Chemoprotective antimalarials identified through quantitative high-throughput screening of Plasmodium blood and liver stage parasites.

The spread of Plasmodium falciparum parasites resistant to most first-line antimalarials creates an imperative to enrich the drug discovery pipeline, preferably with curative compounds that can also act prophylactically. We report a phenotypic quantitative high-throughput screen (qHTS), based on concentration-response curves, which was designed to identify compounds active against Plasmodium liver and asexual blood stage parasites. Our qHTS screened over 450,000 compounds, tested across a range of 5 to 11 concentrations, for activity against Plasmodium falciparum asexual blood stages. Active compounds were then filtered for unique structures and drug-like properties and subsequently screened in a P. berghei liver stage assay to identify novel dual-active antiplasmodial chemotypes. Hits from thiadiazine and pyrimidine azepine chemotypes were subsequently prioritized for resistance selection studies, yielding distinct mutations in P. falciparum cytochrome b, a validated antimalarial drug target. The thiadiazine chemotype was subjected to an initial medicinal chemistry campaign, yielding a metabolically stable analog with sub-micromolar potency. Our qHTS methodology and resulting dataset provides a large-scale resource to investigate Plasmodium liver and asexual blood stage parasite biology and inform further research to develop novel chemotypes as causal prophylactic antimalarials.

Agonist-specific voltage-dependent gating of lysosomal two-pore Na+ channels.

Mammalian two-pore-channels (TPC1, 2; TPCN1, TPCN2) are ubiquitously- expressed, PI(3,5)P2-activated, Na+-selective channels in the endosomes and lysosomes that regulate luminal pH homeostasis, membrane trafficking, and Ebola viral infection. Whereas the channel activity of TPC1 is strongly dependent on membrane voltage, TPC2 lacks such voltage dependence despite the presence of the presumed 'S4 voltage-sensing' domains. By performing high-throughput screening followed by lysosomal electrophysiology, here we identified a class of tricyclic anti-depressants (TCAs) as small-molecule agonists of TPC channels. TCAs activate both TPC1 and TPC2 in a voltage-dependent manner, referred to as Lysosomal Na+ channel Voltage-dependent Activators (LyNa-VAs). We also identified another compound which, like PI(3,5)P2, activates TPC2 independent of voltage, suggesting the existence of agonist-specific gating mechanisms. Our identification of small-molecule TPC agonists should facilitate the studies of the cell biological roles of TPCs and can also readily explain the reported effects of TCAs in the modulation of autophagy and lysosomal functions.

Identification, design and synthesis of novel pyrazolopyridine influenza virus nonstructural protein 1 antagonists.

Nonstructural protein 1 (NS1) plays a crucial function in the replication, spread, and pathogenesis of influenza virus by inhibiting the host innate immune response. Here we report the discovery and optimization of novel pyrazolopyridine NS1 antagonists that can potently inhibit influenza A/PR/8/34 replication in MDCK cells, rescue MDCK cells from cytopathic effects of seasonal influenza A strains, reverse NS1-dependent inhibition of IFN-ß gene expression, and suppress the slow growth phenotype in NS1-expressing yeast. These pyrazolopyridines will enable researchers to investigate NS1 function during infection and how antagonists can be utilized in the next generation of treatments for influenza infection.

Metarrestin, a perinucleolar compartment inhibitor, effectively suppresses metastasis.

Metastasis remains a leading cause of cancer mortality due to the lack of specific inhibitors against this complex process. To identify compounds selectively targeting the metastatic state, we used the perinucleolar compartment (PNC), a complex nuclear structure associated with metastatic behaviors of cancer cells, as a phenotypic marker for a high-content screen of over 140,000 structurally diverse compounds. Metarrestin, obtained through optimization of a screening hit, disassembles PNCs in multiple cancer cell lines, inhibits invasion in vitro, suppresses metastatic development in three mouse models of human cancer, and extends survival of mice in a metastatic pancreatic cancer xenograft model with no organ toxicity or discernable adverse effects. Metarrestin disrupts the nucleolar structure and inhibits RNA polymerase (Pol) I transcription, at least in part by interacting with the translation elongation factor eEF1A2. Thus, metarrestin represents a potential therapeutic approach for the treatment of metastatic cancer.

