Biology subjects

Bush, M. F.

Publications and source records attributed to Bush, M. F..

4 recordsLinked to original sources

bioRxiv2024

An artificial intelligence accelerated virtual screening platform for drug discovery

Structure-based virtual screening is a key tool in early drug discovery, with growing interest in the screening of multi-billion chemical compound libraries. However, the success of virtual screening crucially depends on the accuracy of the binding pose and binding affinity predicted by computational docking. Here we developed a highly accurate structure-based virtual screen method, RosettaVS, for predicting docking poses and binding affinities. Our approach outperforms other state-of-the-art methods on a wide range of benchmarks, partially due to our ability to model receptor flexibility. We incorporate this into a new open-source artificial intelligence accelerated virtual screening platform for drug discovery. Using this platform, we screened multi-billion compound libraries against two unrelated targets, a novel ubiquitin ligase target KLHDC2 and the human voltage-gated sodium channel NaV1.7. On both targets, we discover hits, including seven novel hits (14% hit rate) to KLHDC2 and four novel hits (44% hit rate) to NaV1.7 with single digit micromolar binding affinities. Screening in both cases was completed in less than seven days. Finally, a high resolution X-ray crystallographic structure validates the predicted docking pose for the KLHDC2 ligand complex, demonstrating the effectiveness of our method in lead discovery.

biochemistry↗

bioRxiv2024

Drugs Form Ternary Complexes with Human Liver Fatty Acid Binding Protein (FABP1) and FABP1 Binding Alters Drug Metabolism

Liver fatty acid binding protein (FABP1) binds diverse endogenous lipids and is highly expressed in the human liver. Binding to FABP1 alters the metabolism and homeostasis of endogenous lipids in the liver. Drugs have also been shown to bind to rat FABP1, but limited data is available for human FABP1 (hFABP1). FABP1 has a large binding pocket and multiple fatty acids can bind to FABP1 simultaneously. We hypothesized that drug binding to hFABP1 results in formation of ternary complexes and that FABP1 binding alters drug metabolism. To test these hypotheses native protein mass spectrometry (MS) and fluorescent 11-(dansylamino)undecanoic acid (DAUDA) displacement assays were used to characterize drug binding to hFABP1 and diclofenac oxidation by cytochrome P450 2C9 (CYP2C9) was studied in the presence and absence of hFABP1. DAUDA binding to hFABP1 involved high (Kd,1=0.2 {micro}M) and low affinity (Kd,2 >10 {micro}M) binding sites. Nine drugs bound to hFABP1 with Kd values ranging from 1 to 20 {micro}M. None of the tested drugs completely displaced DAUDA from hFABP1 and fluorescence spectra showed evidence of ternary complex formation. Formation of DAUDA-diclofenac-hFABP1 ternary complex was verified with native MS. Docking placed diclofenac in the portal region of FABP1 with DAUDA in the binding cavity. Presence of hFABP1 decreased the kcat and Km,u of diclofenac with CYP2C9 by [~]50% suggesting that hFABP1 binding in the liver will alter drug metabolism and clearance. Together, these results suggest that drugs form ternary complexes with hFABP1 and that hFABP1 interacts with CYP2C9. Significance statementMany commonly prescribed drugs bind FABP1 forming ternary complexes with FABP1 and the fluorescent fatty acid DAUDA. This suggests that in the human liver drugs will bind to apo-FABP1 and fatty acid bound FABP1. The high expression of FABP1 in the liver and binding of drugs to FABP1 will alter rates of drug metabolism in the liver.

pharmacology and toxicology↗

bioRxiv2023

Elucidating the Impact of Bacterial Lipases, Human Serum Albumin, and FASII Inhibition on the Utilization of Exogenous Fatty Acids by Staphylococcus aureus

