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1.  Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis 
Nature chemical biology  2012;8(10):839-847.
Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. PKM2 interaction with phosphotyrosine-containing proteins inhibits enzyme activity and increases availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small molecule PKM2 activators inhibit growth of xenograft tumors. Structural studies reveal that small molecule activators bind PKM2 at the subunit interaction interface, a site distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small molecule activation of PKM2 can interfere with anabolic metabolism.
doi:10.1038/nchembio.1060
PMCID: PMC3711671  PMID: 22922757
2.  Discovery, SAR, and Biological evaluation of Non-Inhibitory Small Molecule Chaperones of Glucocerebrosidase 
Journal of Medicinal Chemistry  2012;55(12):5734-5748.
A major challenge in the field of Gaucher disease has been the development of new therapeutic strategies including molecular chaperones. All previously described chaperones of glucocerebrosidase are enzyme inhibitors, which complicates their clinical development, because their chaperone activity must be balanced against the functional inhibition of the enzyme. Using a novel high throughput screening methodology, we identified a chemical series that does not inhibit the enzyme, but can still facilitate its translocation to the lysosome as measured by immunostaining of glucocerebrosidase in patient fibroblasts. These compounds provide the basis for the development of a novel approach towards small molecule treatment for patients with Gaucher disease.
doi:10.1021/jm300063b
PMCID: PMC3400126  PMID: 22646221
Gaucher disease; glucocerebrosidase; chaperone; pyrazolopyrimidine; activator
3.  The Synthesis and Evaluation of Dihydroquinazolin-4-ones and Quinazolin-4-ones as Thyroid Stimulating Hormone Receptor Agonists 
MedChemComm  2011;2(10):1016-1020.
We herein describe the rapid synthesis of a diverse set of dihydroquinazolin-4-ones and quinazolin-4-ones, their biological evaluation as thyroid stimulating hormone receptor (TSHR) agonists, and SAR analysis. Among the compounds screened, 8b was 60-fold more potent than the hit compound 1a, which was identified from a high throughput screen of over 73,000 compounds.
doi:10.1039/C1MD00145K
PMCID: PMC3293179  PMID: 22408719
4.  Monitoring compound integrity with cytochrome P450 assays and qHTS 
Journal of biomolecular screening  2009;14(5):538-546.
We describe how room temperature storage of a 1,120 member compound library prepared in either DMSO or in a hydrated DMSO/water (67/33) mixture affects the reproducibility of potency values as monitored using cytochrome P450 1A2 and 2D6 isozyme assays. The bioluminescent assays showed Z′-factors of 0.71 and 0.62, with 18% and 32% of the library found as active against the CYP 1A2 and 2D6 isozymes respectively. We tested the library using quantitative high-throughput screening to generate potency values for every library member which was measured at seven time intervals spanning 37 weeks. We calculated the minimum significant ratio (MSR) from these potency values at each time interval and we found that for the library stored in DMSO, the CYP 1A2 and 2D6 assay MSRs progressed from approximately 2.0 to 5.0. The hydrated conditions showed similar performance in both MSR progression and analytical QC results. Based on this study we recommend that DMSO samples be stored in 1,536-well plates for < 4 months at room temperature. Further, the study shows the magnitude of potency changes that can occur in a robust bioassay due to compound sample storage.
doi:10.1177/1087057109336954
PMCID: PMC3430136  PMID: 19483146
HTS; compound storage; DMSO; quantitative HTS
5.  Discovery of Potent and Selective Inhibitors of Human Platelet type 12-Lipoxygenase 
Journal of medicinal chemistry  2011;54(15):5485-5497.
We report the discovery of novel small molecule inhibitors of platelet type 12-human lipoxygenase, which display nanomolar activity against the purified enzyme, using a quantitative high throughput screen (qHTS) on a library of 153,607 compounds. These compounds also exhibit excellent specificity, >50-fold selectivity vs. the paralogs, 5-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity vs. ovine cyclooxygenase-1 and human cyclooxygenase-2. Kinetic experiments indicate this chemotype is a non-competitive inhibitor that does not reduce the active site iron. Moreover, chiral HPLC separation of two of the racemic lead molecules revealed a strong preference for the (–)-enantiomers (IC50 of 0.43 +/- 0.04 and 0.38 +/- 0.05 μM) compared to the (+)-enantiomers (IC50 of >25 μM for both), indicating a fine degree of selectivity in the active site due to chiral geometry. In addition, these compounds demonstrate efficacy in cellular models, which underscores their relevance to disease modification.
