On the mechanism of sp2 C-H borylation using ortho-N-substituted pyridinium cations.

ortho-N-Substituted pyridinium cations with the weakly coordinating anion [B(C6F5)4]- have been studied and crucial structural features in the sp2 C-H borylation catalysis of 3-methylthiophene have been identified. The electron-deficiency of the aromatic core of the cation is essential for activity together with accessible protons. The spectroscopic yield of the borylation of 3-methylthiophene with catecholborane (CatBH) was optimized up to 86% and the method was further applied to other substrates such as N-alkylbenzenes. A mechanistic DFT study revealed the rate-limiting step in the catalysis to be the liberation of molecular H2 (ΔG‡ = 27.5 kcal mol-1), whereas the overall reaction was found to be exergonic by 5.1 kcal mol-1.

Transgenic overexpression of heat shock protein (HSP83) enhances protein kinase A activity, disrupts GP63 surface protease expression and alters promastigote morphology in Leishmania amazonensis.

Leishmania parasites undergo morphological changes during their infectious life cycle, including developmental transitions within the sandfly vector, culminating in metacyclic stages that are pre-adapted for infection. Upon entering vertebrate host phagocytes, Leishmania differentiate into intracellular amastigotes, the form that is ultimately transmitted back to the vector to complete the life cycle. Although environmental conditions that induce these cellular transitions are well-established, molecular mechanisms governing Leishmania morphologic differentiation in response to these cues remain largely uncharacterized. Previous studies indicate a key role for HSP83 in both promastigote metacyclogenesis and amastigote differentiation. To further elucidate HSP83 functions in the Leishmania lifecycle, we examined the biological impact of experimentally elevating HSP83 gene expression in Leishmania. Significantly, HSP83 overexpression was associated with altered metacyclic morphology, increased protein kinase A (PKA) activity and decreased expression of the Leishmania major surface protease, GP63. Corroborating these findings, overexpression of the L. amazonensis PKA catalytic subunit resulted in a largely similar phenotype. Our findings demonstrate for the first time in Leishmania, a functional link between HSP83 and PKA in the control of Leishmania gene expression, replication and morphogenesis.

Selective Functionalization of Arene C(sp2)-H Bonds by Gold Catalysis: The Role of Carbene Substituents.

The complete regioselective incorporation of carbene units to nonactivated arene rings has been achieved employing gold(I) catalysts bearing alkoxydiaminophosphine ligands, with readily available, nonelaborated ethyl 2-phenyldiazoacetate as the carbene source. These results are in contrast with the scarce precedents which required highly elaborated diazo substrates. Density functional theory (DFT) calculations have revealed the important role of the R group in the C(R)CO2Et fragment, which dramatically affects the energy profile of this transformation.

Dicoordinate Au(I)-Ethylene Complexes as Hydroamination Catalysts.

A series of gold(I)-ethylene π-complexes containing a family of bulky phosphine ligands has been prepared. The use of these sterically congested ligands is crucial to stabilize the gold(I)-ethylene bond and prevent decomposition, boosting up their catalytic performance in the highly underexplored hydroamination of ethylene. The precatalysts bearing the most sterically demanding phosphines showed the best results reaching full conversion to the hydroaminated products under notably mild conditions (1 bar of ethylene pressure at 60 °C). Kinetic analysis together with density functional theory calculations revealed that the assistance of a second molecule of the nucleophile as a proton shuttle is preferred even when using an extremely congested cavity-shaped Au(I) complex. In addition, we have measured a strong primary kinetic isotopic effect that is consistent with the involvement of X-H bond-breaking events in the protodeauration turnover-limiting step.

A dicoordinate gold(I)-ethylene complex.

The use of the exceptionally bulky tris-2-(4,4'-di-tert-butylbiphenylyl)phosphine ligand allows the isolation and complete characterization of the first dicoordinate gold(I)-ethylene adduct, filling a missing fundamental piece on the organometallic chemistry of gold. Besides, the bonding situation of this species has been investigated by means of state-of-the-art Density Functional Theory (DFT) calculations indicating that π-backdonation plays a minor role compared with tricoordinate analogues.

