Helical sulfonyl-γ-AApeptides modulating Aß oligomerization and cytotoxicity by recognizing Aß helix.

In contrast to prevalent strategies which make use of ß-sheet mimetics to block Aß fibrillar growth, in this study, we designed a series of sulfonyl-γ-AApeptide helices that targeted the crucial α-helix domain of Aß13-26 and stabilized Aß conformation to avoid forming the neurotoxic Aß oligomeric ß-sheets. Biophysical assays such as amyloid kinetics and TEM demonstrated that the Aß oligomerization and fibrillation could be greatly prevented and even reversed in the presence of sulfonyl-γ-AApeptides in a sequence-specific and dose-dependent manner. The studies based on circular dichroism, Two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) spectra unambiguously suggested that the sulfonyl-γ-AApeptide Ab-6 could bind to the central region of Aß42 and induce α-helix conformation in Aß. Additionally, Electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) was employed to rule out a colloidal mechanism of inhibitor and clearly supported the capability of Ab-6 for inhibiting the formation of Aß aggregated forms. Furthermore, Ab-6 could rescue neuroblastoma cells by eradicating Aß-mediated cytotoxicity even in the presence of pre-formed Aß aggregates. The confocal microscopy demonstrated that Ab-6 could still specifically bind Aß42 and colocalize into mitochondria in the cellular environment, suggesting the rescue of cell viability might be due to the protection of mitochondrial function otherwise impaired by Aß42 aggregation. Taken together, our studies indicated that sulfonyl-γ-AApeptides as helical peptidomimetics could direct Aß into the off-pathway helical secondary structure, thereby preventing the formation of Aß oligomerization, fibrillation and rescuing Aß induced cell cytotoxicity.

Cardioprotective Effects of 1-(3,6-Dibromo-carbazol-9-yl)-3-Phenylamino-Propan-2-Ol in Diabetic Hearts via Nicotinamide Phosphoribosyltransferase Activation.

Diabetes is associated with increased cardiac injury and sudden death. Nicotinamide phosphoribosyltransferase (Nampt) is an essential enzyme for the NAD+ salvage pathway and is dysregulated in diabetes. Nampt activation results in rescued NADH/NAD+ ratios and provides pharmacological changes necessary for diabetic cardioprotection. Computer docking shows that 1-(3,6-Dibromo-carbazol-9-yl)-3-phenylamino-propan-2-ol (P7C3) allows for enhanced Nampt dimerization and association. To test the pharmacological application, we used male leptin receptor-deficient (db/db) mice and treated them with Nampt activator P7C3. The effects of 4-week P7C3 treatment on cardiac function were evaluated along with molecular signaling changes for phosphorylated protein kinase B (p-AKT), phosphorylated endothelial nitric oxide synthase (p-eNOS), and sirtuin 1 (SIRT1). The cardiac function evaluated by ECG and echocardiography were significantly improved after 4 weeks of P7C3 treatment. Biochemically, higher NADH/NAD+ ratios in diabetic hearts were rescued by P7C3 treatment. Moreover, activities of Nampt and SIRT1 were significantly increased in P7C3-treated diabetic hearts. P7C3 treatment significantly decreased the blood glucose in diabetic mice with 4-week treatment as noted by glucose tolerance test and fasting blood glucose measurements compared with vehicle-treated mice. P7C3 activated Nampt enzymatic activity both in vitro and in the 4-week diabetic mouse hearts, demonstrating the specificity of the small molecule. P7C3 treatment significantly enhanced the expression of cardioprotective signaling of p-AKT, p-eNOS, and Beclin 1 in diabetic hearts. Nampt activator P7C3 allows for decreased infarct size with decreased Troponin I and lactose dehydrogenase (LDH) release, which is beneficial to the heart. Overall, the present study shows that P7C3 activates Nampt and SIRT1 activity and decreases NADH/NAD+ ratio, resulting in improved biochemical signaling providing cardioprotection. SIGNIFICANCE STATEMENT: This study shows that 1-(3,6-Dibromo-carbazol-9-yl)-3-phenylamino-propan-2-ol (P7C3) is effective in treating diabetes and cardiovascular diseases. The novel small molecule is antiarrhythmic and improves the ejection fraction in diabetic hearts. The study successfully demonstrated that P7C3 decreases the infarct size in hearts during myocardial infarction and ischemia-reperfusion injury. Biochemical and cellular signaling show increased NAD+ levels, along with Nampt activity involved in upregulating protective signaling in the diabetic heart. P7C3 has high therapeutic potential for rescuing heart disease.