doi:10.1021/jm2005089
PMCID: PMC3150642  PMID: 21739938
Human 12-lipoxygenase; high-throughput; selective; inhibitor
6.  A high throughput glucocerebrosidase assay using the natural substrate glucosylceramide 
Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide to form ceramide and glucose. A deficiency of lysosomal glucocerebrosidase due to genetic mutations results in Gaucher disease, in which glucosylceramide accumulates in the lysosomes of certain cell types. Although enzyme replacement therapy is currently available for the treatment of type 1 Gaucher disease, the neuronopathic forms of Gaucher disease are still not treatable. Small molecule drugs that can penetrate the blood-brain barrier, such as pharmacological chaperones and enzyme activators, are new therapeutic approaches for Gaucher disease. Enzyme assays for glucocerebrosidase are used to screen compound libraries to identify new lead compounds for drug development for the treatment of Gaucher disease. But the current assays use artificial substrates that are not physiologically relevant. We developed a glucocerebrosidase assay using the natural substrate glucosylceramide coupled to an Amplex-red enzyme reporting system. This assay is in a homogenous assay format and has been miniaturized in a 1,536-well plate format for high throughput screening. The assay sensitivity and robustness is similar to those seen with other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high throughput screening.
doi:10.1007/s00216-011-5496-z
PMCID: PMC3351006  PMID: 22033823
Glucocerebrosidase; Natural substrate; Gaucher disease; Enzyme substrate; Inhibitor
7.  Separation of Betti Reaction Product Enantiomers: Absolute Configuration and Inhibition of Botulinum Neurotoxin A 
ACS Medicinal Chemistry Letters  2011;2(5):396-401.
The racemic product of the Betti reaction of 5-chloro-8-hydroxyquinoline, benzaldehyde, and 2-aminopyridine was separated by chiral HPLC to determine which enantiomer inhibited botulinum neurotoxin serotype A. When the enantiomers unexpectedly proved to have comparable activity, the absolute structures of (+)-(R)-1 and (−)-(S)-1 were determined by comparison of calculated and observed circular dichroism spectra. Molecular modeling studies were undertaken in an effort to understand the observed bioactivity and revealed different ensembles of binding modes, with roughly equal binding energies, for the two enantiomers.
doi:10.1021/ml200028z
PMCID: PMC3217201  PMID: 22102940
Chiral resolution; Betti reaction products; TDDFT CD calculations; molecular docking; inhibition of botulinum neurotoxin
8.  Separation of Betti Reaction Product Enantiomers: Absolute Configuration and Inhibition of Botulinum Neurotoxin A 
ACS medicinal chemistry letters  2011;2(5):396-401.
The racemic product of the Betti reaction of 5-chloro-8-hydroxyquinoline, benzaldehyde and 2-aminopyridine was separated by chiral HPLC to determine which enantiomer inhibited botulinum neurotoxin serotype A. When the enantiomers unexpectedly proved to have comparable activity, the absolute structures of (+)-(R)-1 and (−)-(S)-1 were determined by comparison of calculated and observed circular dichroism spectra. Molecular modeling studies were undertaken in an effort to understand the observed bioactivity and revealed different ensembles of binding modes, with roughly equal binding energies, for the two enantiomers.
doi:10.1021/ml200028z
PMCID: PMC3217201  PMID: 22102940
chiral resolution; Betti reaction products; TDDFT CD calculations; molecular docking; inhibition of botulinum neurotoxin
9.  Evaluation of Quinazoline analogues as Glucocerebrosidase Inhibitors with Chaperone activity 
Journal of medicinal chemistry  2011;54(4):1033-1058.
Gaucher disease is a Lysosomal Storage Disorder (LSD) caused by deficiency in the enzyme glucocerebrosidase (GC). Small molecule chaperones of protein folding and translocation have been proposed as a promising therapeutic approach to this LSD. Most small molecule chaperones described in the literature contain an iminosugar scaffold. Here we present the discovery and evaluation of a new series of GC inhibitors with a quinazoline core. We demonstrate that this series can improve the translocation of GC to the lysosome in patient-derived cells. To optimize this chemical series, systematic synthetic modifications were performed and the SAR was evaluated and compared using three different readouts of compound activity – enzymatic inhibition, enzyme thermostabilization, and lysosomal translocation of GC.
