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1.  Evaluation of Potential Ionizing Irradiation Protectors and Mitigators Using Clonogenic Survival of Human Umbilical Cord Blood Hematopoietic Progenitor Cells 
Experimental hematology  2013;41(11):10.1016/j.exphem.2013.08.001.
We evaluated the use of colony formation (CFU-GM, BFU-E, and CFU-GEMM) by human umbilical cord blood (CB) hematopoietic progenitor cells for testing novel small molecule ionizing irradiation protectors and mitigators. Each of 11 compounds was added before (protection) or after (mitigation) ionizing irradiation including: GS-nitroxides (JP4-039 and XJB-5-131), the bifunctional sulfoxide MMS-350, the phosphoinositol-3-kinase inhibitor (LY294002), TPP-imidazole fatty acid, (TPP-IOA), the nitric oxide synthase inhibitor (MCF-201-89), the p53/mdm2/mdm4 inhibitor (BEB55), methoxamine, isoproterenol, propanolol, and the ATP sensitive potassium channel blocker (glyburide). The drugs XJB-5-131, JP4-039, and MMS-350 were radiation protectors for CFU-GM. JP4-039 was also a radiation protector for CFU-GEMM. The drugs, XJB-5-131, JP4-039, and MMS-350 were radiation mitigators for BFU-E, MMS-350 and JP4-039 were mitigators for CFU-GM, and MMS350 was a mitigator for CFU-GEMM. In contrast, other drugs that were effective in murine assays: TTP-IOA, LY294002, MCF201-89, BEB55, propranolol, isoproterenol, methoxamine, and glyburide showed no significant protection or mitigation in human CB assays. These data support testing of new candidate clinical radiation protectors and mitigators using human CB clonogenic assays early in the drug discovery process, reducing the need for animal experiments.
PMCID: PMC3834150  PMID: 23933481
cord blood; radiosensitivity; radiation mitigation; therapeutics
2.  Drug Discovery Algorithm for Cutaneous Leishmaniasis 
Cutaneous leishmaniasis is clinically widespread but lacks treatments that are effective and well tolerated. Because all present drugs have been grandfathered into clinical use, there are no examples of a pre-clinical product evaluation scheme that lead to new candidates for formal development. To provide oral agents for development targeting cutaneous leishmaniasis, we have implemented a discovery scheme that incorporates in vitro and in vivo testing of efficacy, toxicity, and pharmacokinetics/metabolism. Particular emphasis is placed on in vivo testing, progression from higher-throughput models to those with most clinical relevance, and efficient use of resources.
PMCID: PMC3583307  PMID: 23390221
3.  Compound Acquisition and Prioritization Algorithm for Constructing Structurally Diverse Compound Libraries 
ACS combinatorial science  2011;13(3):223-231.
In the present study, we report a compound acquisition and prioritization algorithm established for rational chemical library purchasing or compound synthesis in order to increase the diversity of an existing compound collection. This method was established based on chemistry-space calculation using BCUT (Burden CAS University of Texas) descriptors. In order to identify the acquisition of compounds from candidate collections into the existing collection, a derived distance-based selection rule was applied, and the results were well supported by pairwise similarity calculations and cell-partition statistics in chemistry space. The correlation between chemistry-space distance and Tanimoto similarity index was also studied to justify the compound acquisition strategy through weighted linear regression. As a rational approach for library design, the distance-based selection rule exhibits certain advantages in prioritizing compound selection to enhance the overall structural diversity of an existing in-house compound collection or virtual combinatorial library for in silico screening, diversity oriented synthesis and high-throughput screening.
PMCID: PMC3909521  PMID: 21480665
BCUT descriptor; Compound Acquisition; Diversity Analysis
4.  Effector Kinase Coupling Enables High-Throughput Screens for Direct HIV-1 Nef Antagonists with Anti-retroviral Activity 
Chemistry & biology  2013;20(1):82-91.
HIV-1 Nef, a critical AIDS progression factor, represents an important target protein for antiretroviral drug discovery. Because Nef lacks intrinsic enzymatic activity, we developed an assay that couples Nef to the activation of Hck, a Src-family member and Nef effector protein. Using this assay, we screened a large, diverse chemical library and identified small molecules that block Nef-dependent Hck activity with low micromolar potency. Of these, a diphenylpyrazolo compound demonstrated sub-micromolar potency in HIV-1 replication assays against a broad range of primary Nef variants. This compound binds directly to Nef via a pocket formed by the Nef dimerization interface and disrupts Nef dimerization in cells. Coupling of non-enzymatic viral accessory factors to host cell effector proteins amenable to high-throughput screening may represent a general strategy for the discovery of new antimicrobial agents.
