Biology subjects

Henes, M.

Publications and source records attributed to Henes, M..

8 recordsLinked to original sources

bioRxiv2026

A Dynamics-Informed Machine Learning Framework for Inhibitor Optimization Against Quickly Evolving Targets to Avoid Resistance

Drug resistance often emerges through mutations that reshape protein conformational ensembles, allowing inhibitor potency to be altered without directly disrupting protein-ligand contacts. Consequently, the molecular mechanisms underlying these effects remain difficult to identify, hindering the design of inhibitors with durable potency against rapidly evolving drug targets. To address this challenge, we developed ROBUST, a physics-informed machine learning framework that integrates experimental measurements, molecular dynamics simulations, and interpretable statistical modeling to identify energetic determinants of inhibitor potency. Using HIV-1 protease as a stringent model system for drug resistance, we determined 157 new inhibition constants and integrated them with prior measurements to establish a comprehensive experimental dataset of 364 protease-inhibitor pairs. Analysis of the corresponding molecular dynamics simulations revealed a compact network of energetic interactions that quantitatively explains how resistance mutations and inhibitor modifications jointly reshape molecular recognition and inhibitor potency across the multidimensional resistance landscape. Models constructed from these energetic signatures predicted binding free energies with high accuracy (RMSE < 1 kcal mol-1) and successfully generalized to unseen darunavir analogs. Importantly, ROBUST accurately prioritized unseen analogs based on their potency across the variant set, demonstrating its utility for identifying compounds with improved resistance profiles. Together, these results uncover the energetic principles through which resistance mutations and inhibitor modifications alter potency and establish ROBUST as a mechanistically interpretable framework for translating these insights into resistance-aware inhibitor optimization against rapidly evolving drug targets.

biophysics↗

bioRxiv2026

Stronger brain responses to acute stress reflect greater everyday stress variability

Laboratory stress tasks are widely used to assess individual differences in acute stress reactivity, yet it remains unclear how these responses correspond to stress experienced in everyday life. Here, we combined the Montreal imaging stress task (MIST) with ecological momentary assessment (EMA) over three months to assess acute and everyday stress in 67 healthy women. Greater within-person variability in everyday stress, but not average stress levels, were associated with stronger overall stress-related brain responses (b = 0.73, p = .039), with a whole-brain association particularly evident in the bilateral caudate (rROI = .32, pcluster.FWE < .001). Greater everyday stress variability was also associated with stronger stress-related functional connectivity between the ventromedial prefrontal cortex (vmPFC) and parietal and posterior medial regions (pcluster.FWE < .001). We conclude that acute neural stress responses relate more closely to fluctuations in perceived stress than to how stressed an individual feels on average. This suggests that laboratory stress tasks capture acute stress responsivity that is distinct from average stress exposure, highlighting the importance of considering what these tasks measure when interpreting individual differences in acute stress responses.

neuroscience↗

bioRxiv2025

Higher glucose levels buffer against everyday stress load

Adaptive stress responses are dependent on the availability of energy and the bodys effectiveness in metabolizing glucose as fuel. However, it is not well understood if glucose levels contribute to the functional regulation of daily stress in healthy individuals. Here, we used 4,307 ratings of stress during a four-week period of ecological momentary assessment (EMA) with concurrent recordings from continuous glucose monitoring (CGM) in 91 participants (47 women). In accordance with the hypothesized metabolic link, we found that higher glucose levels were associated with lower self-reported stress (b = -.038, p = .035, controlling for mood). Crucially, dynamic changes in glucose levels also precipitated subsequent increases in stress levels (0-2h before EMA: p = .013; incremental effect 10-12h before: p < .001), with lower glucose levels associated with subsequent stress increases and elevated glucose with stress relief, highlighting the potential of monitoring physiological biosensor data to improve adaptive stress regulation. We conclude that metabolic signals, such as glucose, play an important role in channeling stress responsiveness in healthy adults, pointing to a pivotal avenue for improved interventions to avoid excessive stress experiences.

