Browsing by Author "Vilseck, Jonah Z."

Now showing 1 - 10 of 18
  • Thumbnail Image
    Item
    A λ-dynamics investigation of insulin Wakayama and other A3 variant binding affinities to the insulin receptor
    (bioRxiv, 2024-03-17) Barron, Monica P.; Vilseck, Jonah Z.; Biochemistry and Molecular Biology, School of Medicine
    Insulin Wakayama is a clinical insulin variant where a conserved valine at the third residue on insulin’s A chain (ValA3) is replaced with a leucine (LeuA3), impairing insulin receptor (IR) binding by 140-500 fold. This severe impact on binding from such a subtle modification has posed an intriguing problem for decades. Although experimental investigations of natural and unnatural A3 mutations have highlighted the sensitivity of insulin-IR binding to minor changes at this site, an atomistic explanation of these binding trends has remained elusive. We investigate this problem computationally using λ-dynamics free energy calculations to model structural changes in response to perturbations of the ValA3 side chain and to calculate associated relative changes in binding free energy (ΔΔGbind). The Wakayama LeuA3 mutation and seven other A3 substitutions were studied in this work. The calculated ΔΔGbind results showed high agreement compared to experimental binding potencies with a Pearson correlation of 0.88 and a mean unsigned error of 0.68 kcal/mol. Extensive structural analyses of λ-dynamics trajectories revealed that critical interactions were disrupted between insulin and the insulin receptor as a result of the A3 mutations. This investigation also quantifies the effect that adding an A3 Cδ atom or losing an A3 Cγ atom has on insulin’s binding affinity to the IR. Thus, λ-dynamics was able to successfully model the effects of subtle modifications to insulin’s A3 side chain on its protein-protein interactions with the IR and shed new light on a decades-old mystery: the exquisite sensitivity of hormone-receptor binding to a subtle modification of an invariant insulin residue.
  • Thumbnail Image
    Item
    Absolute Free Energy of Binding Calculations for Macrophage Migration Inhibitory Factor in Complex with a Druglike Inhibitor
    (ACS, 2019-09) Qian, Yue; Cabeza de Vaca, Israel; Vilseck, Jonah Z.; Cole, Daniel J.; Tirado-Rives, Julian; Jorgensen, William L.; Biochemistry and Molecular Biology, School of Medicine
    Calculation of the absolute free energy of binding (ΔGbind) for a complex in solution is challenging owing to the need for adequate configurational sampling and an accurate energetic description, typically with a force field (FF). In this study, Monte Carlo (MC) simulations with improved side-chain and backbone sampling are used to assess ΔGbind for the complex of a druglike inhibitor (MIF180) with the protein macrophage migration inhibitory factor (MIF) using free energy perturbation (FEP) calculations. For comparison, molecular dynamics (MD) simulations were employed as an alternative sampling method for the same system. With the OPLS-AA/M FF and CM5 atomic charges for the inhibitor, the ΔGbind results from the MC/FEP and MD/FEP simulations, −8.80 ± 0.74 and −8.46 ± 0.85 kcal/mol, agree well with each other and with the experimental value of −8.98 ± 0.28 kcal/mol. The convergence of the results and analysis of the trajectories indicate that sufficient sampling was achieved for both approaches. Repeating the MD/FEP calculations using current versions of the CHARMM and AMBER FFs led to a 6 kcal/mol range of computed ΔGbind. These results show that calculation of accurate ΔGbind for large ligands is both feasible and numerically equivalent, within error limits, using either methodology.
