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1.  Imidazole - Derived Agonists for the Neurotensin 1 Receptor 
A scaffold-hop program seeking full agonists of the neurotensin-1 (NTR1) receptor identified the probe molecule ML301 (1) and associated analogs, including its naphthyl analog (14) which exhibited similar properties. Compound 1 showed full agonist behavior (79 – 93%) with an EC50 of 2.0 – 4.1 μM against NTR1. Compound 1 also showed good activity in a Ca mobilization FLIPR assay (93% efficacy at 298 nM), consistent with it functioning via the Gq coupled pathway, and good selectivity relative to NTR2 and GPR35. In further profiling, 1 showed low potential for promiscuity and good overall pharmacological data. This report describes the discovery, synthesis, and SAR of 1 and associated analogs. Initial in vitro pharmacologic characterization is also presented.
doi:10.1016/j.bmcl.2013.11.026
PMCID: PMC3898338  PMID: 24332089
2.  Preparative Microfluidic Electrosynthesis of Drug Metabolites 
ACS Medicinal Chemistry Letters  2013;4(11):1119-1123.
In vivo, a drug molecule undergoes its first chemical transformation within the liver via CYP450-catalyzed oxidation. The chemical outcome of the first pass hepatic oxidation is key information to any drug development process. Electrochemistry can be used to simulate CYP450 oxidation, yet it is often confined to the analytical scale, hampering product isolation and full characterization. In an effort to replicate hepatic oxidations, while retaining high throughput at the preparative scale, microfluidic technology and electrochemistry are combined in this study by using a microfluidic electrochemical cell. Several commercial drugs were subjected to continuous-flow electrolysis. They were chosen for their various chemical reactivity: their metabolites in vivo are generated via aromatic hydroxylation, alkyl oxidation, glutathione conjugation, or sulfoxidation. It is demonstrated that such metabolites can be synthesized by flow electrolysis at the 10 to 100 mg scale, and the purified products are fully characterized.
doi:10.1021/ml400316p
PMCID: PMC4027460  PMID: 24900614
Drug metabolites; electrochemistry; microfluidic synthesis; continuous-flow oxidation
3.  Discovery of ML314, a Brain Penetrant Non-Peptidic β-Arrestin Biased Agonist of the Neurotensin NTR1 Receptor 
ACS medicinal chemistry letters  2013;4(9):846-851.
The neurotensin 1 receptor (NTR1) is an important therapeutic target for a range of disease states including addiction. A high throughput screening campaign, followed by medicinal chemistry optimization, led to the discovery of a non-peptidic β-arrestin biased agonist for NTR1. The lead compound, 2-cyclopropyl-6,7-dimethoxy-4-(4-(2-methoxyphenyl)- piperazin-1-yl)quinazoline, 32 (ML314), exhibits full agonist behavior against NTR1 (EC50 = 2.0 μM) in the primary assay and selectivity against NTR2. The effect of 32 is blocked by the NTR1 antagonist SR142948A in a dose dependent manner. Unlike peptide based NTR1 agonists, compound 32 has no significant response in a Ca2+ mobilization assay and is thus a biased agonist that activates the β-arrestin pathway rather than the traditional Gq coupled pathway. This bias has distinct biochemical and functional consequences that may lead to physiological advantages. Compound 32 displays good brain penetration in rodents, and studies examining its in vivo properties are underway.
doi:10.1021/ml400176n
PMCID: PMC3940307  PMID: 24611085
NTR1; neurotensin; GPCR; SR142948A; quinazoline; agonist; β-arrestin bias
4.  Exploratory Analysis of Kinetic Solubility Measurements of a Small Molecule Library 
Bioorganic & medicinal chemistry  2011;19(13):4127-4134.
Kinetic solubility measurements using prototypical assay buffer conditions are presented for a ~58,000 member library of small molecules. Analyses of the data based upon physical and calculated properties of each individual molecule were performed and resulting trends were considered in the context of commonly held opinions of how physicochemical properties influence aqueous solubility. We further analyze the data using a decision tree model for solubility prediction and via a multi-dimensional assessment of physicochemical relationships to solubility in the context of specific ‘rule-breakers’ relative to common dogma. The role of solubility as a determinant of assay outcome is also considered based upon each compound’s cross-assay activity score for a collection of publicly available screening results. Further, the role of solubility as a governing factor for colloidal aggregation formation within a specified assay setting is examined and considered as a possible cause of a high cross-assay activity score. The results of this solubility profile should aid chemists during library design and optimization efforts and represents a useful training set for computational solubility prediction.