neuroscience↗

bioRxiv2024

Differential roles of putative arginine fingers of AAA+ ATPases Rvb1 and Rvb2

The evolutionarily conserved AAA+ ATPases Rvb1 and Rvb2 proteins form a heteromeric complex (Rvb1/2) required for assembly or remodeling of macromolecular complexes in essential cellular processes ranging from chromatin remodeling to ribosome biogenesis. Rvb1 and Rvb2 have a high degree of sequence and structural similarity, and both contain the classical features of ATPases of their clade, including an N-terminal AAA+ subdomain with the Walker A motif, an insertion domain that typically interacts with various binding partners, and a C-terminal AAA+ subdomain containing a Walker B motif, the Sensor I and II motifs, and an arginine finger. In this study, we find that despite the high degree of structural similarity, Rvb1 and Rvb2 have distinct active sites that impact their activities and regulation within the Rvb1/2 complex. Using a combination of biochemical and genetic approaches, we show that replacing the homologous arginine fingers of Rvb1 and Rvb2 with different amino acids not only has distinct effects on the catalytic activity of the complex, but also impacts cell growth, and the Rvb1/2 interactions with binding partners. Using molecular dynamics simulations, we find that changes near the active site of Rvb1 and Rvb2 cause long-range effects on the protein dynamics in the insertion domain, suggesting a molecular basis for how enzymatic activity within the catalytic site of ATP hydrolysis can be relayed to other domains of the Rvb1/2 complex to modulate its function. Further, we show the impact that the arginine finger variants have on snoRNP biogenesis and validate the findings from molecular dynamics simulations using a targeted genetic screen. Together, our results reveal new aspects of the regulation of the Rvb1/2 complex by identifying a relay of long-range molecular communication from the ATPase active site of the complex to the binding site of cofactors. Most importantly, our findings suggest that despite high similarity and cooperation within the same protein complex, the two proteins have evolved with unique properties critical for the regulation and function of the Rvb1/2 complex. SignificanceAAA ATPases constitute a large family of proteins involved in various essential cellular functions in living organisms in all kingdoms of life. Members of this family typically form homo or hetero multimers that convert the energy from ATP hydrolysis to mechanical work. How the conserved features of AAA ATPases relay the energy from ATP hydrolysis to other functional domains of the complex remains largely unknown. Here, using arginine finger variants of Rvb1 and Rvb2, two evolutionarily conserved closely related AAA+ ATPases that form a heterohexameric complex, we reveal how individual protomers in a heteromeric complex can uniquely contribute to the overall function of the complex and how changes in the ATP binding site can be relayed to distal functional domains.

biochemistry↗

bioRxiv2021

Deciphering the Molecular Mechanism of HCV Protease Inhibitor Fluorination as a General Approach to Avoid Drug Resistance

Third generation Hepatitis C virus (HCV) NS3/4A protease inhibitors (PIs), glecaprevir and voxilaprevir, are highly effective across genotypes and against many resistant variants. Unlike earlier PIs, these compounds have fluorine substitutions on the P2-P4 macrocycle and P1 moieties. Fluorination has long been used in medicinal chemistry as a strategy to improve physicochemical properties and potency. However, the molecular basis by which fluorination improves potency and resistance profile of HCV NS3/4A PIs is not well understood. To systematically analyze the contribution of fluorine substitutions to inhibitor potency and resistance profile, we used a multi-disciplinary approach involving inhibitor design and synthesis, enzyme inhibition assays, co-crystallography, and structural analysis. A panel of inhibitors in matched pairs were designed with and without P4 cap fluorination, tested against WT protease and the D168A resistant variant, and a total of 22 high-resolution co-crystal structures were determined. While fluorination did not significantly improve potency against the WT protease, PIs with fluorinated P4 caps retained much better potency against the D168A protease variant. Detailed analysis of the co-crystal structures revealed that PIs with fluorinated P4 caps can sample alternate binding conformations that enable adapting to structural changes induced by the D168A substitution. Our results elucidate molecular mechanisms of fluorine-specific inhibitor interactions that can be leveraged in avoiding drug resistance.