Inhibition of PIP4Kγ ameliorates the pathological effects of mutant huntingtin protein.

The discovery of the causative gene for Huntington's disease (HD) has promoted numerous efforts to uncover cellular pathways that lower levels of mutant huntingtin protein (mHtt) and potentially forestall the appearance of HD-related neurological defects. Using a cell-based model of pathogenic huntingtin expression, we identified a class of compounds that protect cells through selective inhibition of a lipid kinase, PIP4Kγ. Pharmacological inhibition or knock-down of PIP4Kγ modulates the equilibrium between phosphatidylinositide (PI) species within the cell and increases basal autophagy, reducing the total amount of mHtt protein in human patient fibroblasts and aggregates in neurons. In two Drosophila models of Huntington's disease, genetic knockdown of PIP4K ameliorated neuronal dysfunction and degeneration as assessed using motor performance and retinal degeneration assays respectively. Together, these results suggest that PIP4Kγ is a druggable target whose inhibition enhances productive autophagy and mHtt proteolysis, revealing a useful pharmacological point of intervention for the treatment of Huntington's disease, and potentially for other neurodegenerative disorders.

Non-iminosugar glucocerebrosidase small molecule chaperones.

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.

Pharmacokinetics of TDP223206 following intravenous and oral administration to intact rats and intravenous administration to bile duct-cannulated rats.

The pharmacokinetics of TDP223206 was studied following single intravenous and oral administrations in rats. A mixture of TDP223206 and (14)C-TDP223206 were administered to intact and bile duct-cannulated rats. Following intravenous administration, plasma concentrations declined biphasically. The AUC(inf) increased linearly with dose but was not dose proportional. The PK parameters of TDP223206 indicated low clearance (254-386 ml/h/kg) and a moderate volume of distribution (968-1883 ml/kg). The bioavailability was 32.95% and 24.46% for 10 and 50 mg/kg oral doses, respectively. (14)C-TDP223206 was distributed widely into different tissues with small intestine, liver, kidneys and large intestine having large tissue to plasma ratios. (14)C-TDP223206 was the major circulating component in the plasma. A total of 91.2% of administered radioactivity of (14)C-TDP223206 was recovered in bile indicating that biliary excretion was the major pathway for drug elimination. (14)C-TDP223206-acyl glucuronides were the major metabolites in bile. The oxo-(14)C-TDP223206 was the major metabolite in plasma and an important metabolite in bile. Two forms of diastereomeric acyl glucuronides of (14)C-TDP223206 were detected in bile with similar LC/MS intensities suggesting a similar biotransformation capacity. Only one form of these (14)C-TDP223206-acyl glucuronides was detected in plasma suggesting that enterohepatic recirculation was related to the nature of the stereo-isomers.

Identification of novel short chain 4-substituted indoles as potent alphavbeta3 antagonist using structure-based drug design.

