Biology subjects

Mack, A. R.

Publications and source records attributed to Mack, A. R..

3 recordsLinked to original sources

bioRxiv2026

Six years of clinical herpes simplex virus genotypic acyclovir resistance testing confirms common resistance mechanisms and identifies novel mutations

Identification of acyclovir-resistant herpes simplex virus (HSV) infections is critical for directing appropriate antiviral therapy, particularly for immunocompromised patients where resistance rates can reach 30%. In 2020, the University of Washington Clinical Virology Laboratory launched the first clinical genotypic HSV drug resistance test in the United States. While genotypic testing offers significantly faster turnaround times than traditional phenotypic assays, interpretation depends on established mutational databases and remains challenging when novel variants are identified. Here, we retrospectively reviewed all HSV acyclovir resistance Sanger sequencing tests performed from January 2020 to November 2025 at this primary national reference laboratory. Mutations identified via clinical sequencing were compared against published databases of HSV UL23 mutations to determine their phenotypic effects. Over the nearly six-year study period, 136 samples were sequenced with a median turnaround time of 10.6 days. Among these, 65 samples (47.8%) harbored acyclovir resistance mutations, including 45 frameshift mutations. Notably, across the 100 samples (73.5%) displaying mutations not known to cause acyclovir resistance at the time of clinical testing, we identified 56 distinct mutations, including 23 without prior characterization. Our national experience demonstrates that genotypic testing accelerates actionable results in clinical practice and confirms that frameshift mutations remain a primary driver of acyclovir resistance. Furthermore, by uncovering these 23 novel variants, this work provides critical targets for future biochemical and phenotypic characterization of HSV UL23 mutations. IMPORTANCEAcyclovir-resistant herpes simplex virus infections remain an important clinical concern, especially for immunocompromised patients, but testing for acyclovir resistance has traditionally been limited to lengthy and laborious phenotypic testing. Since 2020, the University of Washington Clinical Virology Laboratory has provided genotypic acyclovir resistance testing. This testing provides faster turnaround times but relies on prior mutation characterization to identify resistance making it important to continually evaluate and report identified mutations. The clinical experience with this testing provides valuable insight into resistance mechanisms, identifies common polymorphisms, and highlights novel mutations for further characterization.

microbiology↗

bioRxiv2024

{Omega}-Loop mutations control the dynamics of the active site by modulating a network of hydrogen bonds in PDC-3 β-lactamase

The expression of antibiotic-inactivating enzymes, such as Pseudomonas-derived cephalosporinase-3 (PDC-3), is a major mechanism of intrinsic resistance in bacteria. Using reinforcement learning-driven molecular dynamics simulations and constant pH MD, we investigate how clinically observed mutations in the {Omega}-loop (at residues V211, G214, E219, and Y221) alter the structure and function of PDC-3. Our findings reveal that these substitutions modulate the dynamic flexibility of the {Omega}-loop and the R2-loop, reshaping the cavity of the active site. In particular, E219K and Y221A disrupt the tridentate hydrogen bond network around K67, thus lowering its pKa and promoting proton transfer to the catalytic residue S64. Markov state models reveal that E219K achieves enhanced catalysis by adopting stable, long-lived active conformations, whereas Y221A facilitates activity by rapidly toggling between bond-formed and bond-broken states. In addition, substitutions influence key hydrogen bonds that control the opening and closure of the active-site pocket, consequently influencing the overall size. The pocket expands in all nine clinically identified variants, creating additional space to accommodate bulkier R1 and R2 cephalosporin side chains. Taken together, these results provide a mechanistic basis for how single residue substitutions in the {Omega}-loop affect catalytic activity. Insights into the structural dynamics of the catalytic site advance our understanding of emerging {beta}-lactamase variants and can inform the rational design of novel inhibitors to combat drug-resistant P. aeruginosa.

biophysics↗

bioRxiv2021

Allosteric communication in Class A β-lactamases occurs via Cooperative Coupling of Loop Dynamics

Allosteric effects control protein (e.g. enzyme) activity in ways that are not fully understood. Better understanding of allosteric effects, and tools to identify them, would offer promising alternative strategies to inhibitor development. Through a combination of equilibrium and nonequilibrium molecular dynamics simulations, we identify allosteric effects and communication pathways from two distant ligand binding sites to important active site structural elements that control enzymatic activity in two prototypical class A {beta}-lactamases, TEM-1 and KPC-2. Both of these enzymes are important determinants of antibiotic resistance in widespread bacterial pathogens. The simulations show that the allosteric sites are connected to the active site in both enzymes, (e.g. affecting the conformation of the {Omega}-loop) highlighting how allosteric inhibitors may exert their effects. Nonequilibrium simulations reveal pathways of communication operating over distances of 30 [A] or more. In these identified signaling pathways, the propagation of the signal occurs through cooperative coupling of loop dynamics. Notably, 50% or more clinically relevant amino acid substitutions in each enzyme map onto the identified signal transduction pathways. This suggests that clinically important variation may affect, or be driven by, differences in allosteric behavior, providing a mechanism by which amino acid substitutions may affect the relationship between spectrum of activity, catalytic turnover and potential allosteric behavior in this clinically important enzyme family. Simulations of the type presented here will help in identifying and analyzing such differences.

biophysics↗