个人简介

Ph.D. New York University

研究领域

Chemical Biology, Biochemistry, Medicinal Chemistry, Synthetic Biologics

Research in the Holub Laboratory focuses on the development of chemical tools to investigate biological processes. More specifically, we use multidisciplinary approaches to design and synthesize non-natural molecules, such as synthetic biologics and profluorescent ligands, to study and manipulate protein-protein interactions. There are currently two main areas of active research in the Holub Laboratory: Targeting Bcl-2 family proteins with synthetic biologics. Proteins that function through non-covalent interactions with other biomolecules (nucleic acids, lipids, carbohydrates or other proteins) constitute a significant portion of the proteome, but are often difficult to target with traditional, small molecule ligands. Recent evidence indicates, however, that these interactions can effectively be inhibited using peptides and small folded proteins. Indeed, the natural folding of peptide and proteins allows for specific recognition elements to be displayed in patterns that are complementary to native biopolymer surfaces. Such “synthetic biologics” potentially have enormous value as tools for fundamental and applied research and can, in theory, be elaborated into therapeutics that target protein interaction interfaces. The B-cell lymphoma 2 (Bcl-2) proteins are a family of apoptosis regulators that govern mitochondrial outer membrane permeabilization. There currently are 25 known genes in the Bcl-2 family, several of which have been implicated in diseases such as breast and lung cancer. Proteins in the Bcl-2 family control the release of cytochrome c from the mitochondria and may have either pro- or anti-apoptotic function. Bcl-2 signaling pathways are complex (due to cross talk between family members), and there is considerable interest in developing ligands that are capable of selectively modulating Bcl-2 activity. In an effort to enhance our understanding of Bcl-2 mediated signaling, a primary goal of the Holub Laboratory is the design of miniature protein ligands that will bind to and selectively modulate proteins in the Bcl-2 family. My research group uses a complementary, structure-based approach to generate protein ligands that have been engineered to mimic the functional epitope of pro-apoptotic Bcl-2-binding proteins. Using these ligands, our lab studies their ability to potentiate cytochrome c release from mitochondria isolated from cell lines that display anti-apoptotic phenotypes. Our goal for this project is to gain an in depth understanding of the factors that govern molecular recognition of anti-apoptotic proteins within the Bcl-2 family and to use this knowledge to develop therapeutic synthetic biologics. Monitoring steroid hormone receptor dimerization kinetics using profluorescent ligands. The Holub Research Group is also interested in using small molecule “profluorescent” dyes to study the dimerization kinetics of nuclear hormone receptors. Biarsenical dyes selectively label peptides or proteins containing tetracysteine motifs via thiol-arsenic exchange reactions, converting the non-fluorescent 1,2-ethanedithiol (EDT)-bound forms into highly fluorescent tracers. Due to their small size, these molecules impart little steric bulk to the protein of interest, improving overall spatial and kinetic resolution. Importantly, folding of the peptide chain or protein-protein complexation can bring cysteine pairs into close proximity, facilitating the labeling of the tetracysteine motif. Moreover, due to the profluorescent nature of the biarsenical dyes, protein interaction kinetics can be quantified as a function of fluorescence intensity Nuclear hormone receptors (NHRs) are a class of ligand-mediated transcription factors that are modulated by natural steroid hormones (i.e. estrogens or androgens). Following hormone binding, NHRs dimerize and facilitate the expression of specific genes, including genes responsible for cell division. Notably, over-expression of NHRs has been linked to several human diseases, including cancer. The efficiency of NHR dimerization is influenced by several factors including ligand selectivity and structural conformation of the receptor upon ligand binding. Therapies targeting NHRs often antagonize NHR-mediated transcription by causing significant perturbations within the receptor structure. A critical step in the design of potentially therapeutic NHR modulators involves evaluating their influence on receptor dimerization kinetics - however, techniques for screening ligand-mediated NHR dimerization in real time are currently limited. Reliable methods to screen these events would enhance our understanding of ligand-mediated NHR dimerization and would be advantageous when developing novel therapeutics that target specific NHRs. Using highly interdisciplinary techniques, from organic synthesis to protein engineering, the Holub Lab seeks to apply biarsenical profluorophores to monitor NHR dimerization events in real time. We expect that this technology will be amenable to high throughput screening methods capable of rapidly testing large numbers of potential hormone-based therapeutics.

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J. M. Holub, J. R. LaRochelle, J. S. Appelbaum, A. Schepartz. “Improved assays for determining the cytosolic access of peptides, proteins and their mimetics.” Biochemistry, 2013, 52, 9036-9046. P. M. Levine, T. P. Carberry, J. M. Holub, K. Kirshenbaum. "Crafting precise multivalent architectures. Med. Chem. Commun.,,2013, 4, 493-509. J. S. Appelbaum, J. R. LaRochelle, B. A. Smith, D. M. Balkin, J. M. Holub, A. Schepartz. "Arginine topology controls escape of minimally cationic proteins form early endosomes to the cytoplasm." Chem. Biol.,,2012,7, 819-830. J. M. Holub, M. J. Garabedian, K. Kirshenbaum. "Modulation of the human estrogen receptor by estradiol-peptidomimetic conjugates." Mol. Biosys.,,2011,7, 337-345. B. S. Burnham, J. D. Berkowitz, B. McCranor, M. Cooper, K. McCann, K. Pillitteri, J. Hallback, L. Bergh, L. Gofman, M. Melendez, J. M. Holub, K. Morie, C. E. Carraher, Jr. "Lipid-lowering effects of polymers derived from halophenyl pyrroles." Lett. Drug Des. Discov.,,2011,8, 544-549. J. M. Holub, K. Kirshenbaum. "Tricks with clicks: Modification of peptidomimetic oligomers via copper-catalyzed azide-alkyne [3+2] cycloaddition" Chem. Soc. Rev.,,2010,39, 1325-1337. J. M. Holub, H. Jang, K. Kirshenbaum. "Fit to be tied: conformation-directed macrocyclization of peptoid foldamers." Org. Lett.,,2007,9, 3275-3278. J. B. Weidhaas, D. M. Eisenmann, J. M. Holub, S. V. Nallur. "A conserved RAS/Mitogen-Activated Protein Kinase pathway regulates DNA damage-induced cell death postirradiation in Radelegans." Cancer Res.,,2006 66, 10434-10438 J. M. Holub, H. Jang, K. Kirshenbaum. "Clickity-click: highly functionalized peptoid oligomers generated by sequential conjugation reactions on solid phase support." Org. Biomol. Chem.,,2006,4, 1497-1502. J. M. Holub, K. O’Toole-Colin, A. Getzel, A. Argenti, M. Evans, D. Smith, G. Dalglish, S. Rifat, D. Wilson, B. Taylor, U. Miott, J. Glersaye, S. Lam, B. J. McCranor, J. Berkowitz, R. Miller, L. Lukens, K. Krumpte, J. T. Gupton, B. S. Burnham. "Lipid-lowering effects of ethyl 2-phenacyl-3-aryl-1H-pyrrole-4-carboxylates in rodents." Molecules.,,2004,9, 134-157.