Brian K Shoichet

University of Toronto, Toronto, Ontario, Canada

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Publications (208)1460.59 Total impact

  • Marcus Fischer, Brian Shoichet, James Fraser
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    ABSTRACT: Interrogating fragment libraries by X-ray crystallography is a powerful strategy for discovering allosteric ligands for protein targets. Cryocooling crystals should increase the fraction of occupied binding sites and decrease radiation damage. However, it may also perturb protein conformations accessed at room temperature.Using data from crystals consecutively measured at room and cryogenic temperatures, we find that transient binding sites can be abolished at the cryogenic temperatures employed by standard approaches. Shifting the crystallographic data collection temperature can provide a deliberate perturbation of the protein conformational equilibrium to visualize hidden sites that have great potential to allosterically modulate protein function. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    ChemBioChem 05/2015; DOI:10.1002/cbic.201500196 · 3.06 Impact Factor
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    ABSTRACT: Conformational change in protein-ligand complexes is widely modeled, but the protein accommodation expected on binding a congeneric series of ligands has received less attention. Given their use in medicinal chemistry, there are surprisingly few substantial series of congeneric ligand complexes in the Protein Data Bank (PDB). Here we determine the structures of eight alkyl benzenes, in single-methylene increases from benzene to n-hexylbenzene, bound to an enclosed cavity in T4 lysozyme. The volume of the apo cavity suffices to accommodate benzene but, even with toluene, larger cavity conformations become observable in the electron density, and over the series two other major conformations are observed. These involve discrete changes in main-chain conformation, expanding the site; few continuous changes in the site are observed. In most structures, two discrete protein conformations are observed simultaneously, and energetic considerations suggest that these conformations are low in energy relative to the ground state. An analysis of 121 lysozyme cavity structures in the PDB finds that these three conformations dominate the previously determined structures, largely modeled in a single conformation. An investigation of the few congeneric series in the PDB suggests that discrete changes are common adaptations to a series of growing ligands. The discrete, but relatively few, conformational states observed here, and their energetic accessibility, may have implications for anticipating protein conformational change in ligand design.
    Proceedings of the National Academy of Sciences 04/2015; 112(16). DOI:10.1073/pnas.1500806112 · 9.81 Impact Factor
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    ABSTRACT: Traditional Chinese Medicine (TCM) has been the sole source of therapeutics in China for two millennia. In recent drug discovery efforts, purified components of TCM formulations have shown activity in many in vitro assays, raising concerns of promiscuity. Here, we investigated fourteen bioactive small molecules isolated from TCMs for colloidal aggregation. At concentrations commonly used in cell-based or biochemical assay conditions, eight of these compounds formed particles detectable by dynamic light scattering and showed detergent-reversible inhibition against β-lactamase and malate dehydrogenase, two counter-screening enzymes. When tested against their literature-reported molecular targets, three of these eight compounds showed similar reversal of their inhibitory activity in the presence of detergent. For three of the most potent aggregators, contributions to promiscuity via oxidative cycling were investigated - addition of 1 mM DTT had no effect on their activity, which is inconsistent with an oxidative mechanism. TCMs are often active at micromolar concentrations; this study suggests that care must be taken to control for artifactual activity when seeking their primary targets. Implications for the formulation of these molecules are considered.
    ACS Chemical Biology 01/2015; 10(4). DOI:10.1021/cb5009487 · 5.36 Impact Factor
  • Journal of Medicinal Chemistry 01/2015; 58(3). DOI:10.1021/acs.jmedchem.5b00053 · 5.48 Impact Factor
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    ABSTRACT: Enzyme function prediction remains an important open problem. Though structure-based modeling, such as metabolite docking, can identify substrates of some enzymes, it is ill suited to reactions that progress through a covalent intermediate. Here we investigated the ability of covalent docking to identify substrates that pass through such a covalent intermediate, focusing particularly on the Haloalkanoate Dehalogenase superfamily. In retrospective assessments, covalent docking recapitulated substrate binding-modes of known co-crystal structures, and identified experimental substrates from a set of putative phosphorylated metabolites. In comparison, non-covalent docking of high-energy intermediates yielded non-productive poses. In prospective predictions against seven enzymes, a substrate was identified for five. For one of those cases, a covalent docking prediction, confirmed by empirical screening, and combined with genomic context analysis, suggested the identity of the enzyme that catalyzes the orphan phosphatase reaction in the riboflavin biosynthetic pathway of Bacteroides.
