Discordant skeletal muscle gene and protein responses to exercise.

The ability of skeletal muscle to adapt to repeated contractile stimuli is one of the most intriguing aspects of physiology. The molecular bases underpinning these adaptations involve increased protein activity and/or expression, mediated by an array of pre- and post-transcriptional processes, as well as translational and post-translational control. A longstanding dogma assumes a direct relationship between exercise-induced increases in mRNA levels and subsequent changes in the abundance of the proteins they encode. Drawing on the results of recent studies, we dissect and question the common assumption of a direct relationship between changes in the skeletal muscle transcriptome and proteome induced by repeated muscle contractions (e.g., exercise).

Small peptides: could they have a big role in metabolism and the response to exercise?

Exercise is a powerful non-pharmacological intervention for the treatment and prevention of numerous chronic diseases. Contracting skeletal muscles provoke widespread perturbations in numerous cells, tissues and organs, which stimulate multiple integrated adaptations that ultimately contribute to the many health benefits associated with regular exercise. Despite much research, the molecular mechanisms driving such changes are not completely resolved. Technological advancements beginning in the early 1960s have opened new avenues to explore the mechanisms responsible for the many beneficial adaptations to exercise. This has led to increased research into the role of small peptides (<100 amino acids) and mitochondrially derived peptides in metabolism and disease, including those coded within small open reading frames (sORFs; coding sequences that encode small peptides). Recently, it has been hypothesized that sORF-encoded mitochondrially derived peptides and other small peptides play significant roles as exercise-sensitive peptides in exercise-induced physiological adaptation. In this review, we highlight the discovery of mitochondrially derived peptides and newly discovered small peptides involved in metabolism, with a specific emphasis on their functions in exercise-induced adaptations and the prevention of metabolic diseases. In light of the few studies available, we also present data on how both single exercise sessions and exercise training affect expression of sORF-encoded mitochondrially derived peptides. Finally, we outline numerous research questions that await investigation regarding the roles of mitochondrially derived peptides in metabolism and prevention of various diseases, in addition to their roles in exercise-induced physiological adaptations, for future studies.

Dose-fractionation studies of a Plasmodium phosphatidylinositol 4-kinase inhibitor in a humanized mouse model of malaria.

UCT594 is a 2-aminopyrazine carboxylic acid Plasmodium phosphatidylinositol 4-kinase inhibitor with potent asexual blood-stage activity, the potential for interrupting transmission, as well as liver-stage activities. Herein, we investigated pharmacokinetic/pharmacodynamic (PK/PD) relationships relative to blood-stage activity toward predicting the human dose. Dose-fractionation studies were conducted in the Plasmodium falciparum NSG mouse model to determine the PK/PD indices of UCT594, using the in vivo minimum parasiticidal concentration as a threshold. UCT594 demonstrated concentration-dependent killing in the P. falciparum-infected NSG mouse model. Using this data and the preclinical pharmacokinetic data led to a low predicted human dose of <50 mg. This makes UCT594 an attractive potential antimalarial drug.

Total Synthesis of Tosyl-Samroiyotmycin A and Its Biological Profiling.

A total synthesis of the enantiopure syn,syn-tosyl-samroiyotmycin A, a C2-symmetric 20-membered antimalarial macrodiolide with syn,syn-configuration of the 8,24-dihydroxy-9,25-dimethyl units and it's enantiopure anti,anti-derivative is described. The synthesis was accomplished utilizing a linear approach in 7 steps and 3 % overall yield via a sequence of diastereoselective methylation of SuperQuat oxazolidinone auxiliary, cross metathesis and Yamaguchi macrolactonization of fully functionalized seco-acids. By a similar approach we gained access to several samroiyotmycin analogues and precursors. Antimalarial activity was tested on multi-resistant (K1) and sensitive (Nf54) P. falciparum strains providing insight into structure activity relationships. Both tosyl-oxazol unit as well as the syn-configuration of the two contiguous stereogenic centers turned out to be beneficial for antiplasmodial activity. For instance, syn,syn-tosyl-samroiyotmycin A showed 3.4 times higher activities than the "tosyl-free" natural product.