The p.E152K-STIM1 mutation deregulates Ca2+ signaling contributing to chronic pancreatitis.

Since deregulation of intracellular Ca2+ can lead to intracellular trypsin activation, and stromal interaction molecule-1 (STIM1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile α-motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1-sarco/endoplasmic reticulum calcium transport ATPase (SERCA) conformational change and enhanced SERCA pump activity leading to increased store-operated Ca2+ entry (SOCE). In pancreatic AR42J cells expressing the p.E152K variant, Ca2+ signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation.This article has an associated First Person interview with the first author of the paper.

Bioactivity improvement via display of the hydrophobic core of HYD1 in a cyclic ß-hairpin-like scaffold, MTI-101.

HYD1 is an all D-amino acid linear 10-mer peptide that was discovered by one-bead-one-compound screening. HYD1 has five hydrophobic amino acids flanked by polar amino acids. Alanine scanning studies showed that alternating hydrophobic amino acid residues and N- and C-terminal lysine side chains were contributors to the biological activity of the linear 10-mer analogs. This observation led us to hypothesize that display of the hydrophobic pentapeptide sequence of HYD1 in a cyclic beta-hairpin-like scaffold could lead to better bioavailability and biological activity. An amphipathic pentapeptide sequence was used to form an antiparallel strand and those strands were linked via dipeptide-like sequences selected to promote ß-turns. Early cyclic analogs were more active but otherwise mimicked the biological activity of the linear HYD1 peptide. The cyclic peptidomimetics were synthesized using standard Fmoc solid phase synthesis to form linear peptides, followed by solution phase or on-resin cyclization. SAR studies were carried out with an aim to increase the potency of these drug candidates for the killing of multiple myeloma cells in vitro. The solution structures of 1, 5, and 10 were elucidated using NMR spectroscopy. 1H NMR and 2D TOCSY studies of these peptides revealed a downfield Hα proton chemical shift and 2D NOE spectral analysis consistent with a ß-hairpin-like structure.

An Allosteric Binding Site on Sortilin Regulates the Trafficking of VLDL, PCSK9, and LDLR in Hepatocytes.

ApoB lipoproteins (apo B-Lp) are produced in hepatocytes, and their secretion requires the cargo receptor sortilin. We examined the secretion of apo B-Lp-containing very low-density lipoprotein (VLDL), an LDL progenitor. Sortilin also regulates the trafficking of the subtilase PCSK9, which when secreted binds the LDL receptor (LDLR), resulting in its endocytosis and destruction at the lysosome. We show that the site 2 binding compound (cpd984) has multiple effects in hepatocytes, including (1) enhanced Apo-Lp secretion, (2) increased cellular PCSK9 retention, and (3) augmented levels of LDLR at the plasma membrane. We postulate that cpd984 enhances apo B-Lp secretion in part through binding the lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3), which is present at higher levels on circulating VLDL form fed rats relative to after fasting. We attribute the enhanced VLDL secretion to its increased binding affinity for sortilin site 1 induced by cpd984 binding site 2. This hinders PCSK9 binding and secretion, which would subsequently prevent its binding to LDLR leading to its degradation. This suggests that site 2 is an allosteric regulator of site 1 binding. This effect is not limited to VLDL, as cpd984 augments binding of the neuropeptide neurotensin (NT) to sortilin site 1. Molecular dynamics simulations demonstrate that the C-terminus of NT (Ct-NT) stably binds site 1 through an electrostatic interaction. This was bolstered by the ability of Ct-NT to disrupt lower-affinity interactions between sortilin and the site 1 ligand PIP3. Together, these data show that binding cargo at sortilin site 1 is allosterically regulated through site 2 binding, with important ramifications for cellular lipid homeostasis involving proteins such as PCSK9 and LDLR.

A small-molecule competitive inhibitor of phosphatidic acid binding by the AAA+ protein NSF/Sec18 blocks the SNARE-priming stage of vacuole fusion.

