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1.  Small molecule inhibition of group I p21-activated kinases in breast cancer induces apoptosis and potentiates the activity of microtubule stabilizing agents 
Breast cancer, the most common cause of cancer-related deaths worldwide among women, is a molecularly and clinically heterogeneous disease. Extensive genetic and epigenetic profiling of breast tumors has recently revealed novel putative driver genes, including p21-activated kinase (PAK)1. PAK1 is a serine/threonine kinase downstream of small GTP-binding proteins, Rac1 and Cdc42, and is an integral component of growth factor signaling networks and cellular functions fundamental to tumorigenesis.
PAK1 dysregulation (copy number gain, mRNA and protein expression) was evaluated in two cohorts of breast cancer tissues (n = 980 and 1,108). A novel small molecule inhibitor, FRAX1036, and RNA interference were used to examine PAK1 loss of function and combination with docetaxel in vitro. Mechanism of action for the therapeutic combination, both cellular and molecular, was assessed via time-lapse microscopy and immunoblotting.
We demonstrate that focal genomic amplification and overexpression of PAK1 are associated with poor clinical outcome in the luminal subtype of breast cancer (P = 1.29 × 10−4 and P = 0.015, respectively). Given the role for PAK1 in regulating cytoskeletal organization, we hypothesized that combination of PAK1 inhibition with taxane treatment could be combined to further interfere with microtubule dynamics and cell survival. Consistent with this, administration of docetaxel with either a novel small molecule inhibitor of group I PAKs, FRAX1036, or PAK1 small interfering RNA oligonucleotides dramatically altered signaling to cytoskeletal-associated proteins, such as stathmin, and induced microtubule disorganization and cellular apoptosis. Live-cell imaging revealed that the duration of mitotic arrest mediated by docetaxel was significantly reduced in the presence of FRAX1036, and this was associated with increased kinetics of apoptosis.
Taken together, these findings further support PAK1 as a potential target in breast cancer and suggest combination with taxanes as a viable strategy to increase anti-tumor efficacy.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-015-0564-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4445529  PMID: 25902869
2.  A novel class of ion displacement ligands as antagonists of the αIIbβ3 receptor that limit conformational reorganization of the receptor 
A collection of αIIbβ3 integrin receptor antagonists possessing a unique MIDAS metal ion displacement mechanism of action is presented. Insight into these agents’ structure-activity relationships, binding modality, and pharmacokinetic and pharmacodynamic profiles highlight the potential of these small molecule ion displacement ligands as attractive candidates for clinical development.
PMCID: PMC3951875  PMID: 24461295
3.  Chemical signatures and new drug targets for gametocytocidal drug development 
Scientific Reports  2014;4:3743.
Control of parasite transmission is critical for the eradication of malaria. However, most antimalarial drugs are not active against P. falciparum gametocytes, responsible for the spread of malaria. Consequently, patients can remain infectious for weeks after the clearance of asexual parasites and clinical symptoms. Here we report the identification of 27 potent gametocytocidal compounds (IC50 < 1 μM) from screening 5,215 known drugs and compounds. All these compounds were active against three strains of gametocytes with different drug sensitivities and geographical origins, 3D7, HB3 and Dd2. Cheminformatic analysis revealed chemical signatures for P. falciparum sexual and asexual stages indicative of druggability and suggesting potential targets. Torin 2, a top lead compound (IC50 = 8 nM against gametocytes in vitro), completely blocked oocyst formation in a mouse model of transmission. These results provide critical new leads and potential targets to expand the repertoire of malaria transmission-blocking reagents.
PMCID: PMC3894558  PMID: 24434750
4.  Identification of Potent and Selective Diphenylpropanamide RORγ Inhibitors 
Retinoic acid-related orphan receptor RORγt plays a pivotal role in the differentiation of TH17 cells. Antagonizing RORγt transcriptional activity is a potential means to treat TH17-related autoimmune diseases. Herein, we describe the identification of a series of diphenylpropanamides as novel and selective RORγ antagonists. Diphenylpropanamide 4n inhibited transcriptional activity of RORγt, but not RORα, in cells. In addition, it suppressed human TH17 cell differentiation at sub-micromolar concentrations.