doi:10.1021/jm1008902
PMCID: PMC3103057  PMID: 21250698
10.  2-oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamides as activators of the tumor cell specific M2 isoform of pyruvate kinase 
Compared to normal differentiated cells, cancer cells have altered metabolic regulation to support biosynthesis and the expression of the M2 isozyme of pyruvate kinase (PKM2) plays an important role in this anabolic metabolism. While the M1 isoform is a highly active enzyme, the alternatively spliced M2 variant is considerably less active and expressed in tumors. While the exact mechanism by which decreased pyruvate kinase activity contributes to anabolic metabolism remains unclear, it is hypothesized that activation of PKM2 to levels seen with PKM1 may promote a metabolic program that is not conducive to cell proliferation. Here we report the third chemotype in a series of PKM2 activators based on the 2-oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamide scaffold. The synthesis, structure activity relationships, selectivity and notable physiochemical properties are described.
doi:10.1016/j.bmcl.2011.08.114
PMCID: PMC3224553  PMID: 21958545
PKM2; pyruvate kinase; cellular metabolism; anti-cancer strategies; small molecule activators
11.  Discovery of Potent and Selective Inhibitors of Human Reticulocyte 15- Lipoxygenase-1 
Journal of medicinal chemistry  2010;53(20):7392-7404.
There are a variety of lipoxygenases in the human body (hLO), each having a distinct role in cellular biology. Human reticulocyte 15-Lipoxygenase-1 (15-hLO-1), which catalyzes the dioxygenation of 1,4-cis,cis-pentadiene-containing polyunsaturated fatty acids, is implicated in a number of diseases including cancer, atherosclerosis, and neurodegenerative conditions. Despite the potential therapeutic relevance of this target, few inhibitors have been reported that are both potent and selective. To this end, we have employed a quantitative high-throughput (qHTS) screen against ~74,000 small molecules in search of reticulocyte 15-hLO-1 selective inhibitors. This screen led to the discovery of a novel chemotype for 15-hLO-1 inhibition, which displays nM potency and is >7,500-fold selective against the related isozymes, 5-hLO, platelet 12-hLO, epithelial 15-hLO-2, ovine cyclooxygenase-1 and human cyclooxygenase-2. In addition, kinetic experiments were performed which indicate that this class of inhibitor is tight binding, reversible, and appears not to reduce the active-site ferric ion.
doi:10.1021/jm1008852
PMCID: PMC2966298  PMID: 20866075
Human 15-lipoxygenase-1; high-throughput; inhibitor
12.  Evaluation of Thieno[3,2-b]pyrrole[3,2-d]pyridazinones as Activators of the Tumor Cell Specific M2 Isoform of Pyruvate Kinase 
Cancer cells have distinct metabolic needs that are different from normal cells and can be exploited for development of anti-cancer therapeutics. Activation of the tumor specific M2 form of pyruvate kinase (PKM2) is a potential strategy for returning cancer cells to a metabolic state characteristic of normal cells. Here, we describe activators of PKM2 based upon a substituted thieno[3,2-b]pyrrole[3,2-d]pyridazinone scaffold. The synthesis of these agents, structure activity relationships, analysis of activity at related targets (PKM1, PKR and PKL) and examination of aqueous solubility are investigated. These agents represent the second reported chemotype for activation of PKM2.
doi:10.1016/j.bmcl.2010.04.015
PMCID: PMC2874658  PMID: 20451379
Warburg effect; pyruvate kinase; cellular metabolism; anti-cancer strategies; small molecule activators
13.  Identification of Known Drugs that Act as Inhibitors of NF-κB Signaling and their Mechanism of Action 
Biochemical pharmacology  2010;79(9):1272-1280.
Nuclear factor-kappa B (NF-κB) is a transcription factor that plays a critical role across many cellular processes including embryonic and neuronal development, cell proliferation, apoptosis, immune responses to infection, and inflammation. Dysregulation of NF-κB signaling is associated with inflammatory diseases and certain cancers. Constitutive activation of NF-κB signaling has been found in some types of tumors including breast, colon, prostate, skin and lymphoid, hence therapeutic blockade of NF-κB signaling in cancer cells provides an attractive strategy for the development of anticancer drugs. To identify small molecule inhibitors of NF-κB signaling, we screened approximately 2,800 clinically approved drugs and bioactive compounds from the NIH Chemical Genomics Center Pharmaceutical Collection (NPC) in a NF-κB mediated β-lactamase reporter gene assay. Each compound was tested at fifteen different concentrations in a quantitative high throughput screening format. We identified nineteen drugs that inhibited NF-κB signaling, with potencies as low as 20 nM. Many of these drugs, including emetine, fluorosalan, sunitinib malate, bithionol, narasin, tribromsalan, and lestaurtinib, inhibited NF-κB signaling via inhibition of IκBα phosphorylation. Others, such as ectinascidin 743, chromomycin A3 and bortezomib utilized other mechanisms. Furthermore, many of these drugs induced caspase 3/7 activity and had an inhibitory effect on cervical cancer cell growth. Our results indicate that many currently approved pharmaceuticals have previously unappreciated effects on NF-κB signaling, which may contribute to anticancer therapeutic effects. Comprehensive profiling of approved drugs provides insight into their molecular mechanisms, thus providing a basis for drug repurposing.