PMCID: PMC3559019  PMID: 23352142
5.  Alkylation sensitivity screens reveal a conserved cross-species functionome 
Molecular cancer research : MCR  2012;10(12):10.1158/1541-7786.MCR-12-0168.
To identify genes that contribute to chemotherapy resistance in glioblastoma, we conducted a synthetic lethal screen in a chemotherapy-resistant glioblastoma derived cell line with the clinical alkylator temozolomide (TMZ) and an siRNA library tailored towards “druggable” targets. Select DNA repair genes in the screen were validated independently, confirming the DNA glycosylases UNG and MYH as well as MPG to be involved in the response to high dose TMZ. The involvement of UNG and MYH is likely the result of a TMZ-induced burst of reactive oxygen species. We then compared the human TMZ sensitizing genes identified in our screen with those previously identified from alkylator screens conducted in E. coli and S. cerevisiae. The conserved biological processes across all three species composes an Alkylation Functionome that includes many novel proteins not previously thought to impact alkylator resistance. This high-throughput screen, validation and cross-species analysis was then followed by a mechanistic analysis of two essential nodes: base excision repair (BER) DNA glycosylases (UNG, human and mag1, S. cerevisiae) and protein modification systems, including UBE3B and ICMT in human cells or pby1, lip22, stp22 and aim22 in S. cerevisiae. The conserved processes of BER and protein modification were dual targeted and yielded additive sensitization to alkylators in S. cerevisiae. In contrast, dual targeting of BER and protein modification genes in human cells did not increase sensitivity, suggesting an epistatic relationship. Importantly, these studies provide potential new targets to overcome alkylating agent resistance.
PMCID: PMC3877719  PMID: 23038810
Temozolomide; chemotherapy resistance; alkylation response; siRNA; DNA Repair
6.  Development and Validation of a High-Content Screening Assay to Identify Inhibitors of Cytoplasmic Dynein-Mediated Transport of Glucocorticoid Receptor to the Nucleus 
Rapid ligand-induced trafficking of glucocorticoid nuclear hormone receptor (GR) from the cytoplasm to the nucleus is an extensively studied model for intracellular retrograde cargo transport employed in constructive morphogenesis and many other cellular functions. Unfortunately, potent and selective small-molecule disruptors of this process are lacking, which has restricted pharmacological investigations. We describe here the development and validation of a 384-well high-content screening (HCS) assay to identify inhibitors of the rapid ligand-induced retrograde translocation of cytoplasmic glucocorticoid nuclear hormone receptor green fluorescent fusion protein (GR-GFP) into the nuclei of 3617.4 mouse mammary adenocarcinoma cells. We selected 3617.4 cells, because they express GR-GFP under the control of a tetracycline (Tet)-repressible promoter and are exceptionally amenable to image acquisition and analysis procedures. Initially, we investigated the time-dependent expression of GR-GFP in 3617.4 cells under Tet-on and Tet-off control to determine the optimal conditions to measure dexamethasone (Dex)-induced GR-GFP nuclear translocation on the ArrayScan-VTI automated imaging platform. We then miniaturized the assay into a 384-well format and validated the performance of the GR-GFP nuclear translocation HCS assay in our 3-day assay signal window and dimethylsulfoxide validation tests. The molecular chaperone heat shock protein 90 (Hsp90) plays an essential role in the regulation of GR steroid binding affinity and ligand-induced retrograde trafficking to the nucleus. We verified that the GR-GFP HCS assay captured the concentration-dependent inhibition of GR-GFP nuclear translocation by 17-AAG, a benzoquinone ansamycin that selectively blocks the binding and hydrolysis of ATP by Hsp90. We screened the 1280 compound library of pharmacologically active compounds set in the Dex-induced GR-GFP nuclear translocation assay and used the multi-parameter HCS data to eliminate cytotoxic compounds and fluorescent outliers. We identified five qualified hits that inhibited the rapid retrograde trafficking of GR-GFP in a concentration-dependent manner: Bay 11-7085, 4-phenyl-3-furoxancarbonitrile, parthenolide, apomorphine, and 6-nitroso-1,2-benzopyrone. The data presented here demonstrate that the GR-GFP HCS assay provides an effective phenotypic screen and support the proposition that screening a larger library of diversity compounds will yield novel small-molecule probes that will enable the further exploration of intracellular retrograde transport of cargo along microtubules, a process which is essential to the morphogenesis and function of all cells.
PMCID: PMC3464420  PMID: 22830992
7.  Targeting sub-cellular localization through the Polo-Box Domain: non-ATP competitive Inhibitors recapitulate a PLK1 phenotype 
Molecular cancer therapeutics  2012;11(8):1683-1692.