PMCID: PMC3770298  PMID: 24040486
Retinoic acid-related orphan receptor; RORγ antagonist; diphenylpropanamide; TH17-related autoimmune diseases
5.  Identification of Potent and Selective Diphenylpropanamide RORγ Inhibitors 
Retinoic acid-related orphan receptor RORγt plays a pivotal role in the differentiation of TH17 cells. Antagonizing RORγt transcriptional activity is a potential means to treat TH17-related autoimmune diseases. Herein, we describe the identification of a series of diphenylpropanamides as novel and selective RORγ antagonists. Diphenylpropanamide 4n inhibited the transcriptional activity of RORγt, but not RORα, in cells. In addition, it suppressed human TH17 cell differentiation at submicromolar concentrations.
PMCID: PMC3770298  PMID: 24040486
retinoic acid-related orphan receptor; RORγ antagonist; diphenylpropanamide; TH17-related autoimmune diseases
6.  Synthesis and Evaluation of Quinazolin-4-ones as Hypoxia-inducible Factor-1α Inhibitors 
Quinazolin-4-one 1 was identified as an inhibitor of the HIF-1α transcriptional factor from a high-throughput screen. HIF-1α up-regulation is common in many cancer cells. In this paper, we describe an efficient one-pot sequential reaction for the synthesis of quinazolin-4-one 1 analogues. The structure-activity relationship (SAR) study led to the 5-fold more potent analogue, 16.
PMCID: PMC3681418  PMID: 21831635
hypoxia-inducible factor-1α; quinazolin-4-ones; parallel synthesis
7.  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.
PMCID: PMC3293179  PMID: 22408719
8.  Structure-Guided Design of a High-Affinity Platelet Integrin αIIbβ3 Receptor Antagonist That Disrupts Mg2+ Binding to the MIDAS 
Science Translational Medicine  2012;4(125):125ra32.
An integrin found on platelets, αIIbβ3 mediates platelet aggregation, and αIIbβ3 antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg2+) located in the β subunit metal ion–dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the αIIb subunit and a carboxyl group that coordinates the MIDAS Mg2+ in the β3 subunits. They induce conformational changes in the β3 subunit that may have negative consequences such as exposing previously hidden epitopes and inducing the active conformation of the receptor. We recently reported an inhibitor of αIIbβ3 (RUC-1) that binds exclusively to the αIIb subunit; here, we report the structure-based design and synthesis of RUC-2, a RUC-1 derivative with a ~100-fold higher affinity. RUC-2 does not induce major conformational changes in β3 as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2–IIbβ3 headpiece complex in 1 mM calcium ion (Ca2+)/5 mM Mg2+ at 2.6 Å revealed that RUC-2 binds to αIIb the way RUC-1 does, but in addition, it binds to the β3 MIDAS residue glutamic acid 220, thus displacing Mg2+ from the MIDAS. When the Mg2+ concentration was increased to 20 mM, however, Mg2+ was identified in the MIDAS and RUC-2 was absent. RUC-2′s ability to inhibit ligand binding and platelet aggregation was diminished by increasing the Mg2+ concentration. Thus, RUC-2 inhibits ligand binding by a mechanism different from that of all other αIIbβ3 antagonists and may offer advantages as a therapeutic agent.
PMCID: PMC3390238  PMID: 22422993
9.  A Quantitative High Throughput Screen Identifies Novel Inhibitors of the Interaction of Thyroid Receptor β with a Peptide of Steroid Receptor Coactivator 2 
Journal of biomolecular screening  2011;16(6):618-627.