biochemistry↗

bioRxiv2019

Selection of HIV-1 for Resistance to Fourth Generation Protease Inhibitors Reveals Two Independent Pathways to High-Level Resistance

Darunavir (DRV) is exceptional among potent HIV-1 protease inhibitors (PIs) in the high drug concentrations that are achived in vivo. Little is known about the de novo resistance pathway for DRV. We have selected for resistance to high drug concentrations against ten PIs and their structural precursor DRV. Mutations accumulated through two pathways (anchored by protease mutations I50V or I84V). Small chemical changes in the inhibitor P1 side chain led to preferential use of one pathway over the other. Changes in the inhbitor P2 side chain determined differences in potency that were retained in the resistant viruses. Viral variants from the two pathways showed differential selection of compensatory mutations in Gag cleavage sites. These results reveal the high level of selective pressure that is attainable with fourth generation PIs, and how features of the inhibitor affect both the resistance pathway and the residual potency in the face of resistance.

microbiology↗

bioRxiv2019

Molecular and structural mechanism of pan-genotypic HCV NS3/4A protease inhibition by glecaprevir

Hepatitis C virus (HCV), causative agent of chronic viral hepatitis, infects 71 million people worldwide and is divided into seven genotypes and multiple subtypes with sequence identities between 68 to 82%. While older generation direct-acting antivirals (DAAs) had varying effectiveness against different genotypes, the newest NS3/4A protease inhibitors including glecaprevir (GLE) have pan-genotypic activity. The structural basis for pan-genotypic inhibition and effects of polymorphisms on inhibitor potency were not well known due to lack of crystal structures of GLE-bound NS3/4A or genotypes other than 1. In this study, we determined the crystal structures of NS3/4A from genotypes 1a, 3a, 4a and 5a in complex with GLE. Comparison with the highly similar grazoprevir (GZR) indicated the mechanism of GLEs drastic improvement in potency. We found that while GLE is highly potent against wild type NS3/4A of all genotypes, specific resistance-associated substitutions (RASs) confer orders of magnitude loss in inhibition. Our crystal structures reveal molecular mechanisms behind pan-genotypic activity of GLE, including potency loss due to RASs at D168. Our structures permit for the first time analysis of changes due to polymorphisms among genotypes, providing insights into design principles that can aid future drug development and potentially can be extended to other proteins.

biochemistry↗

bioRxiv2018

Structural Adaptation of Darunavir Analogs Against Primary Resistance Mutations in HIV-1 Protease

HIV-1 protease is one of the prime targets of agents used in antiretroviral therapy against HIV. However, under selective pressure of protease inhibitors, primary mutations at the active site weaken inhibitor binding to confer resistance. Darunavir (DRV) is the most potent HIV-1 protease inhibitor in clinic; resistance is limited, as DRV fits well within the substrate envelope. Nevertheless, resistance is observed due to hydrophobic changes at residues including I50, V82 and I84 that line the S1/S1 pocket within the active site. Through enzyme inhibition assays and a series of 12 crystal structures, we interrogated susceptibility of DRV and two potent analogs to primary S1 mutations. The analogs had modifications at the hydrophobic P1 moiety to better occupy the unexploited space in the S1 pocket where the primary mutations were located. Considerable losses of potency were observed against protease variants with I84V and I50V mutations for all three inhibitors. The crystal structures revealed an unexpected conformational change in the flap region of I50V protease bound to the analog with the largest P1 moiety, indicating interdependency between the S1 subsite and the flap region. Collective analysis of protease-inhibitor van der Waals (vdW) interactions in the crystal structures using principle component analysis indicated I84V mutation underlying the largest variation in the vdW contacts. Interestingly, the principle components were able to distinguish inhibitor identity and relative potency solely based on vdW interactions of active site residues in the crystal structures. Our results reveal the interplay between inhibitor P1 moiety and primary S1 mutations, as well as suggesting a novel method for distinguishing the interdependence of resistance through principle component analyses.

molecular biology↗