The polo-box domain (PBD) has critical roles in the mitotic functions of PLK1. The REPLACE strategy to develop inhibitors of protein-protein interactions has identified alternatives for the N-terminal tripeptide of a Cdc25C substrate. In addition, a peptide structure activity relationship described key determinants and novel information useful for drug design. Fragment ligated inhibitory peptides (FLIPs) were generated with comparable affinity to peptide PBD inhibitors and possessed anti-proliferative phenotypes in cells consistent with the observed decrease in PLK1 centrosomal localization. These FLIPs demonstrated evidence of enhanced PLK1 inhibition in cells relative to peptides and induced monopolar and multipolar spindles, which stands in contrast to previously reported small molecule PBD inhibitors that display phenotypes only partially representative of PLK1 knockdown. Progress obtained applying REPLACE validates this approach for identifying fragment alternatives for determinants of the Cdc25C binding motif and extends its applicability of the strategy for discovering protein-protein interaction inhibitors. In addition, the described PBD inhibitors retain high specificity for PLK1 over PLK3 and therefore show promise as isotype selective, non-ATP competitive kinase inhibitors that provide new impetus for the development of PLK1 selective anti-tumor therapeutics.
PMCID: PMC3711794  PMID: 22848093
Phosphorylation and proteolysis in cell cycle control; Protein serine-threonine kinases; Molecular modelling; In silico evaluation of targets and design of libraries; Protein/protein interactions; Cell cycle mechanisms of anticancer drug action; Kinase and phosphatase inhibitors; Novel antitumor agents
8.  Targeted Deletion of the Metastasis-Associated Phosphatase Ptp4a3 (PRL-3) Suppresses Murine Colon Cancer 
PLoS ONE  2013;8(3):e58300.
Ptp4a3 (commonly known as PRL-3) is an enigmatic member of the Ptp4a family of prenylated protein tyrosine phosphatases that are highly expressed in many human cancers. Despite strong correlations with tumor metastasis and poor patient prognosis, there is very limited understanding of this gene family's role in malignancy. Therefore, we created a gene-targeted murine knockout model for Ptp4a3, the most widely studied Ptp4a family member. Mice deficient for Ptp4a3 were grossly normal. Fewer homozygous-null males were observed at weaning, however, and they maintained a decreased body mass. Although Ptp4a3 is normally associated with late-stage cancer and metastasis, we observed increased Ptp4a3 expression in the colon of wildtype mice immediately following treatment with the carcinogen azoxymethane. To investigate the role of Ptp4a3 in malignancy, we used the most commonly studied murine colitis-associated colon cancer model. Wildtype mice treated with azoxymethane and dextran sodium sulfate developed approximately 7–10 tumors per mouse in the distal colon. The resulting tumor tissue had 4-fold more Ptp4a3 mRNA relative to normal colon epithelium and increased PTP4A3 protein. Ptp4a3-null mice developed 50% fewer colon tumors than wildtype mice after exposure to azoxymethane and dextran sodium sulfate. Tumors from the Ptp4a3-null mice had elevated levels of both IGF1Rβ and c-MYC compared to tumors replete with Ptp4a3, suggesting an enhanced cell signaling pathway engagement in the absence of the phosphatase. These results provide the first definitive evidence implicating Ptp4a3 in colon tumorigenesis and highlight the potential value of the phosphatase as a therapeutic target for early stage malignant disease.
PMCID: PMC3610886  PMID: 23555575
9.  Mutagenesis and Computer Modeling Studies of A GPCR Conserved Residue W5.43(194) in Ligand Recognition and Signal Transduction for CB2 Receptor 
International immunopharmacology  2011;11(9):1303-1310.
W5.43(194), a conserved tryptophan residue among G-protein coupled receptors (GPCRs) and cannabinoid receptors (CB), was examined in the present report for its significance in CB2 receptor ligand binding and adenylyl cyclase (AC) activity. Computer modeling postulates that this site in CB2 may be involved in the affinity of WIN55212-2 and SR144528 through aromatic contacts. In the present study, we reported that a CB2 receptor mutant, W5.43(194)Y, which had a tyrosine (Y) substitution for tryptophan (W), retained the binding affinity for CB agonist CP55940, but reduced binding affinity for CB2 agonist WIN55212-2 and inverse agonist SR144528 by 8-fold and 5-fold, respectively; the CB2 W5.43(194)F and W5.43(194)A mutations significantly affect the binding activities of CP55940, WIN55212-2 and SR144528. Furthermore, we found that agonist-mediated inhibition of the forskolin-induced cAMP production was dramatically diminished in the CB2 mutant W5.43(194)Y, whereas W5.43(194)F and W5.43(194)A mutants resulted in complete elimination of downstream signaling, suggesting that W5.43(194) was essential for the full activation of CB2. These results indicate that both aromatic interaction and hydrogen bonding are involved in ligand binding for the residue W5.43(194), and the mutations of this tryptophan site may affect the conformation of the ligand binding pocket and therefore control the active conformation of the wild type CB2 receptor. W5.43(194)Y/F/A mutations also displayed noticeable enhancement of the constitutive activation probably attributed to the receptor conformational changes resulted from the mutations.