Another decade of antimalarial drug discovery: New targets, tools and molecules.

Malaria remains a devastating but preventable infectious disease that disproportionately affects the African continent. Emerging resistance to current frontline therapies means that not only are new treatments urgently required, but also novel validated antimalarial targets to circumvent cross-resistance. Fortunately, tremendous efforts have been made by the global drug discovery community over the past decade. In this chapter, we will highlight some of the antimalarial drug discovery and development programmes currently underway across the globe, charting progress in the identification of new targets and the development of new classes of drugs to prosecute them. These efforts have been complemented by the development of valuable tools to accelerate target validation such as the NOD scid gamma (NSG) humanized mouse efficacy model and progress in predictive modelling and open-source software. Among the medicinal chemistry programmes that have been conducted over the past decade are those targeting Plasmodium falciparum ATPase4 (ATP4) and acetyl-CoA synthetase (AcAS) as well as proteins disrupting parasite protein translation such as the aminoacyl-tRNA synthetases (aaRSs) and eukaryotic elongation factor 2 (eEF2). The benefits and challenges of targeting Plasmodium kinases will be examined, with a focus on Plasmodium cyclic GMP-dependent protein kinase (PKG), cyclin-dependent-like protein kinase 3 (CLK3) and phosphatidylinositol 4-kinase (PI4K). The chapter concludes with a survey of incipient drug discovery centres in Africa and acknowledges the value of recent international meetings in galvanizing and uniting the antimalarial drug discovery community.

Transcription Factor Movement and Exercise-Induced Mitochondrial Biogenesis in Human Skeletal Muscle: Current Knowledge and Future Perspectives.

In response to exercise, the oxidative capacity of mitochondria within skeletal muscle increases through the coordinated expression of mitochondrial proteins in a process termed mitochondrial biogenesis. Controlling the expression of mitochondrial proteins are transcription factors-a group of proteins that regulate messenger RNA transcription from DNA in the nucleus and mitochondria. To fulfil other functions or to limit gene expression, transcription factors are often localised away from DNA to different subcellular compartments and undergo rapid movement or accumulation only when required. Although many transcription factors involved in exercise-induced mitochondrial biogenesis have been identified, numerous conflicting findings and gaps exist within our knowledge of their subcellular movement. This review aims to summarise and provide a critical analysis of the published literature regarding the exercise-induced movement of transcription factors involved in mitochondria biogenesis in skeletal muscle.

The in Vitro Antiplasmodial and Antiproliferative Activity of New Ferrocene-Based α-Aminocresols Targeting Hemozoin Inhibition and DNA Interaction.

The conjugation of organometallic complexes to known bioactive organic frameworks is a proven strategy revered for devising new drug molecules with novel modes of action. This approach holds great promise for the generation of potent drug leads in the quest for therapeutic chemotypes with the potential to overcome the development of clinical resistance. Herein, we present the in vitro antiplasmodial and antiproliferative investigation of ferrocenyl α-aminocresol conjugates assembled by amalgamation of the organometallic ferrocene unit and an α-aminocresol scaffold possessing antimalarial activity. The compounds pursued in the study exhibited higher toxicity towards the chemosensitive (3D7) and -resistant (Dd2) strains of the Plasmodium falciparum parasite than to the human HCC70 triple-negative breast cancer cell line. Indication of cross-resistance was absent for the compounds evaluated against the multi-resistant Dd2 strain. Structure-activity analysis revealed that the phenolic hydroxy group and rotatable σ bond between the α-carbon and NH group of the α-amino-o-cresol skeleton are crucial for the biological activity of the compounds. Spectrophotometric techniques and in silico docking simulations performed on selected derivatives suggest that the compounds show a dual mode of action involving hemozoin inhibition and DNA interaction via minor-groove binding. Lastly, compound 9 a, identified as a possible lead, exhibited preferential binding for the plasmodial DNA isolated from 3D7 P. falciparum trophozoites over the mammalian calf thymus DNA, thereby substantiating the enhanced antiplasmodial activity of the compounds. The presented research demonstrates the strategy of incorporating organometallic complexes into known biologically active organic scaffolds as a viable avenue to fashion novel multimodal compounds with potential to counter the development drug resistance.

