Faculty Bibliography

Unbiased identification of individual, immunogenic B-cell epitopes in major antigens of a pathogen remains a technology challenge for vaccine discovery. We therefore developed a platform for rapid phage display screening of deep recombinant libraries consisting of as little as a single major pathogen antigen. Using the bi-component pore-forming leukocidin (Luks) exotoxins of the major pathogen Staphylococcus aureus (Sa) as a prototype, we randomly fragmented and separately ligated the Hemolysin gamma A (HlgA) and LukS genes into a custom-built, phage-display system, termed pComb-Opti8. Deep sequence analysis of barcoded amplimers of the HlgA and LukS gene fragment libraries demonstrated that biopannng against a cross-reactive anti-Luk mAb recovered convergent molecular clones with short overlapping homologous sequences. We thereby identified an 11-amino acid sequence that is highly conserved in four Luk toxin subunits, and is ubiquitous in representation within Sa clinical isolates. The isolated 11-amino acid peptide probe was predicted to retain the native 3D-conformation seen within the Luk holotoxin. Indeed, this peptide was recognized by the selecting anti-Luk mAb, and using mutated peptides we showed that a particular amino acid side-chain was essential for these interactions. Furthermore, murine immunization with this peptide elicited IgG-responses that were highly reactive with both the autologous synthetic peptide and the full-length Luk toxin homologues. Thus, using a gene fragment, phage-display based pipeline, we have identified and validated immunogenic B-cell epitopes that are cross-reactive between members of the pore-forming leukocidin family. This approach could be harnessed to identify novel epitopes for a much needed Sa-protective subunit vaccine.

Aim/UNASSIGNED:High-throughput phenotypic screens have emerged as a promising avenue for small-molecule drug discovery. The challenge faced in high-throughput phenotypic screens is target deconvolution once a small molecule hit is identified. Chemogenomics libraries have emerged as an important tool for meeting this challenge. Here, we investigate their target-specificity by deriving a 'polypharmacology index' for broad chemogenomics screening libraries. Methods/UNASSIGNED:All known targets of all the compounds in each library were plotted as a histogram and fitted to a Boltzmann distribution, whose linearized slope is indicative of the overall polypharmacology of the library. Results & conclusion/UNASSIGNED:Comparison of libraries clearly distinguished the most target-specific library, which might be assumed to be more useful for target deconvolution in a phenotypic screen.

[This corrects the article DOI: 10.1016/j.csbj.2018.09.003.].

Despite decades of clinical use and research, the mechanism of action (MOA) of antidepressant medications remains poorly understood. Selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) are the most commonly prescribed antidepressants-atypical antidepressants such as bupropion have also proven effective, while exhibiting a divergent clinical phenotype. The difference in phenotypic profiles presumably lies in the differences among the MOAs of SSRIs/SNRIs and bupropion. We integrated the ensemble of bupropion's affinities for all its receptors with the expression levels of those targets in nervous system tissues. This "combined target tissue" profile of bupropion was compared to those of duloxetine, fluoxetine, and venlafaxine to isolate the unique target tissue effects of bupropion. Our results suggest that the three monoamines-serotonin, norepinephrine, and dopamine-all contribute to the common antidepressant effects of SSRIs, SNRIs, and bupropion. At the same time, bupropion is unique in its action on 5-HT3AR in the dorsal root ganglion and nicotinic acetylcholine receptors in the pineal gland. These unique tissue-specific activities may explain unique therapeutic effects of bupropion, such as pain management and smoking cessation, and, given melatonin's association with nicotinic acetylcholine receptors and depression, highlight the underappreciated role of the melatonergic system in bupropion's MOA.

Skp2 is a member of the F-box family of proteins that serve as substrate-specific adaptors in Skp1-CUL1-ROC1-F-box (SCF) E3 ubiquitin ligases. Skp2 (Fbxl1) directly binds to the tumor suppressor p27 in the context of the SCF^Skp2 E3 ubiquitin ligase to ubiquitylate and target-phosphorylated p27 for proteasomal degradation. As p27 is a powerful suppressor of growth in a variety of cells, and as Skp2 is also overexpressed in many human cancers, Skp2 is considered an oncogene and an intriguing drug target. However, despite 20 years of investigation, a valid chemical inhibitor of Skp2-mediated degradation of p27 has not been identified. Recently, an increasing number of compounds designed to have this bioactivity have been reported. Here, we conduct a meta-analysis of the evidence regarding bioactivity, structure, and medicinal chemistry in order to evaluate and compare these Skp2 inhibitor compounds. Despite chemically diverse compounds with a wide array of Skp2-mediated p27 ubiquitylation inhibition properties reported by several independent groups, no current chemical probe formally qualifies as a validated pharmaceutical hit compound. This finding suggests that our knowledge of the structural biochemistry of the Skp2-p27 complex remains incomplete and highlights the need for novel modes of inquiry.

