Early-Enrichment Hit Discovery via Reversible-Work c(t) Estimation in Metadynamics (CTMD)

Virtual screening for small-molecule binders is often limited by false positives from approximate scoring functions and rigid-receptor assumptions. These can be addressed downstream through accurate but expensive free-energy calculations. At the same time, recent artificial-intelligence-based co-folding methods have been proposed that claim to achieve the accuracy of free-energy methods at much lower cost, but these have not yet delivered consistent improvements in early enrichment and can be confounded by memorization. Here we address this gap by introducing c(t)-based metadynamics (CTMD), a physics-based, high-throughput hit-triaging protocol tailored for early enrichment. CTMD uses the nonequilibrium reversible-work estimator c(t) introduced by Tiwary and Parrinello (Journal of Physical Chemistry B, 2015, 119, 736), computed from a small number of short, independent well-tempered metadynamics trajectories, to rank binding stability without requiring converged binding free energies. We demonstrate that CTMD provides robust early enrichment across diverse targets and chemotypes, while remaining fast and transferable with minimal parameter tuning and resistant to memorization-driven artifacts─underscoring both an immediately deployable physics-based alternative for screening. For these systems, we show how co-folding, particularly Boltz-2, achieves enrichment directly proportional to similarity with the training set and, more worryingly, reproduces this even in the presence of significant modifications to the active site. Given its simplicity of implementation, CTMD should thus be an "embarrassingly″ open-source, early enrichment method available for use by the broad pharmacological and academic community that sits right between approximate but fast docking or AI-based co-folding methods and more expensive but accurate free-energy calculations, and is expected to save significant financial and human capital in drug discovery campaigns.