Synthesis and evaluation of the performance of a small molecule library based on diverse tropane-related scaffolds.

A unified synthetic approach was developed that enabled the synthesis of diverse tropane-related scaffolds. The key intermediates that were exploited were cycloadducts formed by reaction between 3-hydroxy-pyridinium salts and vinyl sulfones or sulfonamides. The diverse tropane-related scaffolds were formed by addition of substituents to, cyclisation reactions of, and fusion of additional ring(s) to the key bicyclic intermediates. A set of 53 screening compounds was designed, synthesised and evaluated in order to determine the biological relevance of the scaffolds accessible using the synthetic approach. Two inhibitors of Hedgehog signalling, and four compounds with weak activity against the parasite P. falciparum, were discovered. Three of the active compounds may be considered to be indotropane or pyrrotropane pseudo natural products in which a tropane is fused with a fragment from another natural product class. It was concluded that the unified synthetic approach had yielded diverse scaffolds suitable for the design of performance-diverse screening libraries.

Investigating Sulfoxide-to-Sulfone Conversion as a Prodrug Strategy for a Phosphatidylinositol 4-Kinase Inhibitor in a Humanized Mouse Model of Malaria.

The in vivo antimalarial efficacies of two phosphatidylinositol 4-kinase (PI4K) inhibitors, a 3,5-diaryl-2-aminopyrazine sulfoxide and its corresponding sulfone metabolite, were evaluated in the NOD-scid IL2Rγnull (NSG) murine malaria disease model of Plasmodium falciparum infection. We hypothesized that the sulfoxide would serve as a more soluble prodrug for the sulfone, which would lead to improved drug exposure with oral dosing. Both compounds had similar efficacy (90% effective dose [ED90], 0.1 mg kg-1 of body weight) across a quadruple-dose regimen. Pharmacokinetic profiling revealed rapid sulfoxide clearance via conversion to sulfone, with sulfone identified as the major active metabolite. When the sulfoxide was dosed, the exposure of the sulfone achieved was as much as 2.9-fold higher than when the sulfone was directly dosed, thereby demonstrating that the sulfoxide served as an effective prodrug for the treatment of malaria.

UCT943, a Next-Generation Plasmodium falciparum PI4K Inhibitor Preclinical Candidate for the Treatment of Malaria.

The 2-aminopyridine MMV048 was the first drug candidate inhibiting Plasmodium phosphatidylinositol 4-kinase (PI4K), a novel drug target for malaria, to enter clinical development. In an effort to identify the next generation of PI4K inhibitors, the series was optimized to improve properties such as solubility and antiplasmodial potency across the parasite life cycle, leading to the 2-aminopyrazine UCT943. The compound displayed higher asexual blood stage, transmission-blocking, and liver stage activities than MMV048 and was more potent against resistant Plasmodium falciparum and Plasmodium vivax clinical isolates. Excellent in vitro antiplasmodial activity translated into high efficacy in Plasmodium berghei and humanized P. falciparum NOD-scid IL-2Rγ null mouse models. The high passive permeability and high aqueous solubility of UCT943, combined with low to moderate in vivo intrinsic clearance, resulted in sustained exposure and high bioavailability in preclinical species. In addition, the predicted human dose for a curative single administration using monkey and dog pharmacokinetics was low, ranging from 50 to 80 mg. As a next-generation Plasmodium PI4K inhibitor, UCT943, based on the combined preclinical data, has the potential to form part of a single-exposure radical cure and prophylaxis (SERCaP) to treat, prevent, and block the transmission of malaria.

Adamantane amine-linked chloroquinoline derivatives as chloroquine resistance modulating agents in Plasmodium falciparum.