Voltage-gated ion channels (VGICs) are associated with hundreds of human diseases. To date, 3D structural models of human VGICs have not been reported. We developed a 3D structural integrity metric to rank the accuracy of all VGIC structures deposited in the PDB. The metric revealed inaccuracies in structural models built from recent single-particle, non-crystalline cryo-electron microscopy maps and enabled the building of highly accurate homology models of human Ca_v channel α₁ subunits at atomic resolution. Human Ca_v Mendelian mutations mostly located to segments involved in the mechanism of voltage sensing and gating within the 3D structure, with multiple mutations targeting equivalent 3D structural locations despite eliciting distinct clinical phenotypes. The models also revealed that the architecture of the ion selectivity filter is highly conserved from bacteria to humans and between sodium and calcium VGICs.

Cellular stress signals activate adaptive signaling pathways of the mammalian integrated stress response (ISR), of which the unfolded protein response (UPR) is a subset. These pathways converge at the phosporylation of eIF2α. Drug-like, potent and selective chemical inhibitors (valid chemical probes) targeting major ISR kinases have been previously identified, with the exception of GCN2. We synthesized and evaluated a series of GCN2 inhibitors based on a triazolo[4,5-d]pyrimidine scaffold. Several compounds potently inhibited GCN2 in vitro and displayed good selectivity over the related kinases PERK, HRI, and IRE1. The compounds inhibited phosporylation of eIF2α in HEK293T cells with an IC₅₀ < 150 nM, validating them as chemical probes for cellular studies. These probes were screened against the National Cancer Institute NCI-60 human cancer cell line panel. Uniform growth inhibition was observed in the leukemia group of cell lines. Growth inhibition in the most sensitive cell lines coincided with high GCN2 mRNA expression levels. Oncomine analysis revealed high GCN2 expression accompanied by lower asparagine synthetase (ASNS) expression in patient-derived acute lymphoblastic leukemias with B-Cell origins (B-ALL) as well. Notably, asparaginase, which depletes amino acids and triggers GCN2 activity, is a licensed, first-line B-ALL treatment. Thus, we hypothesize that leukemias exhibiting high GCN2 expression and low ASNS expression may be susceptible to pharmacologic GCN2 inhibition.

Background: Mucosal immunity may be critical to protection from HIV acquisition. Serum antibodies (Abs) targeted specifically to the region of the V2 loop harboring HIV's a4b7 integrin receptor binding site were associated with protection from HIV acquisition in humans. Mucosal Abs of this type may have been further associated with protection in a matched non-human primate model. Cholera toxin B (CTB) is a powerful mucosal adjuvant. We engineered CTB to display the SIVmac251 region of the V2 loop harboring the a4b7 site for testing in Rhesus macaques.
Method(s): The SIVmac251 a4b7 site was fused to CTB and the integrity of the immunogen was confirmed by X-ray crystallography. Antigenicity of the construct was confirmed with the ITS-12 monoclonal antibody. Serum immunogenicity was tested in rabbits and mucosal immunogenicity in young male Rhesus macaques. The macaque study compares 5 intramuscular (IM) and rectal topical co-administrations of CTB-V2 over 24 weeks to a matched-timeline regimen of two sequential DNA SIV gp120 immunizations followed by three ALVAC-SIV immunizations, the last two of which are coadministered with the IM and mucosal CTB-V2 protein boost.
Result(s): The engineered CTB-V2 protein binds galactose, which approximates mucosal GM1-gangliosides, binds ITS-12 potently and elicits titers >10k against the SIVmac251 a4b7 site in 5 of 5 rabbits. Rhesus macaques were successfully inoculated rectally with 200ug of this protein and tolerated the immunization well. The serum and mucosal responses were profiled.
Conclusion(s): Anti-V2, a4b7-site-targeted antibodies may be elicited by mucosal application of an engineered, validated CTB-V2 immunogen. Comparison to potent ALVAC-based regimens may provide insight into protective immune responses elicited by vaccination