The vitronectin receptor alpha(v)beta(3) has been identified as a promising potential target for the treatment of osteoporosis, diabetic retinopathy and cancer. We have recently reported 5-substituted indoles 3-[5-[2-(5,6,7,8-tetrahydro[1,8]naphthyridin-2-yl)ethoxy]indol-1-yl]-3-(3-pyridyl)propionic acid 3 and 3-[5-[2-(5,6,7,8-tetrahydro[1,8]naphthyridin-2-yl)ethoxy]indol-1-yl]-3-(3,4-methylenedioxyphenyl)propionic acid 4, as an original series of potent alpha(v)beta(3) antagonists with subnanomolar activity. Ligand-protein docking analyses have been performed to generate binding models of three different chemical classes of known alpha(v)beta(3) antagonists with alpha(v)beta(3). Results of this docking study suggested that indoles bearing the basic tetrahydronaphthyridine group at position 4 can easily adopt the correct binding conformation and should be as potent as our current 5-substituted indole leads 3 and 4. This hypothesis was nicely demonstrated by the synthesis of a series of 1,4-disubstituted indoles through a tandem of reactions involving: (i) the N-alkylation of indoles 15 and 22 with propargyl esters and cesium fluoride, and (ii) a Heck coupling reaction between 4-bromoindole and 7-vinyl-3,4-dihydro-2H-[1,8]naphthyridine-1-carboxylic acid tert-butyl ester 12, or (iii) a reductive amination involving the N-substituted-4-aminoindole 23 and the BOC-protected tetrahydro[1,8]naphthyridine aldehyde 13. Among the compounds assayed, 3-(3-pyridyl)-3-[4-[2-(5,6,7,8-tetrahydro[1,8]naphthyridin-2-yl)ethyl]indol-1-yl]propionic acid 21 showed the most promising activity on alpha(v)beta(3) (IC(50)=0.5 nM), and was found to have the same potency as our current leads 3 and 4, while maintaining selectivity over alpha(IIb)beta(IIIa). Moreover, based on the reasonable apparent permeability coefficient in an in vitro CACO-2 cell monolayer assay (P(app) apical/basolateral=2.2 x 10(-6)cm/s, P(app) basolateral/apical=2.5 x 10(-6)cm/s), compound 21 is expected to be absorbed through the intestine in human. Thus, 1,4-disubstituted indole 21 represents a new lead for this novel class of conformationally restricted alpha(v)beta(3) antagonists. Additionally, this study validates the pharmacophore model previously postulated and provides an improved basis for further structure-based drug design in the field of alpha(v)beta(3).

Novel potent and selective alphavbeta3/alphavbeta5 integrin dual antagonists with reduced binding affinity for human serum albumin.

The binding of lead compounds and drugs to human serum albumin (HSA) is a ubiquitous problem in drug discovery since it modulates the availability of the leads and drugs to their intended target, which is linked to biological efficacy. In our continuing efforts to identify small molecule alpha(V)beta(3) and alpha(V)beta(5) dual antagonists, we recently reported indoles 2-4 as potent and selective alpha(V)beta(3)/alpha(V)beta(5) antagonists with good oral bioavailability profile. In spite of subnanomolar binding affinity of these compounds to human alpha(V)beta(3) and alpha(V)beta(5) integrins, high HSA binding (96.5-97.3%) emerged as a limiting feature for these leads. Structure-activity HSA binding data of organic acids reported in the literature have demonstrated that the incorporation of polar groups into a given molecule can dramatically decrease the affinity toward HSA. We sought to apply this strategy by examining the effects of such modifications in both the central core constrain and the substituent beta to the carboxylate. Most of these derivatives were prepared in good yields through a cesium fluoride-catalyzed coupling reaction. This reaction was successful with a variety of nitrogen-containing scaffolds (20, 33, and 43) and selected acetylenic derivatives (16, 19, and 34). Among the compounds synthesized, the 3-[5-[2-(5,6,7,8-tetrahydro [1,8]naphthyridin-2-yl)ethoxy]indol-1-yl]-3-[5-(N,N-dimethylaminomethyl)-3-pyridyl]propionic acid (25) was found to be the most promising derivative within this novel series with a subnanomolar affinity for both alpha(v)beta(3) and alpha(v)beta(5) (IC(50) = 0.29 and 0.16 nM, respectively), similar to our initial lead receptor antagonists 2-4, and exhibiting a low HSA protein binding (40% bound, K(d) = 1.1+/-0.4 x 10(3) microM) and an improved in vitro stability profile toward human and mouse microsomes (99.9% and 98.7% remaining after 10 min). Moreover, the selectivity of 25 toward alpha(5)beta(1) and IIbIIIa integrins was perfectly maintained when compared to the parent leads 2-4. Thus, compound 25 was selected as a new lead with improved drug-like properties for further evaluations in the field of oncology and osteoporosis.

Novel 1,4-benzodiazepine-2,5-diones as Hdm2 antagonists with improved cellular activity.