Differential responses of dwarf cashew clones to salinity are associated to osmotic adjustment mechanisms and enzymatic antioxidative defense.

This study evaluate growth, gas exchange, solute accumulation and activity of antioxidant enzymes in dwarf cashew clones subjected to salinity. Shoot dry mass reduced 26.8% (CCP06) and 41.2% (BRS189) at 16 dS m-1, concerning control. For net photosynthesis, CCP06 and BRS189 presented 69.8% and 34.7% of reduction, respectively. Na+ and Cl- contents increased in leaves and roots, in both clones, although CCP06 leaves presented Na+ concentrations lower than those of BRS189, the first one was the clone that the most accumulated such toxic ion, whereas K+ content remained almost unchanged for both clones. Soluble N-amino was the organic solute that more varied with salinity in cashew seedlings. Salt stress increased the activity of superoxide dismutase in both clones, mainly 16 dS m-1 treatment. Additionally, salinity promoted increases in ascorbate and guaiacol peroxidase activities, and the last enzyme was the main involved in H2O2 removal. Despite the reductions in growth and gas exchange, dwarf cashew seedlings of both clones presented an osmotic adjustment mechanism, and an efficient enzymatic antioxidant system that were able to attenuate the salt and oxidative stress, respectively. Our research suggested that BRS189 clone is more tolerant to salt stress than CCP06.

Reductive C-C Coupling from Molecular Au(I) Hydrocarbyl Complexes: A Mechanistic Study.

Organometallic gold complexes are used in a range of catalytic reactions, and they often serve as catalyst precursors that mediate C-C bond formation. In this study, we investigate C-C coupling to form ethane from various phosphine-ligated gem-digold(I) methyl complexes including [Au2(µ-CH3)(PMe2Ar')2][NTf2], [Au2(µ-CH3)(XPhos)2][NTf2], and [Au2(µ-CH3)(tBuXPhos)2][NTf2] {Ar' = C6H3-2,6-(C6H3-2,6-Me)2, C6H3-2,6-(C6H2-2,4,6-Me)2, C6H3-2,6-(C6H3-2,6-iPr)2, or C6H3-2,6-(C6H2-2,4,6-iPr)2; XPhos = 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl; tBuXPhos = 2-di-tert-butylphosphino-2',4',6'-triisopropylbiphenyl; NTf2 = bis(trifluoromethyl sulfonylimide)}. The gem-digold methyl complexes are synthesized through reaction between Au(CH3)L and Au(L)(NTf2) {L = phosphines listed above}. For [Au2(µ-CH3)(XPhos)2][NTf2] and [Au2(µ-CH3)(tBuXPhos)2][NTf2], solid-state X-ray structures have been elucidated. The rate of ethane formation from [Au2(µ-CH3)(PMe2Ar')2][NTf2] increases as the steric bulk of the phosphine substituent Ar' decreases. Monitoring the rate of ethane elimination reactions by multinuclear NMR spectroscopy provides evidence for a second-order dependence on the gem-digold methyl complexes. Using experimental and computational evidence, it is proposed that the mechanism of C-C coupling likely involves (1) cleavage of [Au2(µ-CH3)(PMe2Ar')2][NTf2] to form Au(PR2Ar')(NTf2) and Au(CH3)(PMe2Ar'), (2) phosphine migration from a second equivalent of [Au2(µ-CH3)(PMe2Ar')2][NTf2] aided by binding of the Lewis acidic [Au(PMe2Ar')]+, formed in step 1, to produce [Au2(CH3)(PMe2Ar')][NTf2] and [Au2(PMe2Ar')]+, and (3) recombination of [Au2(CH3)(PMe2Ar')][NTf2] and Au(CH3)(PMe2Ar') to eliminate ethane.

Alkoxydiaminophosphine Ligands as Surrogates of NHCs in Copper Catalysis.