PMCID: PMC3445262  PMID: 21539938
G-protein coupled receptors; cannabinoid receptor CB2; ligand binding and G-protein signaling; tryptophan; site-directed mutagenesis; Computer modeling
10.  Phenotypic Screening Reveals Topoisomerase I as a Breast Cancer Stem Cell Therapeutic Target 
Oncotarget  2012;3(9):998-1010.
Cancer stem cells (CSCs) are a subpopulation generally thought to be responsible for cancer initiation and progression. Because CSCs are often rare in the total tumor cell population and differentiate rapidly when grown in culture, it has been challenging to uncover compounds that selectively target CSCs. We previously described CSC-emulating cells derived from breast cancer cell lines that maintained a stable undifferentiated state. We optimized a phenotypic assay with these cells and screened 1,280-bioactive compounds, identifying five that preferentially inhibited CSC-like cell proliferation. Using a compound-guided target identification approach, we found high topoisomerase I (Topo I) expression levels in breast CSC-like cells and primary breast CSCs. Structurally unrelated small molecules targeting Topo I preferentially inhibited CSC-like cells. These results illustrate the substantial power of this CSC phenotypic screening platform and promote Topo I as a potential molecular therapeutic target for therapies aimed at expunging CSCs.
PMCID: PMC3660065  PMID: 22948175
cancer stem cell; Topoisomerase I; small molecule inhibitor; compound library screening
11.  A simplified synthesis of novel dictyostatin analogs with in vitro activity against epothilone B resistant cells and antiangiogenic activity in zebrafish embryos 
Molecular cancer therapeutics  2011;10(6):994-1006.
The natural product (−)-dictyostatin is a microtubule stabilizing agent that potently inhibits the growth of human cancer cells including paclitaxel-resistant clones. Extensive structure-activity relationship studies have revealed several regions of the molecule that could be altered without loss of activity. The most potent synthetic dictyostatin analog described to date, 6-epi-dictyostatin, has in vivo antitumor activity against human breast cancer xenografts superior to paclitaxel. Despite their encouraging preclinical activities, the complex chemical structure of the dictyostatins presents a major obstacle in their development into novel antineoplastic therapies. We recently reported a streamlined synthesis of 16-desmethyl-25,26 dihydrodictyostatins and found several agents that compared with 6-epi-dictyostatin retained nanomolar activity in cellular microtubule bundling assays but showed cross-resistance to paclitaxel in cells with mutations in beta-tubulin. Extending these studies, we applied the new, highly convergent synthesis to generate 25,26-dihydrodictyostatin and 6-epi-25,26-dihydrodictyostatin. Both compounds were potent microtubule perturbing agents that induced mitotic arrest and microtubule assembly in vitro and in intact cells. In vitro radioligand binding studies showed that 25,26-dihydrodictyostatin and its C-6 epimer were able to displace [3H]paclitaxel and [14C]epothilone B from microtubules with potencies comparable to (−)-dictyostatin and discodermolide. Both compounds inhibited the growth of paclitaxel- and epothilone B-resistant cell lines at low nanomolar concentrations, synergized with paclitaxel in MDA-MB-231 human breast cancer cells, and had antiangiogenic activity in transgenic zebrafish larvae. The data identify 25,26-dihydrodictyostatin and 6-epi-25,26-dihydrodictyostatin as candidates for scale-up synthesis and further preclinical development.
PMCID: PMC3112307  PMID: 21490306
dictyostatin; high-content screening; multidrug resistance; zebrafish; angiogenesis
12.  Synthesis and Structure-Activity Relationships of Benzothienothiazepinone Inhibitors of Protein Kinase D 
ACS medicinal chemistry letters  2011;2(2):154-159.
