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Dive into the research topics where Allison K. Doak is active.

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Featured researches published by Allison K. Doak.


Nature | 2012

Large Scale Prediction and Testing of Drug Activity on Side-Effect Targets

Eugen Lounkine; Michael J. Keiser; Steven Whitebread; Dmitri Mikhailov; Jacques Hamon; Jeremy L. Jenkins; Paul Lavan; Eckhard Weber; Allison K. Doak; Serge Côté; Brian K. Shoichet; Laszlo Urban

Discovering the unintended ‘off-targets’ that predict adverse drug reactions is daunting by empirical methods alone. Drugs can act on several protein targets, some of which can be unrelated by conventional molecular metrics, and hundreds of proteins have been implicated in side effects. Here we use a computational strategy to predict the activity of 656 marketed drugs on 73 unintended ‘side-effect’ targets. Approximately half of the predictions were confirmed, either from proprietary databases unknown to the method or by new experimental assays. Affinities for these new off-targets ranged from 1 nM to 30 μM. To explore relevance, we developed an association metric to prioritize those new off-targets that explained side effects better than any known target of a given drug, creating a drug–target–adverse drug reaction network. Among these new associations was the prediction that the abdominal pain side effect of the synthetic oestrogen chlorotrianisene was mediated through its newly discovered inhibition of the enzyme cyclooxygenase-1. The clinical relevance of this inhibition was borne out in whole human blood platelet aggregation assays. This approach may have wide application to de-risking toxicological liabilities in drug discovery.


Journal of Medicinal Chemistry | 2015

An Aggregation Advisor for Ligand Discovery

John J. Irwin; Da Duan; Hayarpi Torosyan; Allison K. Doak; Kristin T. Ziebart; Teague Sterling; Gurgen Tumanian; Brian K. Shoichet

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 past decade, over 12 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, five of seven new molecules with Tanimoto coefficients (Tcs) between 0.95 and 0.99 to known aggregators aggregated at relevant concentrations. Ten of 19 with Tcs between 0.94 and 0.90 and three of seven 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 9-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 ( http://advisor.bkslab.org ), free to the community, that may help distinguish between fruitful and artifactual screening hits acting by this mechanism.


Proceedings of the National Academy of Sciences of the United States of America | 2012

Structure-based ligand discovery for the protein–protein interface of chemokine receptor CXCR4

Michael M. Mysinger; Dahlia R. Weiss; Joshua J. Ziarek; Stéphanie Gravel; Allison K. Doak; Joel Karpiak; Nikolaus Heveker; Brian K. Shoichet; Brian F. Volkman

G-protein–coupled receptors (GPCRs) are key signaling molecules and are intensely studied. Whereas GPCRs recognizing small-molecules have been successfully targeted for drug discovery, protein-recognizing GPCRs, such as the chemokine receptors, claim few drugs or even useful small molecule reagents. This reflects both the difficulties that attend protein–protein interface inhibitor discovery, and the lack of structures for these targets. Imminent structure determination of chemokine receptor CXCR4 motivated docking screens for new ligands against a homology model and subsequently the crystal structure. More than 3 million molecules were docked against the model and then against the crystal structure; 24 and 23 high-scoring compounds from the respective screens were tested experimentally. Docking against the model yielded only one antagonist, which resembled known ligands and lacked specificity, whereas the crystal structure docking yielded four that were dissimilar to previously known scaffolds and apparently specific. Intriguingly, several were potent and relatively small, with IC50 values as low as 306 nM, ligand efficiencies as high as 0.36, and with efficacy in cellular chemotaxis. The potency and efficiency of these molecules has few precedents among protein–protein interface inhibitors, and supports structure-based efforts to discover leads for chemokine GPCRs.


ACS Chemical Biology | 2012

Colloidal aggregation affects the efficacy of anticancer drugs in cell culture

Shawn C. Owen; Allison K. Doak; Pascal Wassam; Molly S. Shoichet; Brian K. Shoichet

Many small molecules, including bioactive molecules and approved drugs, spontaneously form colloidal aggregates in aqueous solution at micromolar concentrations. Though it is widely accepted that aggregation leads to artifacts in screens for ligands of soluble proteins, the effects of colloid formation in cell-based assays have not been studied. Here, seven anticancer drugs and one diagnostic reagent were found to form colloids in both biochemical buffer and in cell culture media. In cell-based assays, the antiproliferative activities of three of the drugs were substantially reduced when in colloidal form as compared to monomeric form; a new formulation method ensured the presence of drug colloids versus drug monomers in solution. We also found that Evans Blue, a dye classically used to measure vascular permeability and to demonstrate the “enhanced permeability and retention (EPR) effect” in solid tumors, forms colloids that adsorb albumin, as opposed to older literature that suggested the reverse.


Journal of Medicinal Chemistry | 2013

Colloidal Aggregation Causes Inhibition of G Protein-Coupled Receptors

Maria F. Sassano; Allison K. Doak; Bryan L. Roth; Brian K. Shoichet

Colloidal aggregation is the dominant mechanism for artifactual inhibition of soluble proteins, and controls against it are now widely deployed. Conversely, investigating this mechanism for membrane-bound receptors has proven difficult. Here we investigate the activity of four well-characterized aggregators against three G protein-coupled receptors (GPCRs) recognizing peptide and protein ligands. Each of the aggregators was active at micromolar concentrations against the three GPCRs in cell-based assays. This activity could be attenuated by either centrifugation of the inhibitor stock solution or by addition of Tween-80 detergent. In the absence of agonist, the aggregators acted as inverse agonists, consistent with a direct receptor interaction. Meanwhile, several literature GPCR ligands that resemble aggregators themselves formed colloids, by both physical and enzymological tests. These observations suggest that some GPCRs may be artifactually antagonized by colloidal aggregates, an effect that merits the attention of investigators in this field.


