Brian K Shoichet

University of Toronto, Toronto, Ontario, Canada

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Publications (218)1560.61 Total impact

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    ABSTRACT: At least 120 non-olfactory G-protein-coupled receptors in the human genome are 'orphans' for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs.
    Nature 11/2015; DOI:10.1038/nature15699 · 41.46 Impact Factor
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    ABSTRACT: Finding small molecules that target allosteric sites remains a grand challenge for ligand discovery. In the protein kinase field, only a handful of highly selective allosteric inhibitors have been found. Thus, more general methods are needed to discover allosteric modulators for additional kinases. Here, we use virtual screening against an ensemble of both crystal structures and comparative models to identify ligands for an allosteric peptide-binding site on the protein kinase PDK1 (the PIF pocket). We optimized these ligands through an analog-by-catalog search that yielded compound 4, which binds to PDK1 with eight micromolar affinity. We confirmed the docking poses by determining a crystal structure of PDK1 in complex with 4. Because the PIF pocket appears to be a recurring structural feature of the kinase fold, known generally as the helix αC patch, this approach may enable the discovery of allosteric modulators for other kinases.
    Journal of Medicinal Chemistry 10/2015; 58(20). DOI:10.1021/acs.jmedchem.5b01216 · 5.45 Impact Factor
  • Sarah Barelier · Teague Sterling · Matthew J O'Meara · Brian K Shoichet ·
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    ABSTRACT: The binding of drugs and reagents to off-targets is well known. Whereas many off-targets are related to the primary target by sequence and fold, many ligands bind to unrelated pairs of proteins, and these are harder to anticipate. If the binding site in the off-target can be related to that of the primary target, this challenge resolves into aligning the two pockets. However, other cases are possible: the ligand might interact with entirely different residues and environments in the off-target, or wholly different ligand atoms may be implicated in the two complexes. To investigate these scenarios at atomic resolution, the structures of 59 ligands in 116 complexes (62 pairs in total), where the protein pairs were unrelated by fold but bound an identical ligand, were examined. In almost half of the pairs, the ligand interacted with unrelated residues in the two proteins (29 pairs), and in 14 of the pairs wholly different ligand moieties were implicated in each complex. Even in those 19 pairs of complexes that presented similar environments to the ligand, ligand superposition rarely resulted in the overlap of related residues. There appears to be no single pattern-matching "code" for identifying binding sites in unrelated proteins that bind identical ligands, though modeling suggests that there might be a limited number of different patterns that suffice to recognize different ligand functional groups.
    ACS Chemical Biology 09/2015; DOI:10.1021/acschembio.5b00683 · 5.33 Impact Factor
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    ABSTRACT: Most of the existing antibiotics were discovered through screens of environmental microbes, particularly the streptomycetes, for the capacity to prevent the growth of pathogenic bacteria. This 'activity-guided screening' method has been largely abandoned because it repeatedly rediscovers those compounds that are highly expressed during laboratory culture. Most of these metabolites have already been biochemically characterized. However, the sequencing of streptomycete genomes has revealed a large number of 'cryptic' secondary metabolic genes that are either poorly expressed in the laboratory or that have biological activities that cannot be discovered through standard activity-guided screens. Methods that reveal these uncharacterized compounds, particularly methods that are not biased in favour of the highly expressed metabolites, would provide direct access to a large number of potentially useful biologically active small molecules. To address this need, we have devised a discovery method in which a chemical elicitor called Cl-ARC is used to elevate the expression of cryptic biosynthetic genes. We show that the resulting change in product yield permits the direct discovery of secondary metabolites without requiring knowledge of their biological activity. We used this approach to identify three rare secondary metabolites and find that two of them target eukaryotic cells and not bacterial cells. In parallel, we report the first paired use of cheminformatic inference and chemical genetic epistasis in yeast to identify the target. In this way, we demonstrate that oxohygrolidin, one of the eukaryote-active compounds we identified through activity-independent screening, targets the V1 ATPase in yeast and human cells, and secondarily HSP90.
    ACS Chemical Biology 09/2015; DOI:10.1021/acschembio.5b00612 · 5.33 Impact Factor
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    ABSTRACT: Metformin, an established first-line treatment for patients with type 2 diabetes, has been associated with gastrointestinal (GI) adverse effects that limit its use. Histamine and serotonin have potent effects on the GI tract. The effects of metformin on histamine and serotonin uptake were evaluated in cell lines overexpressing several amine transporters (OCT1, OCT3 and SERT). Metformin inhibited histamine and serotonin uptake by OCT1, OCT3 and SERT in a dose-dependent manner, with OCT1-mediated amine uptake being most potently inhibited (IC50 = 1.5 mM). A chemoinformatics-based method known as Similarity Ensemble Approach predicted diamine oxidase (DAO) as an additional intestinal target of metformin, with an E-value of 7.4 × 10(-5). Inhibition of DAO was experimentally validated using a spectrophotometric assay with putrescine as the substrate. The Ki of metformin for DAO was measured to be 8.6 ± 3.1 mM. In this study, we found that metformin inhibited intestinal amine transporters and DAO at concentrations that may be achieved in the intestine after therapeutic doses. Further studies are warranted to determine the relevance of these interactions to the adverse effects of metformin on the gastrointestinal tract.