The disruption of the p53-Hdm2 protein-protein interaction induces cell growth arrest and apoptosis. We have identified the 1,4-benzodiazepine-2,5-dione scaffold as a suitable template for inhibiting this interaction by binding to the Hdm2 protein. Several compounds have been made with improved potency, solubility, and cell-based activities.

Enantiomerically pure 1,4-benzodiazepine-2,5-diones as Hdm2 antagonists.

The 1,4-benzodiazepine-2,5-dione is a suitable template to disrupt the interaction between p53 and Hdm2. The development of an enantioselective synthesis disclosed the stereochemistry of the active enantiomer. An in vitro p53 peptide displacement assay identified active compounds. These activities were confirmed in several cell-based assays including induction of the p53 regulated gene (PIG-3) and caspase activity.

Enhanced pharmacokinetic properties of 1,4-benzodiazepine-2,5-dione antagonists of the HDM2-p53 protein-protein interaction through structure-based drug design.

Guided by structure-based drug design, modification of the 1,4-benzodiazepin-2,5-dione lead compound 1 resulted in the discovery of 19, a potent and orally bioavailable antagonist of the HDM2-p53 protein-protein interaction (FP IC50 = 0.7 microM, F approximately 100%).

A novel series of arylsulfonylthiophene-2-carboxamidine inhibitors of the complement component C1s.

Inhibiting the classical pathway of complement activation by attenuating the proteolytic activity of the serine protease C1s is a potential strategy for the therapeutic intervention in disease states such as hereditary angioedema, ischemia-reperfusion injury, and acute transplant rejection. A series of arylsulfonylthiophene-2-carboxamidine inhibitors of C1s were synthesized and evaluated for C1s inhibitory activity. The most potent compound had a Ki of 10nM and >1000-fold selectivity over uPA, tPA, FX(a), thrombin, and plasmin.

Benzodiazepinedione inhibitors of the Hdm2:p53 complex suppress human tumor cell proliferation in vitro and sensitize tumors to doxorubicin in vivo.

The activity and stability of the p53 tumor suppressor are regulated by the human homologue of the mouse double minute 2 (Hdm2) oncoprotein. It has been hypothesized that small molecules disrupting the Hdm2:p53 complex would allow for the activation of p53 and result in growth suppression. We have identified small-molecule inhibitors of the Hdm2:p53 interaction using our proprietary ThermoFluor microcalorimetry technology. Medicinal chemistry and structure-based drug design led to the development of an optimized series of benzodiazepinediones, including TDP521252 and TDP665759. Activities were dependent on the expression of wild-type (wt) p53 and Hdm2 as determined by lack of potency in mutant or null p53-expressing cell lines or cells engineered to no longer express Hdm2 and wt p53. TDP521252 and TDP665759 inhibited the proliferation of wt p53-expressing cell lines with average IC(50)s of 14 and 0.7 micromol/L, respectively. These results correlated with the direct cellular dissociation of Hdm2 from wt p53 observed within 15 minutes in JAR choriocarcinoma cells. Additional activities of these inhibitors in vitro include stabilization of p53 protein levels, up-regulation of p53 target genes in a DNA damage-independent manner, and induction of apoptosis in HepG2 cells. Administration of TDP665759 to mice led to an increase in p21(waf1/cip1) levels in liver samples. Finally, TDP665759 synergizes with doxorubicin both in culture and in an A375 xenograft model to decrease tumor growth. Taken together, these data support the potential utility of small-molecule inhibitors of the Hdm2:p53 interaction for the treatment of wt p53-expressing tumors.

A functional radioreceptor assay of alpha-V-beta-3 (alphavbeta3) inhibitors in plasma: application as an ex vivo pharmacodynamic model.

