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1.  4-(3-Chloro-5-(trifluoromethyl)pyridin-2-yl)-N-(4-methoxypyridin-2-yl)piperazine-1-carbothioamide (ML267), a Potent Inhibitor of Bacterial Phosphopantetheinyl Transferase That Attenuates Secondary Metabolism and Thwarts Bacterial Growth 
Journal of Medicinal Chemistry  2014;57(3):1063-1078.
4′-Phosphopantetheinyl transferases (PPTases) catalyze a post-translational modification essential to bacterial cell viability and virulence. We present the discovery and medicinal chemistry optimization of 2-pyridinyl-N-(4-aryl)piperazine-1-carbothioamides, which exhibit submicromolar inhibition of bacterial Sfp-PPTase with no activity toward the human orthologue. Moreover, compounds within this class possess antibacterial activity in the absence of a rapid cytotoxic response in human cells. An advanced analogue of this series, ML267 (55), was found to attenuate production of an Sfp-PPTase-dependent metabolite when applied to Bacillus subtilis at sublethal doses. Additional testing revealed antibacterial activity against methicillin-resistant Staphylococcus aureus, and chemical genetic studies implicated efflux as a mechanism for resistance in Escherichia coli. Additionally, we highlight the in vitro absorption, distribution, metabolism, and excretion and in vivo pharmacokinetic profiles of compound 55 to further demonstrate the potential utility of this small-molecule inhibitor.
PMCID: PMC3983359  PMID: 24450337
2.  Potent and Selective Inhibitors of Human Reticulocyte 12/15-Lipoxygenase as Anti-Stroke Therapies 
Journal of medicinal chemistry  2014;57(10):4035-4048.
A key challenge facing drug discovery today is variability of the drug target between species, such as with 12/15-lipoxygenase (12/15-LOX), which contributes to ischemic brain injury, but its human and rodent isozymes have different inhibitor specificities. In the current work, we have utilized a quantitative high-throughput (qHTS) screen to identify compound 1 (ML351), a novel chemotype for 12/15-LOX inhibition, which has nanomolar potency (IC50 = 200 nM) against human 12/15-LOX and is protective against oxidative glutamate toxicity in mouse neuronal HT-22 cells. In addition, it exhibited greater than 250-fold selectivity versus related LOX isozymes, was a mixed inhibitor, and did not reduce the active-site ferric ion. Finally, 1 significantly reduced infarct size following permanent focal ischemia in a mouse model of ischemic stroke. As such, this represents the first report of a selective inhibitor of human 12/15-LOX with demonstrated in vivo activity in proof-of-concept mouse models of stroke.
PMCID: PMC4033661  PMID: 24684213
Human 15-lipoxygenase-1; high-throughput; inhibitor
3.  Synthesis and Structure–Activity Relationship Studies of 4-((2-Hydroxy-3-methoxybenzyl)amino)benzenesulfonamide Derivatives as Potent and Selective Inhibitors of 12-Lipoxygenase 
Journal of medicinal chemistry  2014;57(2):495-506.
Human lipoxygenases (LOXs) are a family of iron-containing enzymes which catalyze the oxidation of polyunsaturated fatty acids to provide the corresponding bioactive hydroxyeicosatetraenoic acid (HETE) metabolites. These eicosanoid signaling molecules are involved in a number of physiologic responses such as platelet aggregation, inflammation, and cell proliferation. Our group has taken a particular interest in platelet-type 12-(S)-LOX (12-LOX) because of its demonstrated role in skin diseases, diabetes, platelet hemostasis, thrombosis, and cancer. Herein, we report the identification and medicinal chemistry optimization of a 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide-based scaffold. Top compounds, exemplified by 35 and 36, display nM potency against 12-LOX, excellent selectivity over related lipoxygenases and cyclooxygenases, and possess favorable ADME properties. In addition, both compounds inhibit PAR-4 induced aggregation and calcium mobilization in human platelets and reduce 12-HETE in β-cells.
PMCID: PMC3967794  PMID: 24393039
4.  Discovery of a novel non-iminosugar acid alpha glucosidase chaperone series 
Journal of medicinal chemistry  2012;55(17):7546-7559.