    Journal of Pharmacokinetics and Biopharmaceutics 09/2015; 42(5). DOI:10.1007/s10928-015-9436-y · 1.86 Impact Factor
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    ABSTRACT: Colloidal aggregation of organic molecules is the dominant mechanism for artifactual inhibition of proteins, and controls against it are widely deployed. Notwithstanding an increasingly detailed understanding of this phenomenon, a method to reliably predict aggregation has remained elusive. Correspondingly, active molecules that act via aggregation continue to be found in early discovery campaigns, and remain common in the literature. Over the last decade, over twelve thousand aggregating organic molecules have been identified, potentially enabling a precedent-based approach to match known aggregators with new molecules that may be expected to aggregate and lead to artifacts. We investigate an approach that uses lipophilicity, affinity, and similarity to known aggregators to advise on the likelihood that a candidate compound is an aggregator. In prospective experimental testing, 5 of 7 new molecules with Tanimoto coefficients (Tcs) between 0.95 and 0.99 to known aggregators aggregated at relevant concentrations. 10 of 19 with Tcs between 0.94 and 0.90, and 3 of 7 with Tcs between 0.89 and 0.85 also aggregated. Another three of the predicted compounds aggregated at higher concentrations. This method finds that 61,827 or 5.1% of the ligands acting in the 0.1 to 10 μM range in the medicinal chemistry literature are at least 85% similar to a known aggregator with these physical properties, and may aggregate at relevant concentrations. Intriguingly, only 0.73% of all drug-like commercially available compounds resemble the known aggregators, suggesting that colloidal aggregators are enriched in the literature. As a percentage of the literature, aggregator-like compounds have increased nine-fold since 1995, partly reflecting the advent of high-throughput and virtual screens against molecular targets. Emerging from this study is an aggregator advisor database and tool (, free to the community, that may help distinguish between fruitful and artifactual screening hits acting by this mechanism.
    Journal of Medicinal Chemistry 08/2015; 58(17). DOI:10.1021/acs.jmedchem.5b01105 · 5.45 Impact Factor
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    ABSTRACT: Galactofuranose (Galf) is present in glycans critical for virulence and viability of several pathogenic microbes, including Mycobacterium tuberculosis, yet the monosaccharide is absent from mammalian glycans. Uridine 5'-diphosphate-galactopyranose mutase (UGM) catalyzes the formation of UDP-Galf, which is required to produce Galf-containing glycoconjugates. Inhibitors of UGM have therefore been sought as antimicrobial leads and to delineate the roles of Galf in cells. Obtaining cell permeable UGM probes by either design or high throughput screens has been difficult, as has elucidating how UGM binds small molecule, non-carbohydrate inhibitors. To address these issues, we employed structure-based virtual screening to uncover new inhibitor chemotypes, including a triazolothiadiazine series. These compounds are among the most potent antimycobacterial UGM inhibitors described. They also facilitated determination of a UGM-small molecule inhibitor structure, which can guide optimization. A comparison of results from the computational screen and a high-throughput fluorescence polarization screen indicated that the scaffold hits from the former had been evaluated in the FP screen but missed. By focusing on promising compounds, the virtual screen rescued false negatives, providing a blueprint for generating new UGM probes and therapeutic leads.
    ACS Chemical Biology 07/2015; 10(10). DOI:10.1021/acschembio.5b00370 · 5.33 Impact Factor
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    Marcus Fischer · Brian K Shoichet · James S 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; 16(11). DOI:10.1002/cbic.201500196 · 3.09 Impact Factor
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    Matthew Merski · Marcus Fischer · Trent E Balius · Oliv Eidam · Brian K Shoichet ·
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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 · 9.67 Impact Factor
  • Da Duan · Allison K Doak · Lyudmila Petrova · Brian K Shoichet ·
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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 · 5.33 Impact Factor
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    Jürgen Bajorath · Hualiang Jiang · Brian K Shoichet · W Patrick Walters ·

    Journal of Medicinal Chemistry 01/2015; 58(3). DOI:10.1021/acs.jmedchem.5b00053 · 5.45 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 · 3.02 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 · 13.00 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 · 9.57 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 · 5.62 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. · 5.62 Impact Factor
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    Marcus Fischer · Ryan G Coleman · James S Fraser · Brian K Shoichet ·
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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 · 25.33 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 · 3.02 Impact Factor

Publication Stats

18k Citations
1,560.61 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
      • Institute of Biophysics and physical Biochemistry
      Ratisbon, Bavaria, Germany
  • 2007
    • Texas A&M University
      • Department of Chemistry
      College Station, TX, United States
  • 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