TopFor: Automated Preparation of AMBER-Compatible Parameters for Noncanonical Amino Acids in Peptides

Buelbuel, E. F.; Gnanasekar, H.; Benasolo, C.; Rasheed, S.; Mueller, R.; Kalinina, O. V.; Bozhueyuek, K. A. J.

doi:10.64898/2026.10.05.756260

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bioRxiv · 10.64898/2026.10.05.756260

TopFor: Automated Preparation of AMBER-Compatible Parameters for Noncanonical Amino Acids in Peptides

Buelbuel, E. F.·Gnanasekar, H.·Benasolo, C.·Rasheed, S.·Mueller, R.·Kalinina, O. V.·Bozhueyuek, K. A. J.

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Abstract

Peptides containing noncanonical amino acids (ncAAs) are becoming increasingly important in drug discovery, but their molecular dynamics (MD) simulation remains limited by labor-intensive residue parameterization and topology preparation. Here, we present TopFor (Topology and Residue Force-Field Parameter Preparation), an open-source, peptide-oriented workflow that automates the generation of ncAA residue templates and force-field parameter files for MD simulations with AMBER using established charge-derivation methods. TopFor supports single-residue, whole-peptide, and batch parameterization workflows. The workflow was validated using the MDM2-p53 complex by comparing native AMBER residue definitions with residues processed by TopFor using three charge-derivation methods, each evaluated in three independent 100 ns MD simulations. TopFor-generated residue definitions showed structural and conformational behavior comparable to the native AMBER representation. Applicability to more complex peptide architectures was demonstrated using the head-to-tail cyclic peptide cyclosporin A and the cyclic depsipeptide cyclohexylgriselimycin. At library scale, 1,138 of 1,214 cyclohexylgriselimycin derivatives (93.7%) successfully completed TopFor parameterization and the subsequent initial AMBER-based computational workflow across two derivatization campaigns. TopFor was further integrated into a structure-based peptide discovery workflow based on the DnaN-griselimycin complex. Six GM-derived peptides were computationally prioritized and experimentally evaluated, of which four produced SPR responses against Mycobacterium smegmatis (M. smegmatis) DnaN and three showed concentration-dependent responses. TopFor reduces the manual effort required to prepare ncAA-containing peptides for MD simulations and enables their integration into both library-scale computational screening and structure-based peptide discovery workflows. Keywords: noncanonical amino acids; peptide parameterization; molecular dynamics; AMBER; AmberTools; peptide design; high-throughput virtual screening

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Buelbuel, E. F., Gnanasekar, H., Benasolo, C., Rasheed, S., Mueller, R., Kalinina, O. V., Bozhueyuek, K. A. J.. 2026-10-11. TopFor: Automated Preparation of AMBER-Compatible Parameters for Noncanonical Amino Acids in Peptides. https://doi.org/10.64898/2026.10.05.756260

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