Pompe disease is an autosomal recessive lysosomal storage disorder (LSD) caused by deficiency of the lysosomal enzyme acid alpha glucosidase (GAA). Many disease-causing mutated GAA retain enzymatic activity, but are not translocated from endoplasmic reticulum (ER) to lysosomes. Enzyme replacement therapy (ERT) is the only treatment for Pompe disease, but remains expensive, inconvenient and does not reverse all disease manifestations. It was postulated that small molecules which aid in protein folding and translocation to lysosomes could provide an alternate to ERT. Previously, several iminosugars have been proposed as small-molecule chaperones for specific LSDs. Here we identified a novel series of non-iminosugar chaperones for GAA. These moderate GAA inhibitors are shown to bind and thermo-stabilize GAA, and increase GAA translocation to lysosomes in both wild-type and Pompe fibroblasts. AMDE and physical properties studies indicate that this series is a promising lead for further pharmacokinetic evaluation and testing in Pompe disease models.
PMCID: PMC3448374  PMID: 22834902
5.  Synthesis, Biological Evaluation and Structure-Activity Relationships of a Novel Class of Apurinic/Apyrimidinic Endonuclease 1 Inhibitors 
Journal of medicinal chemistry  2012;55(7):3101-3112.
APE1 is an essential protein that operates in the base excision repair (BER) pathway and is responsible for ≥95% of the total apurinic/apyrimidinic (AP) endonuclease activity in human cells. BER is a major pathway that copes with DNA damage induced by several anti-cancer agents, including ionizing radiation and temozolomide. Overexpression of APE1 and enhanced AP endonuclease activity has been linked to increased resistance of tumor cells to treatment with monofunctional alkylators, implicating inhibition of APE1 as a valid strategy for cancer therapy. We report herein the results of a focused medicinal chemistry effort around a novel APE1 inhibitor, N-(3-(benzo[d]thiazol-2-yl)-6-isopropyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)acetamide (3). Compound 3 and related analogs exhibit single-digit µM activity against the purified APE1 enzyme, comparable activity in HeLa whole cell extract assays, and potentiate the cytotoxicity of the alkylating agents methylmethane sulfonate and temozolomide. Moreover, this class of compounds possesses a generally favorable in vitro ADME profile, along with good exposure levels in plasma and brain following intraperitoneal dosing (30 mg/kg body weight) in mice.
PMCID: PMC3515842  PMID: 22455312
APEX1; APE1; apurinic/apyrimidinic endonuclease; high-throughput screen; inhibitor
6.  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.
PMCID: PMC3150642  PMID: 21739938
Human 12-lipoxygenase; high-throughput; selective; inhibitor
7.  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.
PMCID: PMC3103057  PMID: 21250698
8.  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.
PMCID: PMC2966298  PMID: 20866075
Human 15-lipoxygenase-1; high-throughput; inhibitor
9.  Protein Lysine Methyltransferase G9a Inhibitors: Design, Synthesis, and Structure Activity Relationships of 2,4-Diamino-7-aminoalkoxy-quinazolines.† 
Journal of medicinal chemistry  2010;53(15):5844-5857.
Protein lysine methyltransferase G9a, which catalyzes methylation of lysine 9 of histone H3 (H3K9) and lysine 373 (K373) of p53, is over expressed in human cancers. Genetic knockdown of G9a inhibits cancer cell growth and the di-methylation of p53 K373 results in the inactivation of p53. Initial SAR exploration of the 2,4-diamino-6,7-dimethoxyquinazoline template represented by 3a (BIX01294), a selective small molecule inhibitor of G9a and GLP, led to the discovery of 10 (UNC0224) as a potent G9a inhibitor with excellent selectivity. A high resolution X-ray crystal structure of the G9a-10 complex, the first co-crystal structure of G9a with a small molecule inhibitor, was obtained. Based on the structural insights revealed by this co-crystal structure, optimization of the 7-dimethylaminopropoxy side chain of 10 resulted in the discovery of 29 (UNC0321) (Morrison Ki = 63 pM), which is the first G9a inhibitor with picomolar potency and the most potent G9a inhibitor to date.
PMCID: PMC2920043  PMID: 20614940
10.  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.
PMCID: PMC2804034  PMID: 19908842
11.  Quantitative Analyses of Aggregation, Autofluorescence, and Reactivity Artifacts in a Screen for Inhibitors of a Thiol Protease 
Journal of medicinal chemistry  2010;53(1):37-51.