The homeostasis of most organelles requires membrane fusion mediated by soluble N -ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs). SNAREs undergo cycles of activation and deactivation as membranes move through the fusion cycle. At the top of the cycle, inactive cis-SNARE complexes on a single membrane are activated, or primed, by the hexameric ATPase associated with the diverse cellular activities (AAA+) protein, N-ethylmaleimide-sensitive factor (NSF/Sec18), and its co-chaperone α-SNAP/Sec17. Sec18-mediated ATP hydrolysis drives the mechanical disassembly of SNAREs into individual coils, permitting a new cycle of fusion. Previously, we found that Sec18 monomers are sequestered away from SNAREs by binding phosphatidic acid (PA). Sec18 is released from the membrane when PA is hydrolyzed to diacylglycerol by the PA phosphatase Pah1. Although PA can inhibit SNARE priming, it binds other proteins and thus cannot be used as a specific tool to further probe Sec18 activity. Here, we report the discovery of a small-molecule compound, we call IPA (inhibitor of priming activity), that binds Sec18 with high affinity and blocks SNARE activation. We observed that IPA blocks SNARE priming and competes for PA binding to Sec18. Molecular dynamics simulations revealed that IPA induces a more rigid NSF/Sec18 conformation, which potentially disables the flexibility required for Sec18 to bind to PA or to activate SNAREs. We also show that IPA more potently and specifically inhibits NSF/Sec18 activity than does N-ethylmaleimide, requiring the administration of only low micromolar concentrations of IPA, demonstrating that this compound could help to further elucidate SNARE-priming dynamics.

A specific small-molecule inhibitor of protein kinase CδI activity improves metabolic dysfunction in human adipocytes from obese individuals.

The metabolic consequences and sequelae of obesity promote life-threatening morbidities. PKCδI is an important elicitor of inflammation and apoptosis in adipocytes. Here we report increased PKCδI activation via release of its catalytic domain concurrent with increased expression of proinflammatory cytokines in adipocytes from obese individuals. Using a screening strategy of dual recognition of PKCδI isozymes and a caspase-3 binding site on the PKCδI hinge domain with Schrödinger software and molecular dynamics simulations, we identified NP627, an organic small-molecule inhibitor of PKCδI. Characterization of NP627 by surface plasmon resonance (SPR) revealed that PKCδI and NP627 interact with each other with high affinity and specificity, SPR kinetics revealed that NP627 disrupts caspase-3 binding to PKCδI, and in vitro kinase assays demonstrated that NP627 specifically inhibits PKCδI activity. The SPR results also indicated that NP627 affects macromolecular interactions between protein surfaces. Of note, release of the PKCδI catalytic fragment was sufficient to induce apoptosis and inflammation in adipocytes. NP627 treatment of adipocytes from obese individuals significantly inhibited PKCδI catalytic fragment release, decreased inflammation and apoptosis, and significantly improved mitochondrial metabolism. These results indicate that PKCδI is a robust candidate for targeted interventions to manage obesity-associated chronic inflammatory diseases. We propose that NP627 may also be used in other biological systems to better understand the impact of caspase-3-mediated activation of kinase activity.

Mechanistic Studies of 1-Deoxy-D-Xylulose-5-Phosphate Synthase from Deinococcus radiodurans.

The non-mevalonate dependent (NMVA) pathway for the biosynthesis of isopentenyl pyrophosphate and dimethylallyl pyrophosphate is the sole source of these terpenoids for the production of isoprenoids in the apicomplexan parasites, in many eubacteria, and in plants. The absence of this pathway in higher organisms has opened a new platform for the development of novel antibiotics and anti-malarials. The enzyme catalyzing the first step of the NMVA pathway is 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). DXPS catalyzes the thiamine pyrophosphate- and Mg (II)-dependent conjugation of pyruvate and D-glyceraldehyde-3-phosphate to form 1-deoxy-D-xylulose-5-phosphate and CO2. The kinetic mechanism of DXPS from Deinococcus radiodurans most consistent with our data is random sequential as shown using a combination of kinetic analysis and product and dead-end inhibition studies. The role of active site amino acids, identified by sequence alignment to other DXPS proteins, was probed by constructing and analyzing the catalytic efficacy of a set of targeted site-directed mutants.

Ligand-mediated protein degradation reveals functional conservation among sequence variants of the CUL4-type E3 ligase substrate receptor cereblon.

Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteosomal destruction by engaging the DDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase in human cells but not in mouse cells, suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degradation. To examine the effects of defined sequence changes on CRBN's activity, we performed a comprehensive study using complementary theoretical, biophysical, and biological assays aimed at understanding CRBN's nonprimate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants retain their drug-binding properties to both classical immunomodulatory drugs and dBET1, a chemical compound and targeting ligand designed to degrade bromodomain-containing 4 (BRD4) via a CRBN-dependent mechanism. We further show that dBET1 stimulates CRBN's E3 ubiquitin-conjugating function and degrades BRD4 in both mouse and human cells. This insight paves the way for studies of CRBN-dependent proteasome-targeting molecules in nonprimate models and provides a new understanding of CRBN's substrate-recruiting function.