Journal of Biological Chemistry | 2013

Structure-based Discovery of Antagonists of Nuclear Receptor LRH-1

Cindy Benod; Jens Carlsson; Rubatharshini Uthayaruban; Peter H. Hwang; John J. Irwin; Allison K. Doak; Brian K. Shoichet; Elena P. Sablin; Robert J. Fletterick

Background: Liver receptor homolog 1 (LRH-1, NR5A2) regulates functions of liver, intestines, and pancreas; its aberrant activity is associated with tumorigenesis. Results: Our work identifies the first antagonists of LRH-1. Conclusion: The identified ligands inhibit LRH-1 transcriptional activity, diminishing expression of the receptors target genes. Significance: LRH-1 inhibitors could be used for analyses of the receptors biological mechanisms and for development of cancer therapeutics. Liver receptor homolog 1 (nuclear receptor LRH-1, NR5A2) is an essential regulator of gene transcription, critical for maintenance of cell pluripotency in early development and imperative for the proper functions of the liver, pancreas, and intestines during the adult life. Although physiological hormones of LRH-1 have not yet been identified, crystallographic and biochemical studies demonstrated that LRH-1 could bind regulatory ligands and suggested phosphatidylinositols as potential hormone candidates for this receptor. No synthetic antagonists of LRH-1 are known to date. Here, we identify the first small molecule antagonists of LRH-1 activity. Our search for LRH-1 modulators was empowered by screening of 5.2 million commercially available compounds via molecular docking followed by verification of the top-ranked molecules using in vitro direct binding and transcriptional assays. Experimental evaluation of the predicted ligands identified two compounds that inhibit the transcriptional activity of LRH-1 and diminish the expression of the receptors target genes. Among the affected transcriptional targets are co-repressor SHP (small heterodimer partner) as well as cyclin E1 (CCNE1) and G0S2 genes that are known to regulate cell growth and proliferation. Treatments of human pancreatic (AsPC-1), colon (HT29), and breast adenocarcinoma cells T47D and MDA-MB-468 with the LRH-1 antagonists resulted in the receptor-mediated inhibition of cancer cell proliferation. Our data suggest that specific antagonists of LRH-1 could be used as specific molecular probes for elucidating the roles of the receptor in different types of malignancies.


Journal of Medicinal Chemistry | 2010

Colloid Formation by Drugs in Simulated Intestinal Fluid

Allison K. Doak; Holger Wille; Stanley B. Prusiner; Brian K. Shoichet

Many organic molecules form colloidal aggregates in aqueous solution at micromolar concentrations. These aggregates promiscuously inhibit soluble proteins and are a major source of false positives in high-throughput screening. Several drugs also form colloidal aggregates, and there has been speculation that this may affect the absorption and distribution of at least one drug in vivo. Here we investigate the ability of drugs to form aggregates in simulated intestinal fluid. Thirty-three Biopharmaceutics Classification System (BCS) class II and class IV drugs, spanning multiple pharmacological activities, were tested for promiscuous aggregation in biochemical buffers. The 22 that behaved as aggregators were then tested for colloid formation in simulated intestinal fluid, a buffer mimicking conditions in the small intestine. Six formed colloids at concentrations equal to or lower than the concentrations reached in the gut, suggesting that aggregation may have an effect on the absorption and distribution of these drugs, and potentially others, in vivo.


ACS Chemical Biology | 2014

Colloidal drug formulations can explain "bell-shaped" concentration-response curves

Shawn C. Owen; Allison K. Doak; Ahil N. Ganesh; Lyudmila Nedyalkova; Christopher K. McLaughlin; Brian K. Shoichet; Molly S. Shoichet

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 anticancer 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 12 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.


Molecular & Cellular Proteomics | 2015

A High Through-put Platform for Recombinant Antibodies to Folded Proteins

Michael Hornsby; Marcin Paduch; Shane Miersch; Annika Sääf; Tet Matsuguchi; Brian Lee; Karolina Wypisniak; Allison K. Doak; Daniel King; Svitlana Usatyuk; Kimberly Perry; Vince Lu; William I. Thomas; Judy Luke; Jay S. Goodman; Robert J. Hoey; Darson Lai; Carly Griffin; Zhijian Li; Franco J. Vizeacoumar; Debbie Dong; Elliot Campbell; Stephen Anderson; Nan Zhong; Susanne Gräslund; Shohei Koide; Jason Moffat; Sachdev S. Sidhu; Anthony A. Kossiakoff; James A. Wells

Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade.


ACS Chemical Biology | 2015

Colloidal Aggregation and the in Vitro Activity of Traditional Chinese Medicines

Da Duan; Allison K. Doak; Lyudmila Nedyalkova; Brian K. Shoichet

Traditional Chinese Medicines (TCMs) have 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 14 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 three of these compounds were tested against their literature-reported molecular targets, they 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.

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James A. Wells

University of California

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Bryan L. Roth

University of North Carolina at Chapel Hill

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Da Duan

University of California

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Joel Karpiak

University of California

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John J. Irwin

University of California

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