A family of phosphine ligands containing a five-membered ring similar to the popular N-heterocyclic carbene ligands and an alkoxy third substituent has been developed. These alkoxydiaminophosphine ligands (ADAP) can be generated in one pot and reacted with a copper(I) source leading to the high yield isolation of complexes [(ADAP)CuX]2 (X=Cl, Br). The dinuclear nature of these compounds has been established by means of X-ray studies and DOSY experiments. A screening of the catalytic properties of these complexes toward carbene-transfer reactions from diazocompounds to C-H bonds (alkane, arene), olefins or N-H bonds, as well as in CuAAC or nitrene transfer reactions have shown a performance at least similar, if not better, than their (NHC)CuCl analogues, opening a new window in copper catalysis with these readily tunable ADAP ligands.

A Versatile Approach to Access Trimetallic Complexes Based on Trisphosphinite Ligands.

A straightforward method for the preparation of trisphosphinite ligands in one step, using only commercially available reagents (1,1,1-tris(4-hydroxyphenyl)ethane and chlorophosphines) is described. We have made use of this approach to prepare a small family of four trisphosphinite ligands of formula [CH3C{(C6H4OR2)3], where R stands for Ph (1a), Xyl (1b, Xyl = 2,6-Me2-C6H3), iPr (1c), and Cy (1d). These polyfunctional phosphinites allowed us to investigate their coordination chemistry towards a range of late transition metal precursors. As such, we report here the isolation and full characterization of a number of Au(I), Ag(I), Cu(I), Ir(III), Rh(III) and Ru(II) homotrimetallic complexes, including the structural characterization by X-ray diffraction studies of six of these compounds. We have observed that the flexibility of these trisphosphinites enables a variety of conformations for the different trimetallic species.

Design and Synthesis of Fungal-Selective Resorcylate Aminopyrazole Hsp90 Inhibitors.

The molecular chaperone Hsp90, essential in all eukaryotes, plays a multifaceted role in promoting survival, virulence, and drug resistance across diverse pathogenic fungal species. The chaperone is also critically important, however, to the pathogen's human host, preventing the use of known clinical Hsp90 inhibitors in antifungal applications due to concomitant host toxicity issues. With the goal of developing Hsp90 inhibitors with acceptable therapeutic indices for the treatment of invasive fungal infections, we initiated a program to design and synthesize potent inhibitors with selective activity against fungal Hsp90 isoforms over their human counterparts. Building on our previously reported derivatization of resorcylate natural products to produce fungal-selective compounds, we have developed a series of synthetic aminopyrazole-substituted resorcylate amides with broad, potent, and fungal-selective Hsp90 inhibitory activity. Herein we describe the synthesis of this series, as well as biochemical structure-activity relationships driving selectivity for the Hsp90 isoforms expressed by Cryptococcus neoformans and Candida albicans, two pathogenic fungi of major clinical importance.

Disruption of the Putative Ribosome-Binding Motif of a Scaffold Protein Impairs Cytochrome c Oxidase Subunit Expression in Leishmania major.

During their parasitic life cycle, through sandflies and vertebrate hosts, Leishmania parasites confront strikingly different environments, including abrupt changes in pH and temperature, to which they must rapidly adapt. These adaptations include alterations in Leishmania gene expression, metabolism, and morphology, allowing them to thrive as promastigotes in the sandfly and as intracellular amastigotes in the vertebrate host. A critical aspect of Leishmania metabolic adaptation to these changes is maintenance of efficient mitochondrial function in the hostile vertebrate environment. Such functions, including generation of ATP, depend upon the expression of many mitochondrial proteins, including subunits of cytochrome c oxidase (COX). Significantly, under mammalian temperature conditions, expression of Leishmania major COX subunit IV (LmCOX4) and virulence are dependent upon two copies of LACK, a gene that encodes the ribosome-associated scaffold protein, LACK (Leishmania ortholog of RACK1 [receptor for activated C kinase]). Targeted replacement of an endogenous LACK copy with a putative ribosome-binding motif-disrupted variant (LACKR34D35G36→LACKD34D35E36) resulted in thermosensitive parasites that showed diminished LmCOX4 expression, mitochondrial fitness, and replication in macrophages. Surprisingly, despite these phenotypes, LACKD34D35E36 associated with monosomes and polysomes and showed no major impairment of global protein synthesis. Collectively, these data suggest that wild-type (WT) LACK orchestrates robust LmCOX4 expression and mitochondrial fitness to ensure parasite virulence, via optimized functional interactions with the ribosome.IMPORTANCELeishmania parasites are trypanosomatid protozoans that persist in infected human hosts to cause a spectrum of pathologies, from cutaneous and mucocutaneous manifestations to visceral leishmaniasis caused by Leishmania donovani The latter is usually fatal if not treated. Persistence of L. major in the mammalian host depends upon maintaining gene-regulatory programs to support essential parasite metabolic functions. These include expression and assembly of mitochondrial L. major cytochrome c oxidase (LmCOX) subunits, important for Leishmania ATP production. Significantly, under mammalian conditions, WT levels of LmCOX subunits require threshold levels of the Leishmania ribosome-associated scaffold protein, LACK. Unexpectedly, we find that although disruption of LACK's putative ribosome-binding motif does not grossly perturb ribosome association or global protein synthesis, it nonetheless impairs COX subunit expression, mitochondrial function, and virulence. Our data indicate that the quality of LACK's interaction with Leishmania ribosomes is critical for LmCOX subunit expression and parasite mitochondrial function in the mammalian host. Collectively, these findings validate LACK's ribosomal interactions as a potential therapeutic target.

