Modeling nucleic acids, particularly RNA, presents challenges due to their dynamic nature, with biologically relevant conformations often differing significantly between unbound and bound states. This variability complicates predictions in silico, making efficient conformational sampling crucial. In this presentation, we investigate the efficacy of the LowModeMD method for enhancing conformational sampling in DNA and RNA systems compared to traditional molecular dynamics approaches.By focusing on low-frequency vibrational modes, LowModeMD enables rapid exploration of conformational space, facilitating the identification of biologically relevant structures. We demonstrate the utility of LowModeMD in analyzing bulged residues, sampling terminal and internal loops, cryptic pockets and large conformational changes caused by complexation.

Davide Bianchi¹, Simona Saporiti², Omar Ben Mariem¹, Fabio Centola² and Ivano Eberini<sup>1

1</sup> Dipartimento di Scienze Farmacologiche e Biomolecolari “Rodolfo Paoletti”, Università degli Studi di Milano, via Giuseppe Balzaretti 9, 2013, Milan, Italy
² Analytical Excellence and Program Management, Merck Serono S.p.A., Rome, Italy

The activity of monoclonal antibodies (mAbs) emerges from a finely tuned interplay between structural dynamics, post-translational modifications, and immune receptor recognition. In collaboration with Merck Serono S.p.A., we have employed advanced molecular dynamics (MD) simulations to unravel how glycosylation patterns, light- chain (LC) isotype, and antigen binding collectively shape the conformational landscape and effector function of therapeutic IgG1 antibodies.

In our initial study (Biophysical Journal, 2021 https://doi.org/10.1016/j.bpj.2021.10.026), we explored the structural basis by which Fc N-glycans modulate antibody flexibility. Classical MD simulations revealed that core fucosylation not only influences local Fc dynamics but also governs large-scale antibody motions, thereby modulating Fc accessibility to effector receptors. This work offered one of the first atomistic descriptions of glycan-mediated conformational regulation.

Building on these findings, we integrated classical and accelerated molecular dynamics (cMD + aMD) to examine the interplay between glycosylation and light-chain isotype (κ vs λ) in shaping IgG1 flexibility. Our goal was to identify the molecular determinants in FcγR engagement across antibody subclasses (Communications Biology, 2023, 10.1038/s42003-023-04622-7). The simulations uncovered distinct hinge dynamics and Fab-Fc orientations depending on both glycan status and isotypes, suggesting a structural rationale for differential receptor binding. This study marked one of the first applications of enhanced-sampling MD to probe the conformational diversity of full-length antibodies.

Expanding this approach, we investigated the IgG1::FcγRIIIa complex to directly assess how fucosylation and light chain isotype influence receptor recognition (Computational and Structural Biotechnology Journal, 2025, DOI: 10.1016/j.csbr.2025.100034). Our analysis identified novel interfacial residues contributing to complex stability and provided a detailed molecular explanation for the reduced FcγRIIIa affinity observed in afucosylated mAbs.

Most recently, we turned our attention to antigen engagement as a potential allosteric modulator of mAb conformation. Using commercial antibodies in complex with their respective antigens, and comparing G0 and G0F glycoforms via accelerated MD, we uncovered a consistent antigen-induced allosteric network linking Fab and Fc domains. Antigen binding shifted the conformational equilibrium toward more open, Y-shaped Fc states, enhancing receptor accessibility, particularly in afucosylated κ-LC antibodies. This effect was attenuated in λ-LC systems, where stronger CH1-CL contacts constrained Fab motion and limited long-range allosteric propagation. Collectively, these studies establish a detailed mechanistic framework for IgG1 function: from intrinsic Fc dynamics to receptor recognition and antigen-triggered allosteric regulation. This evolving paradigm suggests that Fab engineering, alongside Fc and glycan modifications, may represent a promising frontier for optimizing antibody effector functions.

The clinical success of covalent KRAS^G12C inhibition prompts further expansion of the concept to target non-cysteine oncogenic mutation sites as in KRAS^G12D. This endeavor was hampered by the lack of suitable electrophiles for the selective, covalent engagement of aspartate. Thanks to the recent discovery of β-lactone-bearing covalent inhibitors, new opportunities are emerging. Based on X-ray crystallographic insights and quantum chemical calculations, this presentation will describe the elucidation of structure-activity and -stability correlations to advance such electrophiles for drug discovery. Guided by predictions of transition state barrier heights for the attack of aspartate 12 at the β-lactone electrophile and structure-based design, substituted β-lactones were generated, aiming to balance specific reactivity and chemical and metabolic stability. The optimization strategy is driven by MS-based and cellular covalent target occupancy assays and PD marker analysis, proteome-wide profiling, and synthetic chemistry. With this work, the goal is to expand the use of β-lactones as chemoselective electrophiles in medicinal chemistry.