    Biochemistry 12/2014; 54(2). DOI:10.1021/bi501140k · 3.19 Impact Factor
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    Dataset: tondid2014
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    ABSTRACT: Chemical probes that form a covalent bond with a protein target often show enhanced selectivity, potency and utility for biological studies. Despite these advantages, protein-reactive compounds are usually avoided in high-throughput screening campaigns. Here we describe a general method (DOCKovalent) for screening large virtual libraries of electrophilic small molecules. We apply this method prospectively to discover reversible covalent fragments that target distinct protein nucleophiles, including the catalytic serine of AmpC β-lactamase and noncatalytic cysteines in RSK2, MSK1 and JAK3 kinases. We identify submicromolar to low-nanomolar hits with high ligand efficiency, cellular activity and selectivity, including what are to our knowledge the first reported reversible covalent inhibitors of JAK3. Crystal structures of inhibitor complexes with AmpC and RSK2 confirm the docking predictions and guide further optimization. As covalent virtual screening may have broad utility for the rapid discovery of chemical probes, we have made the method freely available through an automated web server (
    Nature Chemical Biology 10/2014; 11(3). DOI:10.1038/nchembio.1666 · 13.22 Impact Factor
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    ABSTRACT: Assembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella [1]. The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella [2]. The molecular pathways regulating IFT are largely a mystery. Because actin network disruption leads to changes in ciliary length and number, actin has been proposed to have a role in ciliary assembly. However, the mechanisms involved are unknown. In Chlamydomonas reinhardtii, conventional actin is found in both the cell body and the inner dynein arm complexes within flagella [3, 4]. Previous work showed that treating Chlamydomonas cells with the actin-depolymerizing compound cytochalasin D resulted in reversible flagellar shortening [5], but how actin is related to flagellar length or assembly remains unknown. Here we utilize small-molecule inhibitors and genetic mutants to analyze the role of actin dynamics in flagellar assembly in Chlamydomonas reinhardtii. We demonstrate that actin plays a role in IFT recruitment to basal bodies during flagellar elongation and that when actin is perturbed, the normal dependence of IFT recruitment on flagellar length is lost. We also find that actin is required for sufficient entry of IFT material into flagella during assembly. These same effects are recapitulated with a myosin inhibitor, suggesting that actin may act via myosin in a pathway by which flagellar assembly is regulated by flagellar length.
    Current Biology 08/2014; 24(17). DOI:10.1016/j.cub.2014.07.038 · 9.92 Impact Factor
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    ABSTRACT: Despite tremendous successes of GPCR crystallography, the receptors with available structures represent only a small fraction of human GPCRs. An important role of the modeling community is to maximize structural insights for the remaining receptors and complexes. The community-wide GPCR Dock assessment was established to stimulate and monitor the progress in molecular modeling and ligand docking for GPCRs. The four targets in the present third assessment round presented new and diverse challenges for modelers, including prediction of allosteric ligand interaction and activation states in 5-hydroxytryptamine receptors 1B and 2B, and modeling by extremely distant homology for smoothened receptor. Forty-four modeling groups participated in the assessment. State-of-the-art modeling approaches achieved close-to-experimental accuracy for small rigid orthosteric ligands and models built by close homology, and they correctly predicted protein fold for distant homology targets. Predictions of long loops and GPCR activation states remain unsolved problems.