Structural basis for species-selective targeting of Hsp90 in a pathogenic fungus.

New strategies are needed to counter the escalating threat posed by drug-resistant fungi. The molecular chaperone Hsp90 affords a promising target because it supports survival, virulence and drug-resistance across diverse pathogens. Inhibitors of human Hsp90 under development as anticancer therapeutics, however, exert host toxicities that preclude their use as antifungals. Seeking a route to species-selectivity, we investigate the nucleotide-binding domain (NBD) of Hsp90 from the most common human fungal pathogen, Candida albicans. Here we report structures for this NBD alone, in complex with ADP or in complex with known Hsp90 inhibitors. Encouraged by the conformational flexibility revealed by these structures, we synthesize an inhibitor with >25-fold binding-selectivity for fungal Hsp90 NBD. Comparing co-crystals occupied by this probe vs. anticancer Hsp90 inhibitors revealed major, previously unreported conformational rearrangements. These insights and our probe's species-selectivity in culture support the feasibility of targeting Hsp90 as a promising antifungal strategy.

Distinctive regulatory properties of pyruvate kinase 1 from Aedes aegypti mosquitoes.

Female Aedes aegypti mosquitoes are vectors of arboviruses that cause diseases of public health significance. The discovery of new metabolic targets is crucial for improving mosquito control strategies. We recently demonstrated that glucose oxidation supports ammonia detoxification in A. aegypti. Pyruvate kinase (PK, EC 2.7.1.40) catalyzes the last step of the glycolytic pathway. In most organisms, one or more allosteric effectors control PK activity. However, the kinetic properties and structure of PK in mosquitoes have not been previously reported. In this study, two alternatively spliced mRNA variants (AaPK1 and AaPK2) that code for PKs were identified in the A. aegypti genome. The AaPK1 mRNA variant, which encodes a 529 amino acid protein with an estimated molecular weight of ∼57 kDa, was cloned. The protein was expressed in Escherichia coli and purified. The AaPK1 kinetic properties were identified. The recombinant protein was also crystallized and its 3D structure determined. We found that alanine, glutamine, proline, serine and fructose-1-phosphate displayed a classic allosteric activation on AaPK1. Ribulose-5-phosphate acted as an allosteric inhibitor of AaPK1 but its inhibitory effect was reversed by alanine, glutamine, proline and serine. Additionally, the allosteric activation of AaPK1 by amino acids was weakened by fructose-1,6-bisphosphate, whereas the allosteric activation of AaPK1 by alanine and serine was diminished by glucose-6-phosphate. The AaPK1 structure shows the presence of fructose-1,6-bisphosphate in the allosteric site. Together, our results reveal that specific amino acids and phosphorylated sugars tightly regulate conformational dynamics and catalytic changes of AaPK1. The distinctive AaPK1 allosteric properties support a complex role for this enzyme within mosquito metabolism.

Repurposing drugs to target the malaria parasite unfolding protein response.