doi:10.1016/j.bmc.2011.05.005
PMCID: PMC3236531  PMID: 21640593
5.  Discovery of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221) as a functional antagonist of the apelin (APJ) receptor 
The recently discovered apelin/APJ system has emerged as a critical mediator of cardiovascular homeostasis and is associated with the pathogenesis of cardiovascular disease. A role for apelin/APJ in energy metabolism and gastrointestinal function has also recently emerged. We disclose the discovery and characterization of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221), a potent APJ functional antagonist in cell-based assays that is >37-fold selective over the closely related angiotensin II type 1 (AT1) receptor. ML221 was derived from an HTS of the ∼330,600 compound MLSMR collection. This antagonist showed no significant binding activity against 29 other GPCRs, except to the κ-opioid and benzodiazepinone receptors (<50/<70%I at 10 μM). The synthetic methodology, development of structure-activity relationship (SAR), and initial in vitro pharmacologic characterization are also presented.
doi:10.1016/j.bmcl.2012.08.105
PMCID: PMC3729231  PMID: 23010269
GPCRs; HTS; APJ; Apelin; Cardiovascular disease; Antagonists; SAR
6.  Discovery of a Plasmodium falciparum glucose-6-phosphate dehydrogenase 6- phosphogluconolactonase inhibitor (R,Z)-N-((1-ethylpyrrolidin-2-yl)methyl)-2-(2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide (ML276) that reduces parasite growth in vitro 
Journal of medicinal chemistry  2012;55(16):7262-7272.
A high throughput screen of the NIH’s MLSMR collection of ~340,000 compounds was undertaken to identify compounds that inhibit Plasmodium falciparum glucose-6-phosphate dehydrogenase (PfG6PD). PfG6PD is essential for proliferating and propagating P. falciparum and differs structurally and mechanistically from the human ortholog. The reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PD) is the first, rate-limiting step in the pentose phosphate pathway (PPP), a key metabolic pathway sustaining anabolic needs in reductive equivalents and synthetic materials in fastgrowing cells. In P. falciparum the bifunctional enzyme glucose-6-phosphate dehydrogenase-6- phosphogluconolactonase (PfGluPho) catalyzes the first two steps of the PPP. Because P. falciparum and infected host red blood cells rely on accelerated glucose flux, they depend on the G6PD activity of PfGluPho. The lead compound identified from this effort, (R,Z)-N-((1-ethylpyrrolidin-2-yl)methyl)-2- (2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide, 11, (ML276), is a submicromolar inhibitor of PfG6PD (IC50 = 889 nM). It is completely selective for the enzyme’s human isoform, displays micromolar potency (IC50 = 2.6 μM) against P. falciparum in culture, and has good drug-like properties, including high solubility and moderate microsomal stability. Studies testing the potential advantage of inhibiting PfG6PD in vivo are in progress.
doi:10.1021/jm300833h
PMCID: PMC3530835  PMID: 22813531
6-phosphogluconolactonase; glucose-6-phosphate; glucose-6-phosphate dehydrogenase; highthroughput screening; malaria; pentose phosphate pathway; Plasmodium; redox metabolism; benzothiazinone
7.  Identification of Inhibitors of NOD1-Induced Nuclear Factor-κB Activation 
ACS Medicinal Chemistry Letters  2011;2(10):780-785.
NOD1 (nucleotide-binding oligomerization domain 1) protein is a member of the NLR (NACHT and leucine rich repeat domain containing proteins) protein family, which plays a key role in innate immunity as a sensor of specific microbial components derived from bacterial peptidoglycans and induction of inflammatory responses. Mutations in NOD proteins have been associated with various inflammatory diseases that affect NF-κB (nuclear factor κB) activity, a major signaling pathway involved in apoptosis, inflammation, and immune response. A luciferase-based reporter gene assay was utilized in a high-throughput screening program conducted under the NIH-sponsored Molecular Libraries Probe Production Center Network program to identify the active scaffolds. Herein, we report the chemical synthesis, structure–activity relationship studies, downstream counterscreens, secondary assay data, and pharmacological profiling of the 2-aminobenzimidazole lead (compound 1c, ML130) as a potent and selective inhibitor of NOD1-induced NF-κB activation.
doi:10.1021/ml200158b
PMCID: PMC3193285  PMID: 22003428
NOD1; NF-κB activation; 2-aminobenzimidazole; hit-to-probe; ML130; MLPCN
8.  Discovery and characterization of 2-aminobenzimidazole derivatives as selective NOD1 inhibitors 
Chemistry & biology  2011;18(7):825-832.