Protein kinase D (PKD) is a member of a novel family of serine/threonine kinases that regulate fundamental cellular processes. PKD is implicated in the pathogenesis of several diseases, including cancer. Progress in understanding the biological functions and therapeutic potential of PKD has been hampered by the lack of specific inhibitors. The benzoxoloazepinolone CID755673 was recently identified as the first potent and selective PKD inhibitor. The study of structure-activity relationships (SAR) of this lead structure led to further improvements in PKD1 potency. We describe herein the synthesis and biological evaluation of novel benzothienothiazepinone analogs. We achieved a ten-fold increase in the in vitro PKD1 inhibitory potency for the second generation lead kb-NB142-70 and accomplished a transition to an almost equally potent novel pyrimidine scaffold, while maintaining excellent target selectivity. These promising results will guide the design of pharmacological tools to dissect PKD function and pave the way for the development of potential anti-cancer agents.
PMCID: PMC3100199  PMID: 21617763
Protein kinase D; small molecule inhibitor; benzothienothiazepinone; pyrimidines; CID755673
13.  Synthesis and Structure−Activity Relationships of Benzothienothiazepinone Inhibitors of Protein Kinase D 
ACS Medicinal Chemistry Letters  2010;2(2):154-159.
Protein kinase D (PKD) is a member of a novel family of serine/threonine kinases that regulate fundamental cellular processes. PKD is implicated in the pathogenesis of several diseases, including cancer. Progress in understanding the biological functions and therapeutic potential of PKD has been hampered by the lack of specific inhibitors. The benzoxoloazepinolone CID755673 was recently identified as the first potent and selective PKD inhibitor. The study of structure−activity relationships (SAR) of this lead compound led to further improvements in PKD1 potency. We describe herein the synthesis and biological evaluation of novel benzothienothiazepinone analogues. We achieved a 10-fold increase in the in vitro PKD1 inhibitory potency for the second generation lead kb-NB142-70 and accomplished a transition to an almost equally potent novel pyrimidine scaffold, while maintaining excellent target selectivity. These promising results will guide the design of pharmacological tools to dissect PKD function and pave the way for the development of potential anticancer agents.
PMCID: PMC3100199  PMID: 21617763
Protein kinase D; small molecule inhibitor; benzothienothiazepinone; pyrimidines; CID755673
14.  Design, Synthesis, and Biological Evaluation of PKD Inhibitors 
Pharmaceutics  2011;3(2):186-228.
Protein kinase D (PKD) belongs to a family of serine/threonine kinases that play an important role in basic cellular processes and are implicated in the pathogenesis of several diseases. Progress in our understanding of the biological functions of PKD has been limited due to the lack of a PKD-specific inhibitor. The benzoxoloazepinolone CID755673 was recently reported as the first potent and kinase-selective inhibitor for this enzyme. For structure-activity analysis purposes, a series of analogs was prepared and their in vitro inhibitory potency evaluated.
PMCID: PMC3261798  PMID: 22267986
protein kinase D; small molecule inhibitor; benzothienothiazepinone; pyrimidines; CID755673; thiazepinothiophenopyrimidinone
15.  Protein kinase D as a potential new target for cancer therapy 
Biochimica et biophysica acta  2010;1806(2):183-192.
Protein kinase D is a novel family of serine/threonine kinases and diacylglycerol receptors that belongs to the calcium/calmodulin-dependent kinase superfamily. Evidence has established that specific PKD isoforms are dysregulated in several cancer types, and PKD involvement has been documented in a variety of cellular processes important to cancer development, including cell growth, apoptosis, motility, and angiogenesis. In light of this, there has been a recent surge in the development of novel chemical inhibitors of PKD. This review focuses on the potential of PKD as a chemotherapeutic target in cancer treatment and highlights important recent advances in the development of PKD inhibitors.
PMCID: PMC2947595  PMID: 20580776
Protein kinase D; cancer; small molecule inhibitors
16.  Optimization of Ultrasound Contrast Agents with Computational Models to Improve Selection of Ligands and Binding Strength 
Biotechnology and bioengineering  2010;107(5):854-864.