The thyroid hormone receptors (TR) are members of the nuclear hormone receptor (NHR) superfamily that regulate development, growth, and metabolism. Upon ligand binding, TR releases bound corepressors and recruits coactivators to modulate target gene expression. Steroid Receptor Coactivator 2 (SRC2) is an important coregulator that interacts with TRβ to activate gene transcription. To identify novel inhibitors of the TRβ and SRC2 interaction, we performed a quantitative high throughput screen (qHTS) of a TRβ-SRC2 fluorescence polarization assay against more than 290,000 small molecules. The qHTS assayed compounds at six concentrations up to 92 uM to generate titration-response curves and determine the potency and efficacy of all compounds. The qHTS dataset enabled the characterization of actives for structure-activity relationships as well as for potential artifacts such as fluorescence interference. Selected qHTS actives were tested in the screening assay using fluoroprobes labeled with Texas Red or fluorescein. The retest identified 19 series and 4 singletons as active in both assays with 40% or greater efficacy, free of compound interference and not toxic to mammalian cells. Selected compounds were tested as independent samples and a methylsulfonylnitrobenzoate series inhibited the TRβ-SRC2 interaction with 5 uM IC50. This series represents a new class of thyroid hormone receptor-coactivator modulators.
PMCID: PMC3162318  PMID: 21482722
thyroid receptor; small molecule; HTS; coactivator; protein-protein interaction
10.  Non-iminosugar glucocerebrosidase small molecule chaperones 
MedChemComm  2011;3(1):56-60.
Small molecule chaperones are a promising therapeutic approach for the Lysosomal Storage Disorders (LSDs). Here, we report the discovery of a new series of non-iminosugar glucocerebrosidase inhibitors with chaperone capacity, and describe their structure activity relationship (SAR), selectivity, cell activity phamacokinetics.
PMCID: PMC3351140  PMID: 22606365
11.  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.
PMCID: PMC3351006  PMID: 22033823
Glucocerebrosidase; Natural substrate; Gaucher disease; Enzyme substrate; Inhibitor
12.  Novel Substitutions of 1-Alkoxy- and 1-Arylsulfonyloxy-η3-Allylmolybdenum Complexes. A Case for η1-Alkenyl Carbene Complexes as Intermediates‡ 
Organometallics  2010;29(21):5083-5097.
A series of acyclic and cyclic 1-alkoxy- and 1-arylsulfonyloxy-substituted TpMo(CO)2(η3-allyl) complexes was synthesized and characterized, and exchange of the oxygenated substituent was investigated under a variety of reaction conditions. 1-Alkoxy-substituted η3-allyl and η3-butenyl complexes participated in direct, uncatalyzed exchange of the alkoxy substituent with benzylamine, but required a Lewis acid for exchange with alcohols. The 1-alkoxy-substituted η3-cyclohexenyl complex was unreactive towards exchange under all conditions investigated. The corresponding acyclic arylsulfonyloxy-substituted complexes underwent direct, uncatalyzed exchange with both benzylamine and alcohols, while the arylsulfonyloxy-substituted cyclohexenyl compounds participated in direct substitution with benzylamine, but not alcohols. High enantiopurity acyclic and cyclic alkoxy- and arylsulfonyloxy-substituted complexes provided exchange products with predominant, but incomplete, losses in enantiomeric excess in all cases examined. Mechanisms accounting for the observed reactivity trends and for the losses in enantiomeric excess are discussed. Reaction of alkoxy-substituted complexes through an associative mechanism and of arylsulfonyloxy-substituted compounds through a dissociative mechanism is suggested.
PMCID: PMC2992457  PMID: 21116457
13.  Selective Modulation of Gq/Gs pathways by Naphtho Pyrano Pyrimidines as antagonists of the Neuropeptide S Receptor 
ACS chemical neuroscience  2010;1(8):559-574.
Antagonists of the Neuropeptide S Receptor have been postulated as promising therapeutics in the treatment of respiratory, sleep, anxiety, and addictive disorders. Here we present the SAR of a new series of orthosteric antagonists. Neuropeptide S Receptor signaling is coupled to both Gq and Gs proteins, and we observe that different analogues in this structural series can selectively antagonize these two pathways. Many G-protein coupled receptors transduce signals through multiple pathways. Selective antagonism of these pathways may lead the way to the development of more targeted pharmacological profiles and therapies.