  • Thumbnail Image
    Item
    Accurate in silico predictions of modified RNA interactions to a prototypical RNA-binding protein with λ-dynamics
    (bioRxiv, 2024-12-11) Angelo, Murphy; Bhargava, Yash; Kierzek, Elzbieta; Kierzek, Ryszard; Hayes, Ryan L.; Zhang, Wen; Vilseck, Jonah Z.; Aoki, Scott Takeo; Biochemistry and Molecular Biology, School of Medicine
    RNA-binding proteins shape biology through their widespread functions in RNA biochemistry. Their function requires the recognition of specific RNA motifs for targeted binding. These RNA binding elements can be composed of both unmodified and chemically modified RNAs, of which over 170 chemical modifications have been identified in biology. Unmodified RNA sequence preferences for RNA-binding proteins have been widely studied, with numerous methods available to identify their preferred sequence motifs. However, only a few techniques can detect preferred RNA modifications, and no current method can comprehensively screen the vast array of hundreds of natural RNA modifications. Prior work demonstrated that λ-dynamics is an accurate in silico method to predict RNA base binding preferences of an RNA-binding antibody. This work extends that effort by using λ-dynamics to predict unmodified and modified RNA binding preferences of human Pumilio, a prototypical RNA binding protein. A library of RNA modifications was screened at eight nucleotide positions along the RNA to identify modifications predicted to affect Pumilio binding. Computed binding affinities were compared with experimental data to reveal high predictive accuracy. In silico force field accuracies were also evaluated between CHARMM and Amber RNA force fields to determine the best parameter set to use in binding calculations. This work demonstrates that λ-dynamics can predict RNA interactions to a bona fide RNA-binding protein without the requirements of chemical reagents or new methods to experimentally test binding at the bench. Advancing in silico methods like λ-dynamics will unlock new frontiers in understanding how RNA modifications shape RNA biochemistry.
  • Thumbnail Image
    Item
    Addressing Intersite Coupling Unlocks Large Combinatorial Chemical Spaces for Alchemical Free Energy Methods
    (American Chemical Society, 2022) Hayes, Ryan L.; Vilseck, Jonah Z.; Brooks, Charles L., III.; Biochemistry and Molecular Biology, School of Medicine
    Alchemical free energy methods are playing a growing role in molecular design, both for computer-aided drug design of small molecules and for computational protein design. Multisite λ dynamics (MSλD) is a uniquely scalable alchemical free energy method that enables more efficient exploration of combinatorial alchemical spaces encountered in molecular design, but simulations have typically been limited to a few hundred ligands or sequences. Here, we focus on coupling between sites to enable scaling to larger alchemical spaces. We first discuss updates to the biasing potentials that facilitate MSλD sampling to include coupling terms and show that this can provide more thorough sampling of alchemical states. We then harness coupling between sites by developing a new free energy estimator based on the Potts models underlying direct coupling analysis, a method for predicting contacts from sequence coevolution, and find it yields more accurate free energies than previous estimators. The sampling requirements of the Potts model estimator scale with the square of the number of sites, a substantial improvement over the exponential scaling of the standard estimator. This opens up exploration of much larger alchemical spaces with MSλD for molecular design.
  • Thumbnail Image
    Item
    Chemically induced partial unfolding of the multifunctional Apurinic/apyrimidinic endonuclease 1
    (bioRxiv, 2023-06-29) Rai, Ratan; Dawodu, Olabode I.; Johnson, Steven M.; Vilseck, Jonah Z.; Kelley, Mark R.; Ziarek, Joshua J.; Georgiadis, Millie M.; Biochemistry and Molecular Biology, School of Medicine
    Targeting of the multifunctional enzyme apurinic/apyrimidinic endonuclease I/redox factor 1 (APE1) has produced small molecule inhibitors of both its endonuclease and redox activities. While one of the small molecules, the redox inhibitor APX3330, completed a Phase I clinical trial for solid tumors and a Phase II clinical trial for Diabetic Retinopathy/Diabetic Macular Edema, the mechanism of action for this drug has yet to be fully understood. Here, we demonstrate through HSQC NMR studies that APX3330 induces chemical shift perturbations (CSPs) of both surface and internal residues in a concentration-dependent manner, with a cluster of surface residues defining a small pocket on the opposite face from the endonuclease active site of APE1. Furthermore, APX3330 induces partial unfolding of APE1 as evidenced by a time-dependent loss of chemical shifts for approximately 35% of the residues within APE1 in the HSQC NMR spectrum. Notably, regions that are partially unfolded include adjacent strands within one of two beta sheets that comprise the core of APE1. One of the strands comprises residues near the N-terminal region and a second strand is contributed by the C-terminal region of APE1, which serves as a mitochondrial targeting sequence. These terminal regions converge within the pocket defined by the CSPs. In the presence of a duplex DNA substrate mimic, removal of excess APX3330 resulted in refolding of APE1. Our results are consistent with a reversible mechanism of partial unfolding of APE1 induced by the small molecule inhibitor, APX3330, defining a novel mechanism of inhibition.