Drug resistant Plasmodium falciparum parasites represent a major obstacle in our efforts to control malaria, a deadly vector borne infectious disease. This situation creates an urgent need to find and validate new drug targets to contain the spread of the disease. Several genes associated with the unfolded protein response (UPR) including Glucose-regulated Protein 78 kDa (GRP78, also known as BiP) have been deemed potential drug targets. We explored the drug target potential of GRP78, a molecular chaperone that is a regulator of the UPR, for the treatment of P. falciparum parasite infection. By screening repurposed chaperone inhibitors that are anticancer agents, we showed that GRP78 inhibition is lethal to drug-sensitive and -resistant P. falciparum parasite strains in vitro. We correlated the antiplasmodial activity of the inhibitors with their ability to bind the malaria chaperone, by characterizing their binding to recombinant parasite GRP78. Furthermore, we determined the crystal structure of the ATP binding domain of P. falciparum GRP78 with ADP and identified structural features unique to the parasite. These data suggest that P. falciparum GRP78 can be a valid drug target and that its structural differences to human GRP78 emphasize potential to generate parasite specific compounds.

Structure-activity relationship of new antimalarial 1-aryl-3-susbtituted propanol derivatives: Synthesis, preliminary toxicity profiling, parasite life cycle stage studies, target exploration, and targeted delivery.

Design, synthesis, structure-activity relationship, cytotoxicity studies, in silico drug-likeness, genotoxicity screening, and in vivo studies of new 1-aryl-3-substituted propanol derivatives led to the identification of nine compounds with promising in vitro (55, 56, 61, 64, 66, and 70-73) and in vivo (66 and 72) antimalarial profiles against Plasmodium falciparum and Plasmodium berghei. Compounds 55, 56, 61, 64, 66 and 70-73 exhibited potent antiplasmodial activity against chloroquine-resistant strain FCR-3 (IC50s < 0.28 µM), and compounds 55, 56, 64, 70, 71, and 72 showed potent biological activity in chloroquine-sensitive and multidrug-resistant strains (IC50s < 0.7 µM for 3D7, D6, FCR-3 and C235). All of these compounds share appropriate drug-likeness profiles and adequate selectivity indexes (77 < SI < 184) as well as lack genotoxicity. In vivo efficacy tests in a mouse model showed compounds 66 and 72 to be promising candidates as they exhibited significant parasitemia reductions of 96.4% and 80.4%, respectively. Additional studies such as liver stage and sporogony inhibition, target exploration of heat shock protein 90 of P. falciparum, targeted delivery by immunoliposomes, and enantiomer characterization were performed and strongly reinforce the hypothesis of 1-aryl-3-substituted propanol derivatives as promising antimalarial compounds.

Putative role of glutamine in the activation of CBL/CIPK signalling pathways during salt stress in sorghum.

The salt overly sensitive (SOS) pathway is the only mechanism known for Na+ extrusion in plant cells. SOS pathway activation involves Ca2+-sensing proteins, such as calcineurin B-like (CBL) proteins, and CBL-interacting protein kinases (CIPKs). In this signalling mechanism, a transit increase in cytosolic Ca2+ concentration triggered by Na+ accumulation is perceived by CBL (also known as SOS3). Afterward, SOS3 physically interacts with a CIPK (also known as SOS2), forming the SOS2/SOS3 complex, which can regulate the number downstream targets, controlling ionic homeostasis. For instance, the SOS2/SOS3 complex phosphorylates and activates the SOS1 plasmalemma protein, which is a Na+/H+ antiporter that extrudes Na+ out of the cell. The CBL-CIPK networking system displays specificity, complexity and diversity, constituting a critical response against salt stress and other abiotic stresses. In a study reported in the journal Plant and Cell Physiology, we showed that NH4+ induces the robust activation of transporters for Na+ homeostasis in root cells, especially the SOS1 antiporter and plasma membrane H+-ATPase, differently than does NO3-. Despite some studies having shown that external NH4+ ameliorates salt-induced effects on ionic homeostasis, there is no evidence that NH4+ per se or some product of its assimilation is responsible for these responses. Here, we speculate about the signalling role behind glutamine in CBL-CIPK modulation, which could effectively activate the SOS pathway in NH4+-fed stressed plants.