    Structure 08/2014; 22(8):1120–1139. DOI:10.1016/j.str.2014.06.012 · 6.79 Impact Factor
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    ABSTRACT: Despite tremendous successes of GPCR crystallog-raphy, the receptors with available structures repre-sent only a small fraction of human GPCRs. An important role of the modeling community is to maxi-mize structural insights for the remaining receptors and complexes. The community-wide GPCR Dock assessment was established to stimulate and monitor the progress in molecular modeling and ligand docking for GPCRs. The four targets in the present third assessment round presented new and diverse challenges for modelers, including prediction of allosteric ligand interaction and activation states in 5-hydroxytryptamine receptors 1B and 2B, and modeling by extremely distant homology for smooth-ened receptor. Forty-four modeling groups partici-pated in the assessment. State-of-the-art modeling approaches achieved close-to-experimental accu-racy for small rigid orthosteric ligands and models built by close homology, and they correctly predicted protein fold for distant homology targets. Predictions of long loops and GPCR activation states remain un-solved problems. INTRODUCTION
    Structure 08/2014; 22:1-20. · 6.79 Impact Factor
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    ABSTRACT: Proteins fluctuate between alternative conformations, which presents a challenge for ligand discovery because such flexibility is difficult to treat computationally owing to problems with conformational sampling and energy weighting. Here we describe a flexible docking method that samples and weights protein conformations using experimentally derived conformations as a guide. The crystallographically refined occupancies of these conformations, which are observable in an apo receptor structure, define energy penalties for docking. In a large prospective library screen, we identified new ligands that target specific receptor conformations of a cavity in cytochrome c peroxidase, and we confirm both ligand pose and associated receptor conformation predictions by crystallography. The inclusion of receptor flexibility led to ligands with new chemotypes and physical properties. By exploiting experimental measures of loop and side-chain flexibility, this method can be extended to the discovery of new ligands for hundreds of targets in the Protein Data Bank for which similar experimental information is available.
    Nature Chemistry 07/2014; 6(7):575-83. DOI:10.1038/nchem.1954 · 23.30 Impact Factor
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    ABSTRACT: A novel lactonase from Mycoplasma synoviae 53 (MS53_0025) and Mycoplasma agalactiae PG2 (MAG_6390) was characterized by protein structure determination, molecular docking, gene context analysis and library screening. The crystal structure of MS53_0025 was determined to a resolution of 2.06 Å. This protein adopts a typical amidohydrolase (β/α)8-fold and contains a binuclear zinc center located at the C-terminal end of the β-barrel. A phosphate molecule was bound in the active site and hydrogen bonds to Lys217, Lys244, Tyr245, Arg275, and Tyr278. Both docking and gene context analysis were used to narrow down the theoretical substrate profile of the enzyme thus directing empirical screening to identify that MS53_0025 and MAG_6390 catalyze the hydrolysis of D-xylono-1,4-lactone-5-phosphate (2), with kcat/Km values of 4.7 x 104 M-1 s-1 and 5.7 x 104 M-1 s-1; and L-arabino-1,4-lactone-5-phosphate (7) with kcat/Km values of 1.3 x 104 M-1 s-1 and 2.2 x 104 M-1 s-1, respectively. The identification of the substrate profile of these two phospho-furanose lactonases only emerged when all methods were integrated and therefore provides a blueprint for future substrate identification of highly related amidohydrolase superfamily members.
    Biochemistry 06/2014; 53(28). DOI:10.1021/bi500595c · 3.19 Impact Factor
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    Dataset: tondid2014
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    ABSTRACT: Production of β-lactamases (BLs) is the most widespread resistance mechanism adopted by bacteria to fight β-lactam antibiotics. The substrate spectrum of BLs has become increasingly broad, posing a serious health problem. Thus, there is an urgent need for novel BL inhibitors. Boronic acid transition-state analogues are able to reverse the resistance conferred by Class A and C BLs. We describe a boronic acid analog possessing interesting and potent broad-spectrum activity vs Class A and C serine-based BLs. Starting from benzo(b)thiophene-2-boronic acid (BZBTH2B), a nanomolar non-β-lactam inhibitor of AmpC that can potentiate the activity of a third-generation cephalosporin against AmpC-producing resistant bacteria, we designed a novel broad-spectrum nanomolar inhibitor of Class A and C BLs. Structure based drug design (SBDD), synthesis, enzymology data and x-ray crystallography results are discussed. We clarified the inhibitor binding geometry responsible for broad-spectrum activi-ty vs serine-active BLs using double-mutant thermodynamic cycle studies.
