validated a novel force field in the context of the AMBER parameterization for the simulation of cadmium(II)-binding proteins. The proposed force field takes into account the polarization effect produced by the central ion on its surroundings. The new polarized atomic charges for cysteine and histidine residues were derived based on the available structures of cadmium-bearing proteins using QM calculations and QM/MM simulations. The developed force field was validated by performing molecular dynamics simulations on several cadmium(II)-binding proteins. Our model preserves the tetra-coordination of the metal site with remarkable stability, yielding mean distances between Cd 2 + $$ {\mathrm{Cd}}^{2+} $$ ion and S or N atoms of the binding residues in close agreement with experimental data.

FKBP12, a peptidyl-prolyl isomerase implicated in cancer, neurodegenerative diseases, and post-transplant anti-rejection mechanisms, represents a critical biomarker for early diagnosis and monitoring. Here, a novel diagnostic nanoplatform is presented for the detection of FKBP12 at nanomolar to picomolar concentrations in biological fluids. The platform integrates a gold-coated Quartz Crystal Microbalance (QCM) functionalized with a synthetic receptor (GPS-SH1) and spacers within a Self-Assembled Monolayer (SAM), enabling direct and label-free detection of FKBP12 in complex biological samples. A careful strategy for the in-silico design and custom synthesis of the receptor is adopted, ensuring optimal binding affinity and additional chemical functionalities for surface chemisorption. The designed nano-architecture demonstrates exceptional sensitivity, with a detection limit in the picomolar range, and high selectivity, as confirmed by minimal interference from abundant serum proteins such as Serum Albumin and Immune Gamma Globulin. Furthermore, the SAM-functionalized sensors exhibit remarkable stability, retaining functionality for up to six months under storage conditions. This work not only advances the field of nanoscale biosensing but also provides a robust, reusable tool for FKBP12 detection, with potential applications in point-of-care diagnostics and personalized medicine. The platform's ability to operate in biologically relevant environments underscores its promise for real-world healthcare applications, including early disease diagnostics.

Estrogen receptor β (ERβ) is the most highly expressed subtype in the colon epithelium and mediates the protective effect of estrogen against the development of colon cancer. Indeed, the expression of this receptor is inversely related to colorectal cancer progression. Structurally estrogen-like compounds, including vitamin E components, affect cell growth by binding to ERs. In the present study, cell proliferation was measured by cell counting in a Bürker hemocytometer, and ERβ expression was measured by Real-Time qPCR and immunoenzymatic methods. The results obtained show that natural δ-tocopherol (δ-Toc) and two of its semi-synthetic derivatives, bis-δ-tocopheryl sulfide (δ-Toc)2S and bis-δ-tocopheryl disulfide (δ-Toc)2S2, play an antiproliferative role and upregulate ERβ expression, similar to 17-β-estradiol (17β-E2), in human colon adenocarcinoma HCT8 cells engineered to overexpress ERβ protein (HCT8-β8). These events are not present in HCT8-pSV2neo and in HCT8-β8 pretreated with ICI 182,780, suggesting that they are mediated by the binding of compounds to ERβ, as also boosted by an in silico assay. The antiproliferative effect is independent of the intracellular redox state and (δ-Toc)2S and (δ-Toc)2S2 reduce cell proliferation at concentrations lower than that of δ-Toc and all tested compounds are also able to upregulate ERβ expression. Taken together, the data indicate that, through the involvement of ERβ activity and expression, δ-Toc, (δ-Toc)2S, and (δ-Toc)2S2 may provide potential therapeutic support against colorectal cancer.

We assess the performance of the nonequilibrium alchemical fast-growth method in calculating water and 1-octanol solvation free energies, comparing the recently proposed ABCG2 model with other empirical and quantum mechanics (QM)-based approaches for modeling electrostatic interactions in condensed phases using fixed atomic charges. The fixed-charge protocols are tested on the challenging set of drug-like polyfunctional molecules previously used by Vassetti et al., J. Chem. Theory Comput. 2019, 15, 1983-1995, broadly spanning the chemical space and often exhibiting complex conformational landscapes. We find that the cost-effective empirical ABCG2 protocol consistently outperforms the AM1/BCC precursor model and the widely used HF/6-31G* ab initio charge derivation method, achieving solvation free energy accuracy comparable to an expensive QM/MM-based methodology for atomic fixed charge determination. For water-octanol transfer free energies, ABCG2 benefits from systematic error cancellation, yielding remarkable agreement with experimental data, exhibiting excellent Pearson and Kendall rank coefficients and a mean unsigned error below 1 kcal/mol, matching the performance of the costly QM/MM approach. These results suggest that the ABCG2 protocol holds great promise for the high-throughput in silico prediction of ligand-protein binding free energies in drug discovery projects.