doi:10.1016/j.bcp.2009.12.021
PMCID: PMC2834878  PMID: 20067776
caspase 3/7; cervical cancer; IκBα phosphorylation; NCGC Pharmaceutical Collection; NF-κB signaling
14.  A Highly Potent and Selective Caspase 1 Inhibitor that Utilizes a Key 3-Cyanopropanoic Acid Moiety 
ChemMedChem  2010;5(5):730-738.
Herein we examine the potential of a nitrile-containing proprionic acid moiety as an electrophile for covalent attack by the active site cysteine residue of caspase 1. The syntheses of several cyanopropanate containing small molecules based upon the optimized peptidic scaffold of the prodrug VX-765 were accomplished and found to be potent inhibitors of caspase 1 (IC50s ≤ 1 nM). Examination of these novel small molecules versus a caspase panel demonstrated an impressive degree of selectivity for caspase 1 inhibition. Assessment of hydrolytic stability and selected ADME properties highlighted these agents as potentially useful tools for studying caspase 1 down-regulation in various settings including in vivo analyses.
doi:10.1002/cmdc.200900531
PMCID: PMC3062473  PMID: 20229566
Inhibitor; enzymes; prodrugs; peptides; caspase 1 inhibitor; Cysteine proteases; Caspase 1; VX-765; VRT-043198; covalent modifiers,; nitrile caspase inhibitors
15.  Evaluation of Substituted N,N′-Diarylsulfonamides as Activators of the Tumor Cell Specific M2 Isoform of Pyruvate Kinase 
The metabolism of cancer cells is altered to support rapid proliferation. Pharmacological activators of a tumor cell specific pyruvate kinase isozyme (PKM2) may be an approach for altering the classic Warburg effect characteristic of aberrant metabolism in cancer cells yielding a novel anti-proliferation strategy. In this manuscript we detail the discovery of a series of substituted N,N′-diarylsulfonamides as activators of PKM2. The synthesis of numerous analogues and the evaluation of structure activity relationships are presented as well as assessments of mechanism and selectivity. Several agents are found that have good potencies and appropriate solubility for use as chemical probes of PKM2 including 55 (AC50 = 43 nM, maximum response = 84%; solubility = 7.3 μg/mL), 56 (AC50 = 99 nM, maximum response = 84%; solubility = 5.7 μg/mL) and 58 (AC50 = 38 nM, maximum response = 82%; solubility = 51.2 μg/mL). The small molecules described here represent first-in-class activators of PKM2
doi:10.1021/jm901577g
PMCID: PMC2818804  PMID: 20017496
Warburg effect; pyruvate kinase; cellular metabolism; high-throughput screening; small molecule activators
16.  Identification and Optimization of Inhibitors of Trypanosomal Cysteine Proteases: Cruzain, Rhodesain, and TbCatB 
Journal of medicinal chemistry  2010;53(1):52-60.
Trypanosoma cruzi and Trypanosoma brucei are parasites that cause Chagas’ disease and African sleeping sickness, respectively. Both parasites rely on essential cysteine proteases for survival, cruzain for T. cruzi and TbCatB/rhodesain for T. brucei. A recent quantitative high-throughput screen of cruzain identified triazine nitriles, which are known inhibitors of other cysteine proteases, as reversible inhibitors of the enzyme. Structural modifications detailed herein, including core scaffold modification from triazine to purine, improved the in vitro potency against both cruzain and rhodesain by 350-fold, while also gaining activity against T. brucei parasites. Selected compounds were screened against a panel of human cysteine and serine proteases to determine selectivity, and a co-crystal was obtained of our most potent analog bound to Cruzain.
doi:10.1021/jm901069a
PMCID: PMC2804034  PMID: 19908842
17.  Evaluation of substituted 6-arylquinazolin-4-amines as potent and selective inhibitors of cdc2-like kinases (Clk) 
A series of substituted 6-arylquinazolin-4-amines were prepared and analyzed as inhibitors of Clk4. Synthesis, structure activity-relationships and the selectivity of a potent analogue against a panel of 402 kinases are presented. Inhibition of Clk4 by these agents at varied concentrations of assay substrates (ATP and receptor peptide) highly suggests that this chemotype is an ATP competitive inhibitor. Molecular docking provides further evidence that inhibition is the result of binding at the kinase hinge region. Selected compounds represent novel tools capable of potent and selective inhibition of Clk1, Clk4 and Dyrk1A.