Molecular Dynamics Simulations of Membrane-Bound STIM1 to Investigate Conformational Changes during STIM1 Activation upon Calcium Release.

Calcium is involved in important intracellular processes, such as intracellular signaling from cell membrane receptors to the nucleus. Typically, calcium levels are kept at less than 100 nM in the nucleus and cytosol, but some calcium is stored in the endoplasmic reticulum (ER) lumen for rapid release to activate intracellular calcium-dependent functions. Stromal interacting molecule 1 (STIM1) plays a critical role in early sensing of changes in the ER's calcium level, especially when there is a sudden release of stored calcium from the ER. Inactive STIM1, which has a bound calcium ion, is activated upon ion release. Following activation of STIM1, there is STIM1-assisted initiation of extracellular calcium entry through channels in the cell membrane. This extracellular calcium entering the cell then amplifies intracellular calcium-dependent actions. At the end of the process, ER levels of stored calcium are reestablished. The main focus of this work was to study the conformational changes accompanying homo- or heterodimerization of STIM1. For this purpose, the ER luminal portion of STIM1 (residues 58-236), which includes the sterile alpha motif (SAM) domain plus the calcium-binding EF-hand domains 1 and 2 attached to the STIM1 transmembrane region (TM), was modeled and embedded in a virtual membrane. Next, molecular dynamics simulations were performed to study the conformational changes that take place during STIM1 activation and subsequent protein-protein interactions. Indeed, the simulations revealed exposure of residues in the EF-hand domains, which may be important for dimerization steps. Altogether, understanding conformational changes in STIM1 can help in drug discovery when targeting this key protein in intracellular calcium functions.

Phosphatidylinositol (3,4,5)-trisphosphate binds to sortilin and competes with neurotensin: Implications for very low density lipoprotein binding.

Sortilin is a multi-ligand sorting receptor that interacts with B100-containing VLDL and LDL as well as other ligands including neurotensin (NT). The current study investigates the hypothesis that phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generated downstream of insulin action can directly bind to sortilin. NT binds to sortilin at a well characterized site via its carboxy terminus (C-term). Using a crystal structure of human sortilin (hsortilin), PIP3 is predicted to bind at this C-term site. Binding of PIP3 to hsortilin is demonstrated using surface plasmon resonance (SPR) flowing PIP3 nanodiscs over immobilized hsortilin. Studies were performed using SPR where dibutanoyl PIP3 is shown to compete with NT for sortilin binding. Rat VLDL and LDL were evaluated for PIP3 content immunologically using monoclonal antibodies directed against PIP3. Rat plasma VLDL contained three times more immunoreactive PIP3 than LDL per µg of protein. Because VLDL contains additional ligands that bind sortilin, to distinguish specific PIP3 binding, we used PIP3 liposomes. Liposome floatation assays were used to demonstrate PIP3 liposome binding to sortilin. Using SPR and immobilized hsortilin, the C-term NT tetrapeptide (P-Y-I-L) is shown to bind to hsortilin. A compound (cpd984) was identified with strong theoretical binding to the site on sortilin involved in NT N-terminal binding. When cpd984 is co-incubated with the tetrapeptide, the affinity of binding to sortilin is increased. Similarly, the affinity of PIP3 liposome binding increased in the presence of cpd984. Overall, results demonstrate that sortilin is a PIP3 binding protein with binding likely to occur at the C-term NT binding site. The presence of multiple ligands on B100-containing lipoproteins, VLDL and LDL, raises the interesting possibility for increased interaction with sortilin based on the presence of PIP3.

Sortilin facilitates VLDL-B100 secretion by insulin sensitive McArdle RH7777 cells.