NLR family proteins play important roles in innate immune response. NOD1 (NLRC1) activates various signaling pathways including NF-κB in response to bacterial ligands. Hereditary polymorphisms in the NOD1 gene are associated with asthma, inflammatory bowel disease, and other disorders. Using a high throughput screening (HTS) assay measuring NOD1-induced NF-κB reporter gene activity, followed by multiple downstream counter-screens that eliminated compounds impacting other NF-κB pathways, 2-aminobenzimidazole compounds were identified that selectively inhibit NOD1. Mechanistic studies of a prototypical compound, Nodinitib-1 (ML130; CID-1088438), suggest these small molecules cause conformational changes of NOD1 in vitro and alter NOD1 subcellular targeting in cells. Altogether, this inaugural class of inhibitors provides chemical probes for interrogating mechanisms regulating NOD1 activity and tools for exploring the roles of NOD1 in various infectious and inflammatory diseases.
doi:10.1016/j.chembiol.2011.06.009
PMCID: PMC3152441  PMID: 21802003
NOD1; NOD2; NLR; NF-κB; HTS
9.  Chemical Biology Strategy Reveals Pathway-Selective Inhibitor of NF-κB Activation Induced by Protein Kinase C 
ACS chemical biology  2010;5(3):287-299.
Dysregulation of NF-κB activity contributes to many autoimmune and inflammatory diseases. At least nine pathways for NF-κB activation have been identified, most of which converge on the IκB kinases (IKKs). Although IKKs represent logical targets for potential drug discovery, chemical inhibitors of IKKs suppress all known NF-κB activation pathways, and thus lack the selectivity required for safe use. A unique NF-κB activation pathway is initiated by protein kinase C (PKC) that is stimulated by antigen receptors and many growth factor receptors. Using a cell-based high throughput screening (HTS) assay and chemical biology strategy, we identified a 2-aminobenzimidazole compound, CID-2858522, which selectively inhibits the NF-κB pathway induced by PKC, operating downstream of PKC but upstream of IKKβ, without inhibiting other NF-κB activation pathways. In human B cells stimulated through surface immunoglobulin, CID-2858522 inhibited NF-κB DNA-binding activity and expression of endogenous NF-κB-dependent target gene, TRAF1. Altogether, as a selective chemical inhibitor of the NF-κB pathway induced by PKC, CID-2858522 serves as a powerful research tool, and may reveal new paths towards therapeutically useful NF-κB inhibitors.
doi:10.1021/cb9003089
PMCID: PMC2842467  PMID: 20141195
10.  ISOLATION, SYNTHESIS AND BIOLOGICAL ACTIVITY OF APHROCALLISTIN, AN ADENINE SUBSTITUTED BROMOTYRAMINE METABOLITE FROM THE HEXACTINELLIDA SPONGE APHROCALLISTES BEATRIX 
Journal of natural products  2009;72(6):1178-1183.
A new adenine substituted bromotyrosine derived metabolite designated as aphrocallistin (1) has been isolated from the deep-water Hexactinellida sponge Aphrocallistes beatrix beatrix Gray, 1858 (Order Hexactinosida, Family Aphrocallistidae). Its structure was elucidated on the basis of spectral data and confirmed through a convergent, modular total synthetic route that is amenable towards future analog preparation. Aphrocallistin inhibits the growth of a panel of human tumor cell lines with IC50 values ranging from 7.5 to >100 μM and has been shown to induce G1 cell cycle arrest in the PANC-1 pancreatic carcinoma cell line. Aphrocallistin has been fully characterized in the NCI cancer cell line panel and has undergone in vitro ADME pharmacological profiling.
doi:10.1021/np900183v
PMCID: PMC3031448  PMID: 19459694
11.  Chemical genetics approach to restoring p27Kip1 reveals novel compounds with antiproliferative activity in prostate cancer cells 
BMC Biology  2010;8:153.