Development of alphavbeta3-integrin inhibitors has been hampered by a lack of pharmacodynamic endpoints to identify doses that inhibit alphavbeta3 in vivo. To address this need, we developed an alphavbeta3 radioreceptor assay (RRA) that could be performed in 100% plasma. The RRA was based on 125I-echistatin binding to plate-immobilized alphavbeta3. Small molecule alphavbeta3 inhibitors efficiently competed echistatin binding to alphavbeta3 when the assay was carried out in buffer. However, when carried out in 100% plasma, the RRA revealed a 45 to >3000-fold loss in compound potencies. The losses in potency reflected, in part, the high plasma protein binding by the compounds examined. The RRA was adapted as an ex vivo pharmacodynamic model. Echistatin binding was measured in the presence of plasma harvested at timed intervals from rats dosed with select compounds. Using this pharmacodynamic model, compound and dose selection was optimized for further testing in models of corneal angiogenesis. Moderate anti-angiogenic activity was achieved when rats were dosed sufficient to achieve sustained (>50%) plasma inhibition through the trough interval. Thus, the RRA provided a simple technique to rank order compound potency in plasma, and could find general use as an ex vivo pharmacodynamic assay to select compounds and doses for preclinical and clinical proof-of-principle studies.

Non-peptidic alpha(v)beta3 antagonists containing indol-1-yl propionic acids.

We describe the synthesis and structure/activity relationship of RGD mimetics that are potent inhibitors of the integrin alpha(v)beta3. Indol-1-yl propionic acids containing a variety of basic moieties at the 5-position, as well as substitutions alpha and beta to the carboxy terminus were synthesized and evaluated. Novel compounds with improved potency have been identified.

Structure-based design, synthesis, and biological evaluation of novel 1,4-diazepines as HDM2 antagonists.

Crystallographic analysis of ligands bound to HDM2 suggested that 7-substituted 1,4-diazepine-2,5-diones could mimic the alpha-helix of p53 peptide and may represent a promising scaffold to develop HDM2-p53 antagonists. To verify this hypothesis, we synthesized and biologically evaluated 5-[(3S)-3-(4-chlorophenyl)-4-[(R)-1-(4-chlorophenyl)ethyl]-2,5-dioxo-7-phenyl-1,4-diazepin-1-yl]valeric acid (10) and 5-[(3S)-7-(2-bromophenyl)-3-(4-chlorophenyl)-4-[(R)-1-(4-chlorophenyl)ethyl]-2,5-dioxo-1,4-diazepin-1-yl]valeric acid (11). Preliminary in vitro testing shows that 10 and 11 substantially antagonize the binding between HDM2 and p53 with an IC(50) of 13 and 3.6 microM, respectively, validating the modeling predictions. Taken together with the high cell permeability of diazepine 11 determined in CACO-2 cells, these results suggest that 1,4-diazepine-2,5-diones may be useful in the treatment of certain cancers.

Design, synthesis, and biological evaluation of novel potent and selective alphavbeta3/alphavbeta5 integrin dual inhibitors with improved bioavailability. Selection of the molecular core.

A novel series of potent and selective alpha(v)beta(3)/alpha(v)beta(5) dual( )()inhibitors was designed, synthesized, and evaluated against several integrins. These compounds were synthesized through a Mitsunobu reaction between the guanidinium mimetics and the corresponding central templates. Guanidinium mimetics with enhaced rigidity (i.e., (2-pyridylamino)propoxy versus the 2-(6-methylamino-2-pyridyl)ethoxy) led to improved activity toward alpha(v)beta(3). Exemplary oral bioavailability in mice was achieved using the indole central scaffold. Although, oral bioavailability was maintained when the indole molecular core was replace with the bioisosteric benzofuran or benzothiophene ring systems, it was found to not significantly impact the integrin activity or selectivity. However, the indole series displayed the best in vivo pharmacokinetic properties. Thus, the indole series was selected for further structure-activity relationships to obtain more potent alpha(v)beta(3)/alpha(v)beta(5) dual antagonist with improved oral bioavailability.

Design, synthesis, and biochemical evaluation of novel alpha V beta 3 integrin ligands.

Studies on integrin alphaVbeta3 have implicated this receptor in a number of pathologies. In this article we describe some of our initial efforts to design small molecules alphaVbeta3 ligands incorporating an indole core template and an oxyguanidine as basic ending. Synthesis, biochemical activity and pharmacological properties are analyzed.