Studies examining the relationship between cellular sortilin and VLDL-B100 secretion demonstrate inconsistent results. Current studies explore the possibility that discrepancies may be related to insulin sensitivity. McArdle RH7777 cells (McA cells) cultured under serum enriched conditions lose sensitivity to insulin. Following incubation in serum-free DMEM containing 1% BSA, McA cells become insulin responsive and demonstrate reduced apo B secretion. Current studies indicate that insulin sensitive McA cells express lower cellular sortilin that corresponds with reduction in VLDL-B100 secretion without changes in mRNA of either sortilin or apo B. When sortilin expression is further reduced by siRNA knockdown (KD), there are additional decreases in VLDL-B100 secretion. A crystal structure of human sortilin (hsortilin) identifies two binding sites on the luminal domain for the N- and C-termini of neurotensin (NT). A small organic compound (cpd984) was identified that has strong theoretical binding to the N-terminal site. Both cpd984 and NT bind hsortilin by surface plasmon resonance. In incubations with insulin sensitive McA cells, cpd984 was shown to enhance VLDL-B100 secretion at each level of sortilin KD suggesting cpd984 acted through sortilin in mediating its effect. Current results support a role for sortilin to facilitate VLDL-B100 secretion which is limited to insulin sensitive McA cells. Inconsistent reports of the relationship between VLDL-B100 secretion and sortilin in previous studies may relate to differing functions of sortilin in VLDL-B100 secretion depending upon insulin sensitivity.

Cupriphilic compounds to aid in proteasome inhibition.

It has been found that tumor cells and tissues, compared to normal cells, have higher levels of copper and possibly other metal ions. This presents a potential vulnerability of tumor cells that can serve as a physiological difference between cancer cells and normal cells and allows design of compounds that selectively target tumor cells while sparing normal cells. Recently we have identified compounds that have potential to inhibit the proteasome in tumor cells and induce cell death by mobilizing endogenous tumor copper resulting in in cellulo activation of the compound. These compounds hence act as pro-drugs, becoming active drugs in tumor cells with high copper content but remaining essentially inactive in normal cells, thereby greatly reducing adverse effects in patients. Such use would be of significant benefit in early detection and treatment of cancers, in particular, aggressive cancers such as pancreatic cancer which is usually not detected until it has reached an advanced stage. Six compounds were identified following virtual screening of the NCI Diversity Set with our proteasome computer model followed by confirmation with a biochemical assay that showed significant inhibition of the proteasome by the compounds in the presence of copper ions. In a dose response assay, NSC 37408 (6,7-dihydroxy-1-benzofuran-3-one), our best compound, exhibited an IC50 of 3µM in the presence of 100nM copper.

ß-Strand mimics based on tetrahydropyridazinedione (tpd) peptide stitching.

Short peptides featuring a tetrahydropyridazinedione (tpd) backbone tether exhibit reduced conformational flexibility external to the heterocyclic constraint. Analysis by NMR, molecular modeling and X-ray crystallography suggests both covalent and non-covalent stabilization of extended peptide conformations. An efficient solid-phase protocol was developed for the synthesis of a new class of ß-strand mimics based on oligomeric tpd subunits.

A computational design approach for virtual screening of peptide interactions across K(+) channel families.

Ion channels represent a large family of membrane proteins with many being well established targets in pharmacotherapy. The 'druggability' of heteromeric channels comprised of different subunits remains obscure, due largely to a lack of channel-specific probes necessary to delineate their therapeutic potential in vivo. Our initial studies reported here, investigated the family of inwardly rectifying potassium (Kir) channels given the availability of high resolution crystal structures for the eukaryotic constitutively active Kir2.2 channel. We describe a 'limited' homology modeling approach that can yield chimeric Kir channels having an outer vestibule structure representing nearly any known vertebrate or invertebrate channel. These computationally-derived channel structures were tested ""in silico for 'docking' to NMR structures of tertiapin (TPN), a 21 amino acid peptide found in bee venom. TPN is a highly selective and potent blocker for the epithelial rat Kir1.1 channel, but does not block human or zebrafish Kir1.1 channel isoforms. Our Kir1.1 channel-TPN docking experiments recapitulated published in vitro ""findings for TPN-sensitive and TPN-insensitive channels. Additionally, in silico site-directed mutagenesis identified 'hot spots' within the channel outer vestibule that mediate energetically favorable docking scores and correlate with sites previously identified with in vitro thermodynamic mutant-cycle analysis. These 'proof-of-principle' results establish a framework for virtual screening of re-engineered peptide toxins for interactions with computationally derived Kir channels that currently lack channel-specific blockers. When coupled with electrophysiological validation, this virtual screening approach may accelerate the drug discovery process, and can be readily applied to other ion channels families where high resolution structures are available.

Orally bioavailable 6-chloro-7-methoxy-4(1H)-quinolones efficacious against multiple stages of Plasmodium.