Background
The cyclin-dependent kinase (CDK) inhibitor p27Kip1 is downregulated in a majority of human cancers due to ectopic proteolysis by the ubiquitin-proteasome pathway. The expression of p27 is subject to multiple mechanisms of control involving several transcription factors, kinase pathways and at least three different ubiquitin ligases (SCFSKP2, KPC, Pirh2), which regulate p27 transcription, translation, protein stability and subcellular localization. Using a chemical genetics approach, we have asked whether this control network can be modulated by small molecules such that p27 protein expression is restored in cancer cells.
Results
We developed a cell-based assay for measuring the levels of endogenous nuclear p27 in a high throughput screening format employing LNCaP prostate cancer cells engineered to overexpress SKP2. The assay platform was optimized to Z' factors of 0.48 - 0.6 and piloted by screening a total of 7368 chemical compounds. During the course of this work, we discovered two small molecules of previously unknown biological activity, SMIP001 and SMIP004, which increase the nuclear level of p27 at low micromolar concentrations. SMIPs (small molecule inhibitors of p27 depletion) also upregulate p21Cip1, inhibit cellular CDK2 activity, induce G1 delay, inhibit colony formation in soft agar and exhibit preferential cytotoxicity in LNCaP cells relative to normal human fibroblasts. Unlike SMIP001, SMIP004 was found to downregulate SKP2 and to stabilize p27, although neither SMIP is a proteasome inhibitor. Whereas the screening endpoint - nuclear p27 - was robustly modulated by the compounds, SMIP-mediated cell cycle arrest and apoptosis were not strictly dependent on p27 and p21 - a finding that is explained by parallel inhibitory effects of SMIPs on positive cell cycle regulators, including cyclins E and A, and CDK4.
Conclusions
Our data provide proof-of-principle that the screening platform we developed, using endogenous nuclear p27 as an endpoint, presents an effective means of identifying bioactive molecules with cancer selective antiproliferative activity. This approach, when applied to larger and more diverse sets of compounds with refined drug-like properties, bears the potential of revealing both unknown cellular pathways globally impinging on p27 and novel leads for chemotherapeutics targeting a prominent molecular defect of human cancers.
doi:10.1186/1741-7007-8-153
PMCID: PMC3025922  PMID: 21182779
12.  Inhibition of Protein Kinase C-Driven Nuclear Factor-κB Activation: Synthesis, Structure−Activity Relationship, and Pharmacological Profiling of Pathway Specific Benzimidazole Probe Molecules 
Journal of Medicinal Chemistry  2010;53(12):4793-4797.
A unique series of biologically active chemical probes that selectively inhibit NF-κB activation induced by protein kinase C (PKC) pathway activators have been identified through a cell-based phenotypic reporter gene assay. These 2-aminobenzimidazoles represent initial chemical tools to be used in gaining further understanding on the cellular mechanisms driven by B and T cell antigen receptors. Starting from the founding member of this chemical series 1a (notated in PubChem as CID-2858522), we report the chemical synthesis, SAR studies, and pharmacological profiling of this pathway-selective inhibitor of NF-κB activation.
doi:10.1021/jm1000248
PMCID: PMC2887059  PMID: 20481485
13.  Identification of Inhibitors of NOD1-Induced Nuclear Factor-κB Activation 
ACS Medicinal Chemistry Letters  2011;2(10):780-785.
NOD1 (nucleotide-binding oligomerization domain 1) protein is a member of the NLR (NACHT and leucine rich repeat domain containing proteins) protein family, which plays a key role in innate immunity as a sensor of specific microbial components derived from bacterial peptidoglycans and induction of inflammatory responses. Mutations in NOD proteins have been associated with various inflammatory diseases that affect NF-κB (nuclear factor κB) activity, a major signaling pathway involved in apoptosis, inflammation, and immune response. A luciferase-based reporter gene assay was utilized in a high-throughput screening program conducted under the NIH-sponsored Molecular Libraries Probe Production Center Network program to identify the active scaffolds. Herein, we report the chemical synthesis, structure–activity relationship studies, downstream counterscreens, secondary assay data, and pharmacological profiling of the 2-aminobenzimidazole lead (compound 1c, ML130) as a potent and selective inhibitor of NOD1-induced NF-κB activation.
doi:10.1021/ml200158b
PMCID: PMC3193285  PMID: 22003428
NOD1; NF-κB activation; 2-aminobenzimidazole; hit-to-probe; ML130; MLPCN

Results 1-13 (13)