Previously we have shown that pentacycloundecylamine-chloroquinoline (PCU-CQ) conjugates possess significant chemosensitizing abilities and can circumvent the resistance associated with chloroquine (CQ) resistant plasmodia. In order to further explore structurally related polycyclic compounds as reversed CQ agents we synthesized a series of eight aza-adamantanol (1-4) and adamantane-imine (5-8) CQ conjugates. All conjugates showed limited cytotoxicity against CHO cells (IC50 > 37 µM). Compounds 1, 2 and 5 were highly active (K1 IC50 < 100 nM) exhibiting a 3-4-fold increase in antiplasmodial activity against CQ resistant strain K1 compared to CQ. Reduced cross-resistance (resistance index, RI: 2-4.3) relative to CQ (RI = 38) was also observed for these compounds. Compound 1 which showed an 18-fold enhancement at retaining its activity against the K1 strain compared to CQ is a promising candidate to substitute CQ in P. falciparum resistant malaria.

Synthesis of five libraries of 6,5-fused heterocycles to establish the importance of the heterocyclic core for antiplasmodial activity.

Research has indicated that N-myristoyl transferase, an enzyme that catalyzes the addition of a myristate group to the N-terminal glycine residues of proteins, is involved in the myristoylation of more than 100 proteins. Genetic knockdown of the enzyme proved detrimental for the viability of the parasite P. knowlesi. A crystal structure of P. vivax N-myristoyl transferase (pvNMT), containing a 3-methyl benzofuran ligand has made it possible to assess key amino acid residue-ligand interactions. We synthesized five libraries of 6,5-fused heterocycles to establish the importance of the heterocycles as core scaffolds, as well as introduced various aromatic amides and esters to determine which carbonylic group affects the potency of each heterocyclic antiplasmodial agent.

Polycyclic amines as chloroquine resistance modulating agents in Plasmodium falciparum.

Pentacycloundecylamines (PCUs) and adamantane amines, such as NGP1-01 (1) and amantadine, have shown significant channel blocking activities. They are postulated to act as chemosensitizers and circumvent the resistance of the plasmodia parasite against chloroquine (CQ) by inhibiting the p-glycoprotein efflux pump and enabling the accumulation of CQ inside the parasite digestive vacuole. Twelve polycyclic amines containing either a PCU or adamantane amine moiety conjugated to different aromatic functionalities through various tethered linkers were selected based on their channel blocking abilities and evaluated as potential chemosensitizers. Compounds 2, 4, 5 and 10 showed significant voltage-gated calcium channel (VGCC) blocking ability (IC50=0.27-35 µM) and were able to alter the CQ IC50 in differing degrees (45-81%) in the multidrug resistant Plasmodium falciparum Dd2 isolate. Among them, the PCU-dansyl amine compound (4) displayed the best potential to act as a chemosensitizer against the Dd2 strain at a 1 µM concentration (RMI=0.19) while displaying moderate antiplasmodial activity (Dd2 IC50=6.25 µM) and low in vitro cytotoxicity against a mammalian cell line (CHO, IC50=119 µM). Compounds 2 and 10 also showed some promising chemosensitizing abilities (RMI=0.36 and 0.35 respectively). A direct correlation was found between the VGCC blocking ability of these polycyclic amines and their capacity to act as CQ resistance modulating agents.

Antiplasmodial activity, in vivo pharmacokinetics and anti-malarial efficacy evaluation of hydroxypyridinone hybrids in a mouse model.