    Journal of Medicinal Chemistry 05/2014; DOI:10.1021/jm5006572 · 5.48 Impact Factor
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    ABSTRACT: Most libraries for fragment-based drug discovery are restricted to 1,000 to 10,000 compounds, but over 700,000 fragments are commercially available and potentially accessible by virtual screening. Whether this larger set would increase chemotype coverage, and whether a computational screen can pragmatically prioritize them, is debated. To investigate this question, a 1281 fragment library was screened by Nuclear Magnetic Resonance (NMR) against AmpC β-lactamase and hits confirmed by Surface Plasmon Resonance (SPR). Nine hits with novel chemotypes were confirmed biochemically with KI values from 0.2 to low mM. We also computationally docked 290,000 purchasable fragments with chemotypes unrepresented in the empirical library, finding ten that had KI values from 0.03 to low mM. Though less novel than those discovered by NMR, the docking-derived fragments filled chemotype holes from the empirical library. Crystal structures of nine of the fragments in complex with AmpC β-lactamase revealed new binding sites, and explained the relatively high-affinity of the docking-derived fragments. The existence of chemotype holes is likely a general feature of fragment libraries, as calculation suggests that to represent the fragment substructures of even known biogenic molecules, would minimally demand a library of over 32,000 fragments. Combining computational and empirical fragment screens enables the discovery of unexpected chemotypes, here by the NMR screen, while capturing chemotypes missing from the empirical library and tailored to the target, with little extra cost in resources.
    ACS Chemical Biology 05/2014; 9(7). DOI:10.1021/cb5001636 · 5.36 Impact Factor
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    ABSTRACT: Enzymes in the glutathione transferase (GST) superfamily catalyze the conjugation of glutathione (GSH) to electrophilic substrates. As a consequence they are involved in a number of key biological processes, including protection of cells against chemical damage, steroid and prostaglandin biosynthesis, tyrosine catabolism, and cell apoptosis. Although virtual screening has been used widely to discover substrates by docking potential non-covalent ligands into active site clefts of enzymes, docking has been rarely constrained by a covalent bond between the enzyme and ligand. In this study, we investigate the accuracy of docking poses and substrate discovery in the GST superfamily, by docking 6738 potential ligands from the KEGG and MetaCyc compound libraries into 14 representative GST enzymes with known structures and substrates using the PLOP program(1). For X-ray structures as receptors, one of the top 3 ranked models is within 3 Å all-atom RMSD of the native complex in 11 of the 14 cases; the enrichment LogAUC value is better than random in all cases, and better than 25 in 7 of 11 cases. For comparative models as receptors, near native ligand-enzyme configurations are often sampled, but difficult to rank highly. For models based on templates with the highest sequence identity, the enrichment LogAUC is better than 25 in 5 of 11 cases, not significantly different from the crystal structures. In conclusion, we show that covalent docking can be a useful tool for substrate discovery and point out specific challenges for future method improvement. Availability: The scripts, comparative models, benchmark and the docking library are available at
    Journal of Chemical Information and Modeling 05/2014; 54(6). DOI:10.1021/ci5001554 · 4.07 Impact Factor
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    ABSTRACT: Predicting substrates for enzymes of unknown function is a major post-genomic challenge. Substrate discovery, like inhibitor discovery, is constrained by our ability to explore chemotypes; it would be expanded by orders of magnitude if reactive sites could be probed with fragments rather than fully elaborated substrates, as is done for inhibitor discovery. To explore the feasibility of this approach, substrates of six enzymes from three different superfamilies were deconstructed into 41 overlapping fragments that were tested for activity or binding. Surpris-ingly, even those fragments containing the key reactive group had little activity, and most frag-ments did not bind measurably, until they captured most of the substrate features. Removing a single atom from a recognized substrate could often reduce catalytic recognition by six log-orders. To explore recognition at atomic resolution, the structures of three fragment complexes of the β-lactamase substrate cephalothin were determined by X-ray crystallography. Substrate discovery may be difficult to reduce to the fragment level, with implications for function discov-ery and for the tolerance of metabolite promiscuity by enzymes. Pragmatically, this study sup-ports the development of libraries of fully elaborated metabolites as probes for enzyme function, which currently do not exist.