We have assembled a computational pipeline based on virtual screening, docking techniques, and nonequilibrium molecular dynamics simulations, with the goal of identifying possible inhibitors of the SARS-CoV-2 NSP13 helicase, catalyzing by ATP hydrolysis the unwinding of double or single-stranded RNA in the viral replication process inside the host cell. The druggable sites for broad-spectrum inhibitors are represented by the RNA binding sites at the 5' entrance and 3' exit of the central channel, a structural motif that is highly conserved across coronaviruses. Potential binders were first generated using structure-based ligand techniques. Their potency was estimated by using four popular docking scoring functions. Common docking hits for NSP13 were finally tested using advanced nonequilibrium alchemical techniques for binding free energy calculations on a high-performing parallel cluster. Four potential NSP13 inhibitors with potency from submicrimolar to nanomolar were finally identified. The channel for RNA unwinding of NSP13 SARS-CoV-2 helicase is highly conserved in alpha and beta mammals coronavirus. Exploiting a funnel-like computational pipeline, we identified several micromolar or submicromolar NSP13 ligands blocking the central channel of NSP13. These compounds could potentially be good candidates for the development of a broad-spectrum drug for coronavirus infections. image

As a contribution to the understanding and rationalization of methodological and modeling effects in recent host-guest SAMPL challenges, using an alchemical molecular dynamics technique we have examined the impact of force field parameterization and ionic strength in connection with guest charge neutralization on computed dissociation free energies in two typical SAMPL heavily charged macrocyclic hosts encapsulating small protonated amines with disparate binding affinities. We have shown that the methodological treatment for host neutralization, with explicit ions or with the background neutralizing plasma in the context of alchemical calculations under periodic boundary conditions, has a moderate effect on the calculated affinities. On the other hand, we have shown that seemingly small differences in the force field parameterization in highly symmetric hosts can produce systematic effects on the structural features that can have a significant impact on the predicted binding affinities.

Alchemical transformations can be used to quantitatively estimate absolute binding free energies at a reasonable computational cost. However, most of the approaches currently in use require knowledge of the correct (crystallographic) pose. In this paper, we present a combined Hamiltonian replica exchange nonequilibrium alchemical method that allows us to reliably calculate absolute binding free energies, even when starting from suboptimal initial binding poses. Performing a preliminary Hamiltonian replica exchange enhances the sampling of slow degrees of freedom of the ligand and the target, allowing the system to populate the correct binding pose when starting from an approximate docking pose. We apply the method on 6 ligands of the first bromodomain of the BRD4 bromodomain-containing protein. For each ligand, we start nonequilibrium alchemical transformations from both the crystallographic pose and the top-scoring docked pose that are often significantly different. We show that the method produces statistically equivalent binding free energies, making it a useful tool for computational drug discovery pipelines.

We describe a step‐by‐step protocol and toolkit for the computation of the relative dissociation free energy (RDFE) with the GROMACS molecular dynamics package, based on a novel bidirectional nonequilibrium alchemical approach. The proposed methodology does not require any intervention on the code and allows computing with good accuracy the RDFE between small molecules with arbitrary differences in volume, charge, and chemical topology. The procedure is illustrated for the challenging SAMPL9 batch of host–guest pairs. The article is supplemented by a detailed online tutorial, available at https://procacci.github.io/vdssb_gromacs/NE-RDFE and by a public Zenodo repository available at https://zenodo.org/record/6982932 .

Alchemical transformations can be used to quantitatively estimate absolute binding free energies at reasonable computational cost. However, most of the approaches currently in use require knowledge of the correct (crystallographic) bond position. In this paper we present a combined Hamiltonian replica exchange non-equilibrium alchemical method that allows to reliably calculate absolute binding free energies, even when starting from sub-optimal initial binding poses. Performing a preliminary Hamiltonian replica exchange enhances the sampling of slow degrees of freedom of the ligand and the target allowing the system to populate the correct binding pose when starting from an approximate docking pose. We apply the method on 6 ligands of the first bromodomain of the BRD4 bromodomain containing protein. For each ligand, we start non-equilibrium alchemical transformations from both the crystallographic pose and the top-scoring docked pose that are often significantly different. We show that the method produces statistically equivalent binding free energies making it a useful tool for computational drug discovery pipelines.

In this paper we further develop a combined Hamiltonian replica exchange / non-equilibrium alchemical method for absolute binding free energies and test its performance on 11 ligands of the BRD4 bromodomain protein. We compare the results obtained with our approach with those obtained with other equilibrium and non-equilibrium alchemical methods showing their relative strengths, weaknesses, and limits. We show how using an enhanced sampling technique, before the alchemical transformations, allows to get accurate estimates of the binding free energies even when starting with sub-optimal initial binding poses (e.g. from docking). We also study the effect of different restraining mechanisms on the final result, and introduce a new "Loose-Tight" restraining algorithm. The method proves to strike a good balance between ease of use, automation, speed, and accuracy for absolute ligand binding free energy calculations and our scripts make it easy to integrate it into pre-existing computational drug discovery pipelines.

In this study, we have tested the performance of standard molecular dynamics (MD) simulations, replicates of shorter standard MD simulations, and Hamiltonian Replica Exchange (HREM) simulations for the sampling of two macrocyclic hosts for guest delivery, characterized by induced fit (phenyl-based host) and conformation selection (naphthyl-based host) and of the ODR-BRD4(I) drug-receptor system where the ligand can assume two main poses. For the optimization of the HREM simulation, we have proposed and tested an on-the-fly iterative scheme for equalizing the acceptance ratio along the replica progression at a constant replica number resulting in a moderate impact of the sampling efficiency. Concerning standard MD, we have found that, while splitting the total allocated simulation time in short MD replicates can reproduce the sampling efficiency of HREM in the phenyl-based host and in the ODR-BRD4(I) complex, in the naphthyl-based macrocycle, characterized by long-lived metastable states, enhanced sampling techniques are the only viable alternative for a reliable canonical sampling of the rugged conformational landscape.

Supporting material of the paper "A nonequilibrium alchemical method for drug-receptor absolute binding free energy calculations: the role of restraints". The directory is fully documented with README files. Differences of V2.0 with V1.0: Due to a software bug we had to re-parametrize the ligands whose torsions were parametized with ANI-2.X (ligand 6 and 7). During the peer reviewing process we also parametrized with ANI-2.X and docked ligand 8.