Identification and characterization of the antiplasmodial activity of Hsp90 inhibitors.

BACKGROUND: The recent reduction in mortality due to malaria is being threatened by the appearance of Plasmodium falciparum parasites that are resistant to artemisinin in Southeast Asia. To limit the impact of resistant parasites and their spread across the world, there is a need to validate anti-malarial drug targets and identify new leads that will serve as foundations for future drug development programmes targeting malaria. Towards that end, the antiplasmodial potential of several Hsp90 inhibitors was characterized. Because, the Hsp90 chaperone has been suggested as a good drug target against multiple parasitic infections including malaria. RESULTS: Chemically diverse sets of Hsp90 inhibitors, evaluated in clinical trials as anti-cancer agents, were tested against the malaria parasite. Most of the compounds showed strong antiplasmodial activity in growth inhibition assays against chloroquine sensitive and resistant strains. There was a good agreement between the compound in vitro anti-parasitic activity and their affinity against the Plasmodium chaperone. The two most potent Hsp90 inhibitors also showed cytocidal activity against two P. falciparum strains. Their antiplasmodial activity affected all parasite forms during the malaria blood cycle. However, the compounds activity against the parasite showed no synergy when combined with anti-malarial drugs, like chloroquine or DHA. DISCUSSION: The Hsp90 inhibitors anti-parasitic activity correlates with their affinity to their predicted target the P. falciparum chaperone Hsp90. However, the most effective compounds also showed high affinity for a close homologue, Grp94. This association points to a mode of action for Hsp90 inhibitors that correlate compound efficacy with multi-target engagement. Besides their ability to limit parasite replication, two compounds also significantly impacted P. falciparum viability in vitro. Finally, a structural analysis suggests that the best hit represents a promising scaffold to develop parasite specific leads according. CONCLUSION: The results shown that Hsp90 inhibitors are lethal against the malaria parasite. The correlation between biochemical and in vitro data strongly supports Hsp90 as a drug target against the malaria parasite. Furthermore, at least one Hsp90 inhibitor developed as anticancer therapeutics could serve as starting point to generate P. falciparum-specific lead compounds.

Antiplasmodial activity of targeted zinc(II)-dipicolylamine complexes.

This study measured the antiplasmodial activity of nine zinc-dipicolylamine (ZnDPA) complexes against three strains of Plasmodium falciparum, the causative parasite of malaria. Growth inhibition assays showed significant activity against all tested strains, with 50% inhibitory concentrations between 5 and 600nM and almost no toxic effect against host cells including healthy red blood cells. Fluorescence microscopy studies with a green-fluorescent ZnDPA probe showed selective targeting of infected red blood cells. The results suggest that ZnDPA coordination complexes are promising antiplasmodial agents with potential for targeted malaria treatment.

Integrative Control Between Proton Pumps and SOS1 Antiporters in Roots is Crucial for Maintaining Low Na+ Accumulation and Salt Tolerance in Ammonium-Supplied Sorghum bicolor.

An effective strategy for re-establishing K+ and Na+ homeostasis is a challenge for the improvement of plant performance in saline soil. Specifically, attempts to understand the mechanisms of Na+ extrusion from plant cells, the control of Na+ loading in the xylem and the partitioning of the accumulated Na+ between different plant organs are ongoing. Our goal was to provide insight into how an external nitrogen source affects Na+ accumulation in Sorghum bicolor under saline conditions. The NH4+ supply improved the salt tolerance of the plant by restricting Na+ accumulation and improving the K+/Na+ homeostasis in shoots, which was consistent with the high activity and expression of Na+/H+ antiporters and proton pumps in the plasma membrane and vacuoles in the roots, resulting in low Na+ loading in the xylem. Conversely, although NO3--grown plants had exclusion and sequestration mechanisms for Na+, these responses were not sufficient to reduce Na+ accumulation. In conclusion, NH4+ acts as an efficient signal to activate co-ordinately responses involved in the regulation of Na+ homeostasis in sorghum plants under salt stress, which leads to salt tolerance.

Correction: Probing the ATP Site of GRP78 with Nucleotide Triphosphate Analogs.

[This corrects the article DOI: 10.1371/journal.pone.0154862.].