  • Thumbnail Image
    Item
    Examining the Potential of Targeting the HSP60 Chaperonin System as a Broadly Applicable Chemotherapeutic Strategy
    (2023-12) Liechty, Hope Lauren; Johnson, Steven M.; Motea, Edward A.; Turchi, John J.; Vilseck, Jonah Z.
    This study methodically examined our diversity set of GroEL and HSP60 inhibitors to identify lead candidates that exhibited the most potent and selective cytotoxicity to colon cancer cells over non-cancer cells in vitro. While several structurally distinct candidates were identified, we found that our nitrofuran and hydroxyquinoline-containing N-acylhydrazone series (NF-NAH and HQ-NAH, respectively) were among the most potent and selective. Subsequent screenings across an NCI panel of cancer cell lines of different origins revealed the superior efficacy of the NF-NAH and HQ-NAH series as chemotherapeutic candidates, in contrast to the ABK-based inhibitors we previously reported, which showed poor efficacy across the panel. Given the emerging evidence of the role of mis-localized HSP60 in cancer cell survival, this study also compared the structure and function of naive cHSP60 (the aberrant form presumed to be in the cytosol) with that of mHSP60 (the processed and mature form that is in mitochondria). Analytical size exclusion chromatography revealed cHSP60 is a more stable oligomer consisting of both single and double-ring complexes. Intriguingly, cryoEM analyses revealed that cHSP60 formed a unique face-to-face double-ring complex, as opposed to the structures of other double-ring GroEL and HSP60 chaperonins where their rings stack back-to-back with one another. Subsequent assays demonstrated similar ATPase activities for both mHSP60 and cHSP60, with stimulatory effects observed in the presence of HSP10 for both. Despite the apparent engagement of HSP10, cHSP60 was unable to refold the denatured MDH client protein efficiently, suggesting potential functional divergence in vivo. These enticing results offer novel insights into the physiological importance of the cHSP60 complex and its possible role in cancer progression. As our previous studies examined inhibitors that were developed as GroEL-targeting antibacterial candidates, and given the unique structural/functional differences of cHSP60 compared to GroEL and other chaperonins, including mHSP60, the findings from this study underscore the need for future to identify and optimize inhibitors specifically for targeting cHSP60 to enhance chemotherapeutic effectiveness.
  • Thumbnail Image
    Item
    Exploring Unconventional SAM Analogues To Build Cell-Potent Bisubstrate Inhibitors for Nicotinamide N-Methyltransferase
    (Wiley, 2022) Iyamu, Iredia D.; Vilseck, Jonah Z.; Yadav, Ravi; Noinaj, Nicholas; Huang, Rong; Biochemistry and Molecular Biology, School of Medicine
    Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide and has been associated with various diseases. Herein, we report the first cell-potent NNMT bisubstrate inhibitor II399, demonstrating a Ki of 5.9 nM in a biochemical assay and a cellular IC50value of 1.9µM. The inhibition mechanism and cocrystal structure confirmed II399 engages both the substrate and cofactor binding pockets. Computational modeling and binding data reveal a balancing act between enthalpic and entropic components that lead to II399’s low nM binding affinity. Notably, II399 is 1,000-fold more selective for NNMT than closely related methyltransferases. We expect that II399would serve as a valuable probe to elucidate NNMT biology. Furthermore, this strategy provides the first case of introducing unconventional SAM mimics, which can be adopted to develop cell-potent inhibitors for other SAM-dependent methyltransferases.