BACKGROUND: During the erythrocytic stage in humans, malaria parasites digest haemoglobin of the host cell, and the toxic haem moiety crystallizes into haemozoin. Chloroquine acts by forming toxic complexes with haem molecules and interfering with their crystallization. In chloroquine-resistant strains, the drug is excluded from the site of action, which causes the parasites to accumulate less chloroquine in their acid food vacuoles than chloroquine-sensitive parasites. 3-Hydroxylpyridin-4-ones are known to chelate iron; hydroxypyridone-chloroquine (HPO-CQ) hybrids were synthesized in order to determine whether they can inhibit parasites proliferation in the parasitic digestive vacuole by withholding iron from plasmodial parasite metabolic pathway. METHODS: Two HPO-CQ hybrids were tested against Plasmodium falciparum chloroquine-sensitive (D10 and 3D7) and -resistant strains (Dd2 and K1). The pharmacokinetic properties of active compounds were determined using a mouse model and blood samples were collected at different time intervals and analysed using LC-MS/MS. For in vivo efficacy the mice were infected with Plasmodium berghei in a 4-day Peters' test. The parasitaemia was determined from day 3 and the course of the infection was followed by microscopic examination of stained blood films every 2-3 days until a rise in parasitaemia was observed in all test subjects. RESULTS: IC50 values of the two compounds for sensitive and resistant strains were 0.064 and 0.047 µM (compound 1), 0.041 and 0.122 µM (compound 2) and 0.505 and 0.463 µM (compound 1), 0.089 and 0.076 µM (compound 2), respectively. Pharmacokinetic evaluation of these compounds showed low oral bioavailability and this affected in vivo efficacy when compounds were dosed orally. However, when dosed intravenously compound 1 showed a clearance rate of 28 ml/min/kg, an apparent volume of distribution of 20 l/kg and a half-life of 4.3 h. A reduction in parasitaemia was observed when compound 1 was dosed intravenously for four consecutive days in P. berghei-infected mice. However, a rise in parasitaemia levels was observed on day 6 and on day 9 for chloroquine-treated mice. CONCLUSION: The hybrid compounds that were tested were able to reduce parasitaemia levels in P. berghei-infected mice when dosed intravenously, but parasites recrudesced 24 h after the administration of the least dose. Despite low oral bioavailability, the IV data obtained suggests that further structural modifications may lead to the identification of more HPO-CQ hybrids with improved pharmacokinetic properties and in vivo efficacy.

Pentacycloundecylamines and conjugates thereof as chemosensitizers and reversed chloroquine agents.

The control of malaria has been complicated by increased resistance of the malaria parasite to existing antimalarials such as chloroquine (CQ). Herein, we report the ability of NGP1-01, the prototype pentacycloundecylamine (PCU), to reverse CQ resistance (>50%) and act as a chemosensitizer. Based on this finding we set out to synthesize a small series of novel agents comprising of a PCU moiety as the reversal agent conjugated to a CQ-like aminoquinoline (AM) molecule and evaluate the potential of these PCU-AM derivatives as reversed CQ agents. PCU-AM derivatives 1-3 showed anti-plasmodial IC50 values in the ranges of 3.74-17.6 nM and 27.6-253.5 nM against CQ-sensitive (D10) and CQ-resistant strains (Dd2) of Plasmodium falciparum, respectively. Compound 1 presented with the best antiplasmodial activity at low nM concentrations against both strains and was found to be 5 fold more active against the resistant strain than CQ. Compound 1 can be considered as a lead compound to develop reversed CQ agents with improved pharmacodynamic and pharmacokinetic properties.

2,8-Disubstituted-1,5-naphthyridines as Dual Inhibitors of Plasmodium falciparum Phosphatidylinositol-4-kinase and Hemozoin Formation with In Vivo Efficacy.

Structure-activity relationship studies of 2,8-disubstituted-1,5-naphthyridines, previously reported as potent inhibitors of Plasmodium falciparum (Pf) phosphatidylinositol-4-kinase ß (PI4K), identified 1,5-naphthyridines with basic groups at 8-position, which retained Plasmodium PI4K inhibitory activity but switched primary mode of action to the host hemoglobin degradation pathway through inhibition of hemozoin formation. These compounds showed minimal off-target inhibitory activity against the human phosphoinositide kinases and MINK1 and MAP4K kinases, which were associated with the teratogenicity and testicular toxicity observed in rats for the PfPI4K inhibitor clinical candidate MMV390048. A representative compound from the series retained activity against field isolates and lab-raised drug-resistant strains of Pf. It was efficacious in the humanized NSG mouse malaria infection model at a single oral dose of 32 mg/kg. This compound was nonteratogenic in the zebrafish embryo model of teratogenicity and has a low predicted human dose, indicating that this series has the potential to deliver a preclinical candidate for malaria.