Diagnosis of cardiovascular disease is currently limited by the testing modality. Serum tests for biomarkers can provide quantification of severity but lack the ability to localize the source of the cardiovascular disease, while imaging technology such as angiography and ultrasound can only determine areas of reduced flow but not the severity of tissue ischemia. Targeted imaging with ultrasound contrast agents offers the ability to locally image as well as determine the degree of ischemia by utilizing agents that will cause the contrast agent to home to the affected tissue. Ultrasound molecular imaging via targeted microbubbles (MB) is currently limited by its sensitivity to molecular markers of disease relative to other techniques (e.g. radiolabeling). We hypothesize that computational modeling may provide a useful first approach to maximize microbubble binding by defining key parameters governing adhesion. Adhesive Dynamics was used to simulate the fluid dynamic and stochastic molecular binding of microbubbles to inflamed endothelial cells. Sialyl LewisX (sLex), P-selectin aptamer (PSA), and ICAM-1 antibody (abICAM) were modeled as the targeting receptors on the microbubble surface in both single-and dual-targeted arrangements. Microbubble properties (radius [Rc], kinetics [kf, kr] and densities of targeting receptors) and the physical environment (shear rate and target ligand densities) were modeled. The kinetics for sLex and PSA were measured with surface plasmon resonance. Rc, shear rate, and densities of sLex, PSA or abICAM were varied independently to assess model sensitivity. Firm adhesion was defined as MB velocity < 2% of the free stream velocity. Adhesive Dynamics simulations revealed an optimal microbubble radius of 1–2 μm and thresholds for kinf (>102 sec−1) and kor (<10−3 sec−1) for firm adhesion in a multi-targeted system. State diagrams for multi-targeted microbubbles suggest sLex and abICAM microbubbles may require 10-fold more ligand to achieve firm adhesion at higher shear rates than sLex and PSA microbubbles. The Adhesive Dynamics model gives useful insight into the key parameters for stable microbubble binding, and may allow flexible, prospective design and optimization of microbubbles to enhance clinical translation of ultrasound molecular imaging.
PMCID: PMC3225194  PMID: 20665479
Targeted Ultrasound Contrast; Computational Modeling; Adhesive Dynamics; Surface Plasmon Resonance; Stochastic Model; Microbubbles
17.  Discovery of Diverse Small Molecule Chemotypes with Cell-Based PKD1 Inhibitory Activity 
PLoS ONE  2011;6(10):e25134.
Protein kinase D (PKD) is a novel family of serine/threonine kinases regulated by diacylglycerol, which is involved in multiple cellular processes and various pathological conditions. The limited number of cell-active, selective inhibitors has historically restricted biochemical and pharmacological studies of PKD. We now markedly expand the PKD1 inhibitory chemotype inventory with eleven additional novel small molecule PKD1 inhibitors derived from our high throughput screening campaigns. The in vitro IC50s for these eleven compounds ranged in potency from 0.4 to 6.1 µM with all of the evaluated compounds being competitive with ATP. Three of the inhibitors (CID 1893668, (1Z)-1-(3-ethyl-5-methoxy-1,3-benzothiazol-2-ylidene)propan-2-one; CID 2011756, 5-(3-chlorophenyl)-N-[4-(morpholin-4-ylmethyl)phenyl]furan-2-carboxamide; CID 5389142, (6Z)-6-[4-(3-aminopropylamino)-6-methyl-1H-pyrimidin-2-ylidene]cyclohexa-2,4-dien-1-one) inhibited phorbol ester-induced endogenous PKD1 activation in LNCaP prostate cancer cells in a concentration-dependent manner. The specificity of these compounds for PKD1 inhibitory activity was supported by kinase assay counter screens as well as by bioinformatics searches. Moreover, computational analyses of these novel cell-active PKD1 inhibitors indicated that they were structurally distinct from the previously described cell-active PKD1 inhibitors while computational docking of the new cell-active compounds in a highly conserved ATP-binding cleft suggests opportunities for structural modification. In summary, we have discovered novel PKD1 inhibitors with in vitro and cell-based inhibitory activity, thus successfully expanding the structural diversity of small molecule inhibitors available for this important pharmacological target.
PMCID: PMC3187749  PMID: 21998636
18.  Characterization and Optimization of a Novel Protein–Protein Interaction Biosensor High-Content Screening Assay to Identify Disruptors of the Interactions Between p53 and hDM2 
We present here the characterization and optimization of a novel imaging-based positional biosensor high-content screening (HCS) assay to identify disruptors of p53-hDM2 protein–protein interactions (PPIs). The chimeric proteins of the biosensor incorporated the N-terminal PPI domains of p53 and hDM2, protein targeting sequences (nuclear localization and nuclear export sequence), and fluorescent reporters, which when expressed in cells could be used to monitor p53-hDM2 PPIs through changes in the subcellular localization of the hDM2 component of the biosensor. Coinfection with the recombinant adenovirus biosensors was used to express the NH-terminal domains of p53 and hDM2, fused to green fluorescent protein and red fluorescent protein, respectively, in U-2 OS cells. We validated the p53-hDM2 PPI biosensor (PPIB) HCS assay with Nutlin-3, a compound that occupies the hydrophobic pocket on the surface of the N-terminus of hDM2 and blocks the binding interactions with the N-terminus of p53. Nutlin-3 disrupted the p53-hDM2 PPIB in a concentration-dependent manner and provided a robust, reproducible, and stable assay signal window that was compatible with HCS. The p53-hDM2 PPIB assay was readily implemented in HCS and we identified four (4) compounds in the 1,280-compound Library of Pharmacologically Active Compounds that activated the p53 signaling pathway and elicited biosensor signals that were clearly distinct from the responses of inactive compounds. Anthracycline (topoisomerase II inhibitors such as mitoxantrone and ellipticine) and camptothecin (topoisomerase I inhibitor) derivatives including topotecan induce DNA double strand breaks, which activate the p53 pathway through the ataxia telangiectasia mutated-checkpoint kinase 2 (ATM-CHK2) DNA damage response pathway. Although mitoxantrone, ellipticine, camptothecin, and topotecan all exhibited concentration-dependent disruption of the p53-hDM2 PPIB, they were much less potent than Nutlin-3. Further, their corresponding cellular images and quantitative HCS data did not completely match the Nutlin-3 phenotypic profile.