Staphylococcus aureus only synthesizes straight-chain or branched-chain saturated fatty acids (SCFAs or BCFAs) via the type II fatty acid synthesis (FASII) pathway, but as a highly adaptive pathogen, S. aureus can also utilize host-derived exogenous fatty acids (eFAs), including SCFAs and unsaturated fatty acids (UFAs). S. aureus secretes three lipases, Geh, sal1, and SAUSA300_0641, which could perform the function of releasing fatty acids from host lipids. Once released, the FAs are phosphorylated by the fatty acid kinase, FakA, and incorporated into the bacterial lipids. In this study, we determined the substrate specificity of S. aureus secreted lipases, the effect of human serum albumin (HSA) on eFA incorporation, and the effect of FASII inhibitor, AFN-1252, on eFA incorporation using comprehensive lipidomics. When grown with major donors of fatty acids, cholesteryl esters (CEs) and triglycerides (TGs), Geh was found to be the primary lipase responsible for hydrolyzing CEs, but other lipases could compensate for the function of Geh in hydrolyzing TGs. Lipidomics showed that eFAs were incorporated into all major S. aureus lipid classes and that fatty acid-containing HSA can serve as a source of eFAs. Furthermore, S. aureus grown with UFAs displayed decreased membrane fluidity and increased production of reactive oxygen species (ROS). Exposure to AFN-1252 enhanced UFAs in the bacterial membrane, even without a source of eFAs, indicating a FASII pathway modification. Thus, the incorporation of eFAs alters the S. aureus lipidome, membrane fluidity, and ROS formation, which could affect host-pathogen interactions and susceptibility to membrane-targeting antimicrobials. IMPORTANCEIncorporation of host-derived exogenous fatty acids (eFAs), particularly unsaturated fatty acids (UFAs), by Staphylococcus aureus could affect the bacterial membrane fluidity and susceptibility to antimicrobials. In this work, we found that Geh is the primary lipase hydrolyzing cholesteryl esters and, to a less extent, triglycerides (TGs) and that human serum albumin (HSA) could serve as a buffer of eFAs, where low levels of HSA facilitate the utilization of eFAs, but high levels of HSA inhibit it. The fact that the FASII inhibitor, AFN-1252, leads to an increase in UFA content even in the absence of eFA suggests that membrane property modulation is part of its mechanism of action. Thus, Geh and/or the FASII system look to be promising targets to enhance S. aureus killing in a host environment by restricting eFA utilization or modulating membrane property, respectively.

microbiology↗

bioRxiv2022

HSPB5 disease-associated mutations have long-range effects on structure and dynamics through networks of quasi-ordered interactions

Found from bacteria to humans, small heat shock proteins (sHSPs) are the least understood protein chaperones. HSPB5 (or B-crystallin) is among the most widely expressed of the ten human sHSPs, including in muscle, brain, and eye lens where it is constitutively present at very high levels and carries out a myriad of functions. A high content of disorder in HSPB5 has stymied efforts to uncover how its structure gives rise to function. To uncover its mechanisms of action, we compared human HSPB5 and two disease-associated mutants, R120G and D109H. Expecting to learn how the mutations lead to loss of function, we found instead that the mutants are constitutively activated chaperones while wild-type HSPB5 can transition reversibly between non-activated (low activity) and activated (high activity) states in response to changing conditions. Techniques that provide information regarding interactions and accessibility of disordered regions revealed that the disordered N-terminal regions (NTR) that are required for chaperone activity exist in a complicated interaction network within HSPB5 oligomers and are sequestered from solvent in non-activated states. Either mutation or an activating pH change cause rearrangements in the network that expose parts of the NTR, making them more available to bind an aggregating client. While beneficial in the short-term, failure of the mutants to adopt a state with lower activity and lower NTR accessibility leads to increased co-aggregation propensity and, presumably, early cataract. The results support a model where chaperone activity and solubility are modulated through the quasi-ordered NTR and its multiple competing interactions. SignificanceSmall heat shock proteins (sHSPs) are the oldest known protein chaperones, but how they recognize misfolding proteins in early stages of aggregation is unknown. Disordered regions within sHSPs are critical to their function, raising the question of how disorder recognizes disorder. We investigated a human sHSP (HSPB5) and two disease mutants using approaches that provide residue-level information in regions of disorder. The findings reveal that large, heterogeneous oligomers of HSPB5 contain complicated networks of interactions involving their disordered regions that protect them from solvent. Conditions associated with stress or disease mutations cause a rearrangement of the interaction network and enhancement of chaperone activity. The study provides new information regarding how disordered regions are modulated in a network of competing interactions.

biophysics↗