Antimalarial Imidazopyridines Incorporating an Intramolecular Hydrogen Bonding Motif: Medicinal Chemistry and Mechanistic Studies.

We previously identified a novel class of antimalarial benzimidazoles incorporating an intramolecular hydrogen bonding motif. The frontrunner of the series, analogue A, showed nanomolar activity against the chloroquine-sensitive NF54 and multi-drug-resistant K1 strains of Plasmodium falciparum (PfNF54 IC50 = 0.079 µM; PfK1 IC50 = 0.335 µM). Here, we describe a cell-based medicinal chemistry structure-activity relationship study using compound A as a basis. This effort led to the identification of novel antimalarial imidazopyridines with activities of <1 µM, favorable cytotoxicity profiles, and good physicochemical properties. Analogue 14 ( PfNF54 IC50 = 0.08 µM; PfK1 IC50 = 0.10 µM) was identified as the frontrunner of the series. Preliminary mode of action studies employing molecular docking, live-cell confocal microscopy, and a cellular heme fractionation assay revealed that 14 does not directly inhibit the conversion of heme to hemozoin, although it could be involved in other processes in the parasite's digestive vacuole.

A Water-Soluble Polymer-Lumefantrine Conjugate for the Intravenous Treatment of Severe Malaria.

Uncomplicated malaria is effectively treated with oral artemisinin-based combination therapy (ACT). Yet, there is an unmet clinical need for the intravenous treatment of the more fatal severe malaria. There is no combination intravenous therapy for uncomplicated due to the nonavailability of a water-soluble partner drug for the artemisinin, artesunate. The currently available treatment is a two-part regimen split into an intravenous artesunate followed by the conventional oral ACT . In a novel application of polymer therapeutics, the aqueous insoluble antimalarial lumefantrine is conjugated to a carrier polymer to create a new water-soluble chemical entity suitable for intravenous administration in a clinically relevant formulation . The conjugate is characterized by spectroscopic and analytical techniques, and the aqueous solubility of lumefantrine is determined to have increased by three orders of magnitude. Pharmacokinetic studies in mice indicate that there is a significant plasma release of lumefantrine and production its metabolite desbutyl-lumefantrine (area under the curve of metabolite is ≈10% that of the parent). In a Plasmodium falciparum malaria mouse model, parasitemia clearance is 50% higher than that of reference unconjugated lumefantrine. The polymer-lumefantrine shows potential for entering the clinic to meet the need for a one-course combination treatment for severe malaria.

Synthesis and evaluation of hybrid drugs for a potential HIV/AIDS-malaria combination therapy.

Malaria and HIV are among the most important global health problems of our time and together are responsible for approximately 3 million deaths annually. These two diseases overlap in many regions of the world including sub-Saharan Africa, Southeast Asia and South America, leading to a higher risk of co-infection. In this study, we generated and characterized hybrid molecules to target Plasmodium falciparum and HIV simultaneously for a potential HIV/malaria combination therapy. Hybrid molecules were synthesized by the covalent fusion of azidothymidine (AZT) with dihydroartemisinin (DHA), a tetraoxane or a 4-aminoquinoline derivative; and the small library was tested for antiviral and antimalarial activity. Our data suggests that compound 7 is the most potent molecule in vitro, with antiplasmodial activity comparable to that of DHA (IC(50)=26 nM, SI>3000), a moderate activity against HIV (IC(50)=2.9 µM; SI>35) and not toxic to HeLa cells at concentrations used in the assay (CC(50)>100 µM). Pharmacokinetics studies further revealed that compound 7 is metabolically unstable and is cleaved via O-dealkylation. These studies account for the lack of in vivo efficacy of compound 7 against the CQ-sensitive Plasmodium berghei N strain in mice, when administered orally at 20mg/kg.