The perceived and actual burden of false positives in high-throughput screening has received considerable attention; however, few studies exist on the contributions of distinct mechanisms of non-specific effects like chemical reactivity, assay signal interference, and colloidal aggregation. Here, we analyze the outcome of a screen of 197,861 diverse compounds in a concentration-response format against the cysteine protease cruzain, a target expected to be particularly sensitive to reactive compounds and using an assay format with light detection in the short-wavelength region where significant compound autofluorescence is typically encountered. Approximately 1.9% of all compounds screened were detergent-sensitive inhibitors. The contribution from autofluorescence and compounds bearing reactive functionalities was dramatically lower: of all hits, only 1.8% were autofluorescent and 1.48% contained reactive or undesired functional groups. The distribution of false positives was relatively constant across library sources. The simple step of including detergent in the assay buffer suppressed the nonspecific effect of approximately 93% of the original hits.
PMCID: PMC2992957  PMID: 19908840
12.  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.
PMCID: PMC2772170  PMID: 19761212
13.  Discovery of a 2,4-Diamino-7-aminoalkoxy-quinazoline as a Potent and Selective Inhibitor of Histone Lysine Methyltransferase G9a† 
Journal of medicinal chemistry  2009;52(24):7950-7953.
SAR exploration of the 2,4-diamino-6,7-dimethoxyquinazoline template led to the discovery of 8 (UNC0224) as a potent and selective G9a inhibitor. A high resolution X-ray crystal structure of the G9a-8 complex, the first co-crystal structure of G9a with a small molecule inhibitor, was obtained. The co-crystal structure validated our binding hypothesis and will enable structure-based design of novel inhibitors. 8 is a useful tool for investigating the biology of G9a and its roles in chromatin remodeling.
PMCID: PMC2825141  PMID: 19891491
14.  Comprehensive Mechanistic Analysis of Hits from High-Throughput and Docking Screens against β-Lactamase 
Journal of medicinal chemistry  2008;51(8):2502-2511.
High-throughput screening (HTS) is widely used in drug discovery. Especially for screens of unbiased libraries, false positives can dominate “hit lists”; their origins are much debated. Here we determine the mechanism of every active hit from a screen of 70,563 unbiased molecules against β-lactamase using quantitative HTS (qHTS). Of the 1274 initial inhibitors, 95% were detergent-sensitive and were classified as aggregators. Among the 70 remaining were 25 potent, covalent-acting β-lactams. Mass spectra, counter-screens, and crystallography identified 12 as promiscuous covalent inhibitors. The remaining 33 were either aggregators or irreproducible. No specific reversible inhibitors were found. We turned to molecular docking to prioritize molecules from the same library for testing at higher concentrations. Of 16 tested, 2 were modest inhibitors. Subsequent X-ray structures corresponded to the docking prediction. Analog synthesis improved affinity to 8 µM. These results suggest that it may be the physical behavior of organic molecules, not their reactivity, that accounts for most screening artifacts. Structure-based methods may prioritize weak-but-novel chemotypes in unbiased library screens.
PMCID: PMC2655312  PMID: 18333608
15.  Complementarity Between a Docking and a High-Throughput Screen in Discovering New Cruzain Inhibitors† 
Journal of Medicinal Chemistry  2010;53(13):4891-4905.
Virtual and high-throughput screens (HTS) should have complementary strengths and weaknesses, but studies that prospectively and comprehensively compare them are rare. We undertook a parallel docking and HTS screen of 197861 compounds against cruzain, a thiol protease target for Chagas disease, looking for reversible, competitive inhibitors. On workup, 99% of the hits were eliminated as false positives, yielding 146 well-behaved, competitive ligands. These fell into five chemotypes: two were prioritized by scoring among the top 0.1% of the docking-ranked library, two were prioritized by behavior in the HTS and by clustering, and one chemotype was prioritized by both approaches. Determination of an inhibitor/cruzain crystal structure and comparison of the high-scoring docking hits to experiment illuminated the origins of docking false-negatives and false-positives. Prioritizing molecules that are both predicted by docking and are HTS-active yields well-behaved molecules, relatively unobscured by the false-positives to which both techniques are individually prone.
PMCID: PMC2895358  PMID: 20540517

Results 1-15 (15)