PMCID: PMC2929144  PMID: 20662736
19.  Design, Synthesis, and Biological Evaluation of PKD Inhibitors 
Pharmaceutics  2011;3(2):186-228.
Protein kinase D (PKD) belongs to a family of serine/threonine kinases that play an important role in basic cellular processes and are implicated in the pathogenesis of several diseases. Progress in our understanding of the biological functions of PKD has been limited due to the lack of a PKD-specific inhibitor. The benzoxoloazepinolone CID755673 was recently reported as the first potent and kinase-selective inhibitor for this enzyme. For structure-activity analysis purposes, a series of analogs was prepared and their in vitro inhibitory potency evaluated.
PMCID: PMC3261798  PMID: 22267986
protein kinase D; small molecule inhibitor; benzothienothiazepinone; pyrimidines; CID755673; thiazepinothiophenopyrimidinone
20.  Profiling the NIH Small Molecule Repository for Compounds That Generate H2O2 by Redox Cycling in Reducing Environments 
We have screened the Library of Pharmacologically Active Compounds (LOPAC) and the National Institutes of Health (NIH) Small Molecule Repository (SMR) libraries in a horseradish peroxidase–phenol red (HRP-PR) H2O2 detection assay to identify redox cycling compounds (RCCs) capable of generating H2O2 in buffers containing dithiothreitol (DTT). Two RCCs were identified in the LOPAC set, the ortho-naphthoquinone β-lapachone and the para-naphthoquinone NSC 95397. Thirty-seven (0.02%) concentration-dependent RCCs were identified from 195,826 compounds in the NIH SMR library; 3 singleton structures, 9 ortho-quinones, 2 para-quinones, 4 pyrimidotriazinediones, 15 arylsulfonamides, 2 nitrothiophene-2-carboxylates, and 2 tolyl hydrazides. Sixty percent of the ortho-quinones and 80% of the pyrimidotriazinediones in the library were confirmed as RCCs. In contrast, only 3.9% of the para-quinones were confirmed as RCCs. Fifteen of the 251 arylsulfonamides in the library were confirmed as RCCs, and since we screened 17,868 compounds with a sulfonamide functional group we conclude that the redox cycling activity of the arylsulfonamide RCCs is due to peripheral reactive enone, aromatic, or heterocyclic functions. Cross-target queries of the University of Pittsburgh Drug Discovery Institute (UPDDI) and PubChem databases revealed that the RCCs exhibited promiscuous bioactivity profiles and have populated both screening databases with significantly higher numbers of active flags than non-RCCs. RCCs were promiscuously active against protein targets known to be susceptible to oxidation, but were also active in cell growth inhibition assays, and against other targets thought to be insensitive to oxidation. Profiling compound libraries or the hits from screening campaigns in the HRP-PR H2O2 detection assay significantly reduce the timelines and resources required to identify and eliminate promiscuous nuisance RCCs from the candidates for lead optimization.
PMCID: PMC3098569  PMID: 20070233
21.  Strategies for Discovery of Small Molecule Radiation Protectors and Radiation Mitigators 
Mitochondrial targeted radiation damage protectors (delivered prior to irradiation) and mitigators (delivered after irradiation, but before the appearance of symptoms associated with radiation syndrome) have been a recent focus in drug discovery for (1) normal tissue radiation protection during fractionated radiotherapy, and (2) radiation terrorism counter measures. Several categories of such molecules have been discovered: nitroxide-linked hybrid molecules, including GS-nitroxide, GS-nitric oxide synthase inhibitors, p53/mdm2/mdm4 inhibitors, and pharmaceutical agents including inhibitors of the phosphoinositide-3-kinase pathway and the anti-seizure medicine, carbamazepine. Evaluation of potential new radiation dose modifying molecules to protect normal tissue includes: clonogenic radiation survival curves, assays for apoptosis and DNA repair, and irradiation-induced depletion of antioxidant stores. Studies of organ specific radioprotection and in total body irradiation-induced hematopoietic syndrome in the mouse model for protection/mitigation facilitate rational means by which to move candidate small molecule drugs along the drug discovery pipeline into clinical development.