    Journal of the American Chemical Society 05/2014; 136(20). DOI:10.1021/ja501354q · 11.44 Impact Factor
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    ABSTRACT: One drug may suppress the effects of another. Although knowledge of drug suppression is vital to avoid efficacy-reducing drug interactions or discover countermeasures for chemical toxins, drug-drug suppression relationships have not been systematically mapped. Here, we analyze the growth response of Saccharomyces cerevisiae to anti-fungal compound ("drug") pairs. Among 440 ordered drug pairs, we identified 94 suppressive drug interactions. Using only pairs not selected on the basis of their suppression behavior, we provide an estimate of the prevalence of suppressive interactions between anti-fungal compounds as 17%. Analysis of the drug suppression network suggested that Bromopyruvate is a frequently suppressive drug and Staurosporine is a frequently suppressed drug. We investigated potential explanations for suppressive drug interactions, including chemogenomic analysis, coaggregation, and pH effects, allowing us to explain the interaction tendencies of Bromopyruvate.
    Chemistry & biology 04/2014; DOI:10.1016/j.chembiol.2014.02.012 · 6.59 Impact Factor
  • Brian K Shoichet
    Nature Chemical Biology 03/2014; 10(4):244-245. DOI:10.1038/nchembio.1484 · 13.22 Impact Factor
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    ABSTRACT: Drug efficacy does not always increase sigmoidally with concentration, which has puzzled the community for decades. Unlike standard sigmoidal curves, bell-shaped concentration-response curves suggest more complex biological effects, such as multiple-binding sites or multiple targets. Here, we investigate a physical property-based mechanism for bell-shaped curves. Beginning with the observation that some drugs form colloidal aggregates at relevant concentrations, we determined concentration-response curves for three aggregating anti-cancer drugs, formulated both as colloids and as free monomer. Colloidal formulations exhibited bell-shaped curves, losing activity at higher concentrations, while monomeric formulations gave typical sigmoidal curves, sustaining a plateau of maximum activity. Inverting the question, we next asked if molecules with bell-shaped curves, reported in the literature, form colloidal aggregates at relevant concentrations. We selected twelve molecules reported to have bell-shaped concentration-response curves and found that five of these formed colloids. To understand the mechanism behind the loss of activity at concentrations where colloids are present, we investigated the diffusion of colloid-forming dye Evans blue into cells. We found that colloidal species are excluded from cells, which may explain the mechanism behind toxicological screens that use Evans blue, Trypan blue, and related dyes.
    ACS Chemical Biology 01/2014; 9(3). DOI:10.1021/cb4007584 · 5.36 Impact Factor

Publication Stats

16k Citations
1,460.59 Total Impact Points


  • 2013–2015
    • University of Toronto
      • Leslie L. Dan Faculty of Pharmacy
      Toronto, Ontario, Canada
  • 1991–2015
    • University of California, San Francisco
      • Department of Pharmaceutical Chemistry
      San Francisco, California, United States
  • 2012
    • University of Southern California
      Los Ángeles, California, United States
    • CSU Mentor
      Long Beach, California, United States
  • 2009
    • Universität Regensburg
      Ratisbon, Bavaria, Germany
  • 2007
    • Texas A&M University
      • Department of Chemistry
      College Station, TX, United States
  • 2005
    • University of Auvergne
      Clermont, Auvergne, France
  • 1998–2003
    • Northwestern University
      • Department of Molecular Pharmacology and Biological Chemistry
      Evanston, IL, United States
  • 2000
    • Eli Lilly
      • Lilly Research Laboratories
      Indianapolis, Indiana, United States
  • 1999
    • Cornell University
      Итак, New York, United States
  • 1997
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States