PMCID: PMC2992437  PMID: 21116448
Neuropeptide S Receptor antagonist; sleep disorders; addiction disorders; Naphtho Pyrano Pyrimidines; homogeneous time resolved fluorescence; Neuropeptide S radiolabel displacement assay
14.  Selective Modulation of Gq/Gs pathways by Naphtho Pyrano Pyrimidines As Antagonists of the Neuropeptide S Receptor 
ACS Chemical Neuroscience  2010;1(8):559-574.
Antagonists of the neuropeptide S receptor have been postulated as promising therapeutics in the treatment of respiratory, sleep, anxiety, and addictive disorders. Here, we present the SAR of a new series of orthosteric antagonists. Neuropeptide S receptor signaling is coupled to both Gq and Gs proteins, and we observe that different analogues in this structural series can selectively antagonize these two pathways. Many G-protein coupled receptors transduce signals through multiple pathways. Selective antagonism of these pathways may lead the way to the development of more targeted pharmacological profiles and therapies.
PMCID: PMC2992437  PMID: 21116448
Neuropeptide S receptor antagonist; sleep disorders; addiction disorders; naphtho pyrano pyrimidines; homogeneous time-resolved fluorescence; neuropeptide S radiolabel displacement assay
15.  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
16.  The Identification of Aminothienopyridazine Inhibitors of Tau Assembly by Quantitative High-Throughput Screening† 
Biochemistry  2009;48(32):7732-7745.
Inclusions comprised of fibrils of the microtubule (MT)-associated protein tau are found in the brains of those with Alzheimer’s disease (AD) and other neurodegenerative tauopathies. The pathology that is observed in these diseases is believed to result from the formation of toxic tau oligomers or fibrils, and/or from the loss of normal tau function due to its sequestration into insoluble deposits. Hence, small molecules that prevent tau oligomerization and/or fibrillization might have therapeutic value. Indeed, examples of such compounds have been published but nearly all have properties that render them unsuitable as drug candidates. For these reasons, we conducted quantitative high-throughput screening (qHTS) of ~292,000 compounds to identify drug-like inhibitors of tau assembly. The fibrillization of a truncated tau fragment that contains four MT-binding domains was monitored in an assay that employed complementary thioflavine T fluorescence and fluorescence polarization methods. Previously described classes of inhibitors as well as new scaffolds were identified, including novel aminothienopyridazines (ATPZ’s). A number of ATPZ analogs were synthesized and structure-activity relationships were defined. Further characterization of representative ATPZ compounds showed they do not interfere with tau-mediated MT assembly, and they are significantly more effective at preventing the fibrillization of tau than the Aβ(1–42) peptide which forms AD senile plaques. Thus, the ATPZ molecules described here represent a novel class of tau assembly inhibitors that merit further development for testing in animal models of AD-like tau pathology.
PMCID: PMC2773749  PMID: 19580328
17.  A Quantitative High-Throughput Screen Identifies Potential Epigenetic Modulators of Gene Expression 
Analytical biochemistry  2007;375(2):237-248.
Epigenetic regulation of gene expression is essential in embryonic development and contributes to cancer pathology. We used a cell-based imaging assay that measures derepression of a silenced GFP reporter to identify novel classes of compounds involved in epigenetic regulation. This Locus Derepression (LDR) assay was screened against a 69,137-member chemical library using quantitative high-throughput screening (qHTS), a titration-response method that assays compounds at multiple concentrations. From structure-activity relationships of the 411 actives recovered from the qHTS, six distinct chemical series were chosen for further study. Forty-eight qHTS actives and analogs were counter screened using the parental line of the LDR cells, which lack the GFP reporter. Three series, 8-hydroxy quinoline, quinoline-8-thiol and 1,3,5-thiadiazinane-2-thione, were not fluorescent and re-confirmed activity in the LDR cells. The three active series did not inhibit histone deacetylase activity in nuclear extracts or reactivate the expression of the densely methylated p16 gene in cancer cells. However, one series induced expression of the methylated CDH13 gene and inhibited the viability of several lung cancer lines at submicromolar concentrations. These results suggest that the identified small molecules act on epigenetic or transcriptional components and validate our approach of using a cell-based imaging assay in conjunction with qHTS.