doi:10.1016/j.bmcl.2009.09.121
PMCID: PMC2807730  PMID: 19837585
kinase inhibition; pre-mRNA splicing; Clk; Dyrk1A
18.  The Pilot Phase of the NIH Chemical Genomics Center 
The NIH Chemical Genomics Center (NCGC) was the inaugural center of the Molecular Libraries and Screening Center Network (MLSCN). Along with the nine other research centers of the MLSCN, the NCGC was established with a primary goal of bringing industrial technology and experience to empower the scientific community with small molecule compounds for use in their research. We intend this review to serve as 1) an introduction to the NCGC standard operating procedures, 2) an overview of several of the lessons learned during the pilot phase and 3) a review of several of the innovative discoveries reported during the pilot phase of the MLSCN.
PMCID: PMC2989597  PMID: 19807664
19.  Structure-Mechanism Insights and the Role of Nitric Oxide Donation Guide the Development of Oxadiazole-2-Oxides as Therapeutic Agents against Schistosomiasis 
Journal of medicinal chemistry  2009;52(20):6474-6483.
Schistosomiasis is a chronic parasitic disease affecting hundreds of millions of individuals worldwide. Current treatment depends on a single agent, praziquantel, raising concerns of emergence of resistant parasites. Here, we continue our explorations of an oxadiazole-2-oxide class of compounds we recently identified as inhibitors of thioredoxin glutathione reductase (TGR), a selenocysteine-containing flavoenzyme required by the parasite to maintain proper cellular redox balance. Through systematic evaluation of the core molecular structure of this chemotype we define the essential pharmacophore, establish a link between the nitric oxide donation and TGR inhibition, determine the selectivity for this chemotype versus related reductase enzymes and present evidence that these agents can be modified to possess appropriate drug metabolism and pharmacokinetic properties. The mechanistic link between exogenous NO donation and parasite injury is expanded and better defined. The results of these studies verify the utility of oxadiazole-2-oxides as novel inhibitors of TGR and as efficacious anti-schistosomal reagents.
doi:10.1021/jm901021k
PMCID: PMC2772170  PMID: 19761212
20.  Synthesis of Oxadiazole-2-oxide Analogues as Potential Antischistosomal Agents 
Tetrahedron letters  2009;50(15):1710-1713.
The synthesis of several 1,2,5-oxadiazole-2-oxide (Furoxan) analogues is described herein. These compounds were prepared in an effort to probe the SAR around the phenyl substituent and oxadiazole core for our studies toward thioredoxin-glutathione reductase (TGR) inhibition and anti-schistosomal activity.
PMCID: PMC2721338  PMID: 19802367
21.  Examining the Chirality, Conformation and Selective Kinase Inhibition of 3-((3R,4R)-4-methyl-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropanenitrile (CP-690,550) 
Journal of medicinal chemistry  2008;51(24):8012-8018.
Here, we examine the significance that stereochemistry plays within the clinically relevant Janus Kinase 3 (Jak3) inhibitor CP-690,550. A synthesis of all four enantiopure stereoisomers of the drug was carried out and an examination of each compound revealed that only the enantiopure 3R, 4R isomer was capable of blocking Stat5 phosphorylation (Jak3 dependent). Each compound was profiled across a panel of over 350 kinases which revealed a high level of selectivity for the Jak family kinases for these related compounds. Each stereoisomer retained a degree of binding to Jak3 and Jak2 and the 3R, 4S and 3S, 4R stereoisomers were further revealed to have binding affinity for selected members of the STE7 and STE20 subfamily of kinases. Finally, an appraisal of the minimum energy conformation of each stereoisomer and molecular docking at Jak3 was performed in an effort to better understand each compounds selectivity and potency profiles.
doi:10.1021/jm801142b
PMCID: PMC2660606  PMID: 19053756
Janus Kinase 3; CP-690,550; Kinase inhibition; Chiral drugs
22.  Identification of a Potent New Chemotype for the Selective Inhibition of PDE4 
A series of substituted 3,6-diphenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines were prepared and analyzed as inhibitors of phosphodiesterase 4 (PDE4). Synthesis, structure activity relationships and the selectivity of a highly potent analogue against related phosphodiesterase isoforms are presented.
doi:10.1016/j.bmcl.2008.01.028
PMCID: PMC2268978  PMID: 18243697

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