  • Thumbnail Image
    Item
    Exploring Unconventional SAM Analogues To Build Cell-Potent Bisubstrate Inhibitors for Nicotinamide N-Methyltransferase
    (Wiley, 2022) Iyamu, Iredia D.; Vilseck, Jonah Z.; Yadav, Ravi; Noinaj, Nicholas; Huang, Rong; Biochemistry and Molecular Biology, School of Medicine
    Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide and has been associated with various diseases. Herein, we report the first cell-potent NNMT bisubstrate inhibitor II399, demonstrating a Ki of 5.9 nM in a biochemical assay and a cellular IC50 value of 1.9 μM. The inhibition mechanism and cocrystal structure confirmed II399 engages both the substrate and cofactor binding pockets. Computational modeling and binding data reveal a balancing act between enthalpic and entropic components that lead to II399′s low nM binding affinity. Notably, II399 is 1 000-fold more selective for NNMT than closely related methyltransferases. We expect that II399 would serve as a valuable probe to elucidate NNMT biology. Furthermore, this strategy provides the first case of introducing unconventional SAM mimics, which can be adopted to develop cell-potent inhibitors for other SAM-dependent methyltransferases.
  • Thumbnail Image
    Item
    Fast free energy estimates from λ-dynamics with bias-updated Gibbs sampling
    (Springer Nature, 2023-12-21) Robo, Michael T.; Hayes, Ryan L.; Ding, Xinqiang; Pulawski, Brian; Vilseck, Jonah Z.; Biochemistry and Molecular Biology, School of Medicine
    Relative binding free energy calculations have become an integral computational tool for lead optimization in structure-based drug design. Classical alchemical methods, including free energy perturbation or thermodynamic integration, compute relative free energy differences by transforming one molecule into another. However, these methods have high operational costs due to the need to perform many pairwise perturbations independently. To reduce costs and accelerate molecular design workflows, we present a method called λ-dynamics with bias-updated Gibbs sampling. This method uses dynamic biases to continuously sample between multiple ligand analogues collectively within a single simulation. We show that many relative binding free energies can be determined quickly with this approach without compromising accuracy. For five benchmark systems, agreement to experiment is high, with root mean square errors near or below 1.0 kcal mol-1. Free energy results are consistent with other computational approaches and within statistical noise of both methods (0.4 kcal mol-1 or less). Notably, large efficiency gains over thermodynamic integration of 18-66-fold for small perturbations and 100-200-fold for whole aromatic ring substitutions are observed. The rapid determination of relative binding free energies will enable larger chemical spaces to be more readily explored and structure-based drug design to be accelerated.
  • Thumbnail Image
    Item
    Gas-Phase Ion/Ion Chemistry for Structurally Sensitive Probes of Gaseous Protein Ion Structure: Electrostatic and Electrostatic to Covalent Cross-Linking
    (Elsevier, 2021-05) Kit, Melanie Cheung See; Carvalho, Veronica V.; Vilseck, Jonah Z.; Webb, Ian K.; Chemistry and Chemical Biology, School of Science
    Intramolecular interactions within a protein are key in maintaining protein tertiary structure and understanding how proteins function. Ion mobility-mass spectrometry (IM-MS) has become a widely used approach in structural biology since it provides rapid measurements of collision cross sections (CCS), which inform on the gas-phase conformation of the biomolecule under study. Gas-phase ion/ion reactions target amino acid residues with specific chemical properties and the modified sites can be identified by MS. In this study, electrostatically reactive, gas-phase ion/ion chemistry and IM-MS are combined to characterize the structural changes between ubiquitin electrosprayed from aqueous and denaturing conditions. The electrostatic attachment of sulfo-NHS acetate to ubiquitin via ion/ion reactions and fragmentation by electron-capture dissociation (ECD) provide the identification of the most accessible protonated sites within ubiquitin as the sulfonate group forms an electrostatic complex with accessible protonated side chains. The protonated sites identified by ECD from the different solution conditions are distinct and, in some cases, reflect the disruption of interactions such as salt bridges that maintain the native protein structure. This agrees with previously published literature demonstrating that a high methanol concentration at low pH causes the structure of ubiquitin to change from a native (N) state to a more elongated A state. Results using gas-phase, electrostatic cross-linking reagents also point to similar structural changes and further confirm the role of methanol and acid in favoring a more unfolded conformation. Since cross-linking reagents have a distance constraint for the two reactive sites, the data is valuable in guiding computational structures generated by molecular dynamics. The research presented here describes a promising strategy that can detect subtle changes in the local environment of targeted amino acid residues to inform on changes in the overall protein structure.