The continued proliferation of malaria throughout temperate and tropical regions of the world has promoted a push for more efficacious treatments to combat the disease. Unfortunately, more recent remedies such as artemisinin combination therapies have been rendered less effective due to developing parasite resistance, and new drugs are required that target the parasite in the liver to support the disease elimination efforts. Research was initiated to revisit antimalarials developed in the 1940s and 1960s that were deemed unsuitable for use as therapeutic agents as a result of poor understanding of both physicochemical properties and parasitology. Structure-activity and structure-property relationship studies were conducted to generate a set of compounds with the general 6-chloro-7-methoxy-2-methyl-4(1H)-quinolone scaffold which were substituted at the 3-position with a variety of phenyl moieties possessing various properties. Extensive physicochemical evaluation of the quinolone series was carried out to downselect the most promising 4(1H)-quinolones, 7, 62, 66, and 67, which possessed low-nanomolar EC50 values against W2 and TM90-C2B as well as improved microsomal stability. Additionally, in vivo Thompson test results using Plasmodium berghei in mice showed that these 4(1H)-quinolones were efficacious for the reduction of parasitemia at >99% after 6 days.

In silico characterization of an atypical MAPK phosphatase of Plasmodium falciparum as a suitable target for drug discovery.

Plasmodium falciparum, the causative agent of malaria, contributes to significant morbidity and mortality worldwide. Forward genetic analysis of the blood-stage asexual cycle identified the putative phosphatase from PF3D7_1305500 as an important element of intraerythrocytic development expressed throughout the life cycle. Our preliminary evaluation identified it as an atypical mitogen-activated protein kinase phosphatase. Additional bioinformatic analysis delineated a conserved signature motif and three residues with potential importance to functional activity of the atypical dual-specificity phosphatase domain. A homology model of the dual-specificity phosphatase domain was developed for use in high-throughput in silico screening of the available library of antimalarial compounds from ChEMBL-NTD. Seven compounds from this set with predicted affinity to the active site were tested against in vitro cultures, and three had reduced activity against a ∆PF3D7_1305500 parasite, suggesting PF3D7_1305500 is a potential target of the selected compounds. Identification of these compounds provides a novel starting point for a structure-based drug discovery strategy that moves us closer toward the discovery of new classes of clinical antimalarial drugs. These data suggest that mitogen-activated protein kinase phosphatases represent a potentially new class of P. falciparum drug target.

Development of new N-Arylbenzamides as STAT3 Dimerization Inhibitors.

The O-tosylsalicylamide S3I-201 (10) was used as a starting point for design and synthesis of novel STAT-3 dimerization inhibitors with improved drug-like qualities. The phosphonic acid 12d and salicylic acids 13f, 13g with a shorter amide linker lacking the O-tosyl group had improved STAT-3 inhibitory activity. The equivalent potencies observed by the replacement of phosphonic acid moiety of 12d with 5-amino-2-hydroxybenzoic acid group as in 13f further validates 5-amino-2-hydroxybenzoic acid as a phosphotyrosine mimic. The salicylic acid 13f displayed improved whole cell activity. The focused library of salicylic acids 13 with benzamide linker indicated that hydrophobic heptyl and cyclohexyl are the best tolerated R groups and a biphenyl ether (as the Ar group) significantly contributes to STAT3 inhibitory activity. Our docking studies indicated that the acidic groups of 12d, 13f and 13g interact in the p-Tyr-705 binding site in a broadly similar manner, while the phenoxybenzoyl group and the cyclohexylbenzyl group occupying pY+1 and pY-X hydrophobic pockets respectively. The in vitro and cell based potency of 13f warrants further development of this scaffold as STAT3 inhibitors.

Production of recombinant 1-deoxy-d-xylulose-5-phosphate synthase from Plasmodium vivax in Escherichia coli.

Humanity is burdened by malaria as millions are infected with this disease. Although advancements have been made in the treatment of malaria, optimism regarding our fight against malaria must be tempered against the problem of drug resistance in the Plasmodium parasites causing malaria. New targets are required to overcome the resistance problem. The enzymes of the mevalonate-independent pathway of isoprenoid biosynthesis are targets for the development of novel antimalarial drugs. One enzyme in this pathway, 1-deoxy-d-xylulose-5-phosphate synthase (DXS), catalyzes the conversion of 1-deoxy-d-xylulose-5-phosphate to isopentenylpyrophosphate and dimethylallyl phosphate. We demonstrate the use of a step deletion method to identify and eliminate the putative nuclear-encoded and transit peptides from full length DXS to yield a truncated, active, and soluble form of Plasmodium vivax DXS, the DXS catalytic core (DXScc).