PMCID: PMC2330280  PMID: 18211814
epigenetic; small molecule; GFP; HTS; HDAC; cell assay; cancer
18.  Ambient Temperature Synthesis of High Enantiopurity N-Protected Peptidyl Ketones by Peptidyl Thiol Ester–Boronic Acid Cross-Coupling 
α-Amino acid thiol esters derived from N-protected mono-, di-, and tripeptides couple with aryl, π-electron-rich heteroaryl, or alkenyl boronic acids in the presence of stoichiometric Cu(I) thiophene-2-carboxylate (CuTC) and catalytic Pd2(dba)3/triethylphosphite to generate the corresponding N-protected peptidyl ketones in good to excellent yields and in high enantiopurity. Triethylphosphite plays a key role as a supporting ligand by mitigating an undesired palladium-catalyzed decarbonylation-β-elimination of the α-amino thiol esters. The peptidyl ketone synthesis proceeds at room temperature under non-basic conditions and demonstrates a high tolerance to functionality.
PMCID: PMC2652697  PMID: 17263394
19.  Identification of N-(quinolin-8-yl)benzenesulfonamides as Agents Capable of Down-Regulating NFκB Activity within Two Separate High-Throughput Screens of NFκB Activation 
We describe here a series of N-(quinolin-8-yl)benzenesulfonamides capable of suppressing the NFκB pathway identified from two high-throughput screens run at two centers of the NIH Molecular Libraries Initiative. These small molecules were confirmed in both primary and secondary assays of NFκB activation and expanded upon through analogue synthesis. The series exhibited potencies in the cell-based assays as low as 0.6 µM, and several indications suggest that the targeted activity lies within a common region of the NFκB pathway.
PMCID: PMC2275118  PMID: 18024113
20.  Differentiating Alzheimer Disease-Associated Aggregates with Small Molecules 
Neurobiology of disease  2007;28(3):251-260.
Alzheimer disease is diagnosed postmortem by the density and spatial distribution of β-amyloid plaques and tau-bearing neurofibrillary tangles. The major protein component of each lesion adopts cross-β-sheet conformation capable of binding small molecules with submicromolar affinity. In many cases, however, Alzheimer pathology overlaps with Lewy body disease, characterized by the accumulation of a third cross-β-sheet forming protein, α-synuclein. To determine the feasibility of distinguishing tau aggregates from β-amyloid and α-synuclein aggregates with small molecule probes, a library containing 71,975 small molecules was screened for antagonists of tau-aggregate mediated changes in Thioflavin S fluorescence, followed by secondary screens to distinguish the relative affinity for each substrate protein. Results showed that >10-fold binding selectivity among substrates could be achieved, with molecules selective for tau aggregates containing at least three aromatic or rigid moieties connected by two rotatable bonds.
PMCID: PMC2194600  PMID: 17761424
Alzheimer disease; tau; neurofibrillary tangle; contrast agents; diagnosis
21.  N4-Phenyl Modifications of N2-(2-hydroxyl)ethyl-6-(pyrrolidin-1-yl)-1,3,5-triazine-2,4-diamines Enhance Glucocerebrosidase Inhibition by Small Molecules with Potential as Chemical Chaperones for Gaucher Disease 
A series of 1,3,5-triazine-2,4,6-triamines were prepared and analyzed as inhibitors of glucocerebrosidase. Synthesis, structure activity relationships and the selectivity of chosen analogues against related sugar hydrolases enzymes are described.
PMCID: PMC2083578  PMID: 17827006

Results 1-21 (21)