PMCID: PMC3356036  PMID: 22655254
radiation mitigators; radioprotectors; small molecules; mitochondria
22.  Chemical genetics of Plasmodium falciparum 
Nature  2010;465(7296):311-315.
Malaria caused by Plasmodium falciparum is a catastrophic disease worldwide (880,000 deaths yearly). Vaccine development has proved difficult and resistance has emerged for most antimalarials. In order to discover new antimalarial chemotypes, we have employed a phenotypic forward chemical genetic approach to assay 309,474 chemicals. Here we disclose structures and biological activity of the entire library, many of which exhibited potent in vitro activity against drug resistant strains, and detailed profiling of 172 representative candidates. A reverse chemical genetic study identified 19 new inhibitors of 4 validated drug targets and 15 novel binders among 61 malarial proteins. Phylochemogenetic profiling in multiple organisms revealed similarities between Toxoplasma gondii and mammalian cell lines and dissimilarities between P. falciparum and related protozoans. One exemplar compound displayed efficacy in a murine model. Overall, our findings provide the scientific community with new starting points for malaria drug discovery.
PMCID: PMC2874979  PMID: 20485428
23.  Automated High-Content Live Animal Drug Screening Using C. elegans Expressing the Aggregation Prone Serpin α1-antitrypsin Z 
PLoS ONE  2010;5(11):e15460.
The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms.
PMCID: PMC2980495  PMID: 21103396
24.  MicroRNA-21 negatively regulates Cdc25A and cell cycle progression in colon cancer cells 
Cancer research  2009;69(20):8157-8165.
MicroRNAs are small non-coding RNAs that participate in diverse biological processes by suppressing target gene expression. Altered expression of miR-21 has been reported in cancer. To gain insights in its potential role in tumorigenesis, we generated miR-21 knockout colon cancer cells through gene targeting. Unbiased microarray analysis combined with bioinformatics identified cell cycle regulator Cdc25A as a miR-21 target. miR-21 suppressed Cdc25A expression through a defined sequence in its 3′UTR. We found that miR-21 is induced by serum starvation and DNA damage, negatively regulates G1-S transition, and participates in DNA damage-induced G2-M checkpoint through downregulation of Cdc25A. In contrast, miR-21 deficiency did not affect apoptosis induced by a variety of commonly used anticancer agents or cell proliferation under normal cell culture conditions. Furthermore, miR-21 was found to be underexpressed in a subset of Cdc25A overexpressing colon cancers. Our data demonstrated a role of miR-21 in modulating cell cycle progression following stress, providing a novel mechanism of Cdc25A regulation and a potential explanation of miR-21 in tumorigenesis.
PMCID: PMC2763324  PMID: 19826040
MicroRNA-21; Cdc25A; cell cycle; DNA damage; colon cancer
25.  Synthetic Protection Short Interfering RNA Screen Reveals Glyburide as a Novel Radioprotector 
Radiation research  2009;172(4):414-422.
To assist in screening existing drugs for use as potential radioprotectors, we used a human unbiased 16,560 short interfering RNA (siRNA) library targeting the druggable genome. We performed a synthetic protection screen that was designed to identify genes that, when silenced, protected human glioblastoma T98G cells from γ-radiation-induced cell death. We identified 116 candidate protective genes, then identified 10 small molecule inhibitors of 13 of these candidate gene products and tested their radioprotective effects. Glyburide, a clinically used second-generation hypoglycemic drug, effectively decreased radiation-induced cell death in several cell lines including T98G, glioblastoma U-87 MG, and normal lung epithelial BEAS-2B and in primary cultures of astrocytes. Glyburide significantly increased the survival of 32D cl3 murine hematopoietic progenitor cells when administrated before irradiation. Glyburide was radioprotective in vivo (90% of C57BL/6NHsd female mice pretreated with 10 mg/kg glyburide survived 9.5 Gy total-body irradiation compared to 42% of irradiated controls, P = 0.0249). These results demonstrate the power of unbiased siRNA synthetic protection screening with a druggable genome library to identify new radioprotectors.
PMCID: PMC2782411  PMID: 19772462

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