A potent and selective reaction hijacking inhibitor of Plasmodium falciparum tyrosine tRNA synthetase exhibits single dose oral efficacy in vivo.

The Plasmodium falciparum cytoplasmic tyrosine tRNA synthetase (PfTyrRS) is an attractive drug target that is susceptible to reaction-hijacking by AMP-mimicking nucleoside sulfamates. We previously identified an exemplar pyrazolopyrimidine ribose sulfamate, ML901, as a potent reaction hijacking inhibitor of PfTyrRS. Here we examined the stage specificity of action of ML901, showing very good activity against the schizont stage, but lower trophozoite stage activity.

Reaction hijacking inhibition of Plasmodium falciparum asparagine tRNA synthetase.

Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold.

Reaction hijacking inhibition of asparagine tRNA synthetase.

Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold.

Dimeric Artesunate Glycerophosphocholine Conjugate Nano-Assemblies as Slow-Release Antimalarials to Overcome Kelch 13 Mutant Artemisinin Resistance.

Current best practice for the treatment of malaria relies on short half-life artemisinins that are failing against emerging Kelch 13 mutant parasite strains. Here, we introduce a liposome-like self-assembly of a dimeric artesunate glycerophosphocholine conjugate (dAPC-S) as an amphiphilic prodrug for the short-lived antimalarial drug, dihydroartemisinin (DHA), with enhanced killing of Kelch 13 mutant artemisinin-resistant parasites. Cryo-electron microscopy (cryoEM) images and the dynamic light scattering (DLS) technique show that dAPC-S typically exhibits a multilamellar liposomal structure with a size distribution similar to that of the liposomes generated using thin-film dispersion (dAPC-L).

Dimeric artesunate-choline conjugate micelles coated with hyaluronic acid as a stable, safe and potent alternative anti-malarial injection of artesunate.

Artesunate (ARS) is the only artemisinin-based intravenous drug approved for treatment of malaria in the clinic. ARS is rapidly metabolized in vivo to short lived (∼30-45 min) but fast acting, dihydroartemisinin (DHA). The short half-life of DHA necessitates multiple dose administration to circumvent the risk of recrudescence and development of artemisinin resistance.

Design of proteasome inhibitors with oral efficacy in vivo against and selectivity over the human proteasome.

The proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the 20S proteasome (20S) β5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of compared with a human cell line and exhibit high potency against field isolates of and They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity.

Artemisinin kills malaria parasites by damaging proteins and inhibiting the proteasome.

Artemisinin and its derivatives (collectively referred to as ARTs) rapidly reduce the parasite burden in Plasmodium falciparum infections, and antimalarial control is highly dependent on ART combination therapies (ACTs). Decreased sensitivity to ARTs is emerging, making it critically important to understand the mechanism of action of ARTs. Here we demonstrate that dihydroartemisinin (DHA), the clinically relevant ART, kills parasites via a two-pronged mechanism, causing protein damage, and compromising parasite proteasome function.

Substrate Locking Promotes Dimer-Dimer Docking of an Enzyme Antibiotic Target.

Protein dynamics manifested through structural flexibility play a central role in the function of biological molecules. Here we explore the substrate-mediated change in protein flexibility of an antibiotic target enzyme, Clostridium botulinum dihydrodipicolinate synthase. We demonstrate that the substrate, pyruvate, stabilizes the more active dimer-of-dimers or tetrameric form.

Comparison of untagged and his-tagged dihydrodipicolinate synthase from the enteric pathogen Vibrio cholerae.

Given the emergence of multi drug resistant Vibrio cholerae strains, there is an urgent need to characterize new anti-cholera targets. One such target is the enzyme dihydrodipicolinate synthase (DHDPS; EC 4.3.

Nanocrystallography measurements of early stage synthetic malaria pigment.

The recent availability of extremely intense, femtosecond X-ray free-electron laser (XFEL) sources has spurred the development of serial femtosecond nanocrystallography (SFX). Here, SFX is used to analyze nanoscale crystals of β-hematin, the synthetic form of hemozoin which is a waste by-product of the malaria parasite. This analysis reveals significant differences in β-hematin data collected during SFX and synchrotron crystallography experiments.

A Dynamic Stress Model Explains the Delayed Drug Effect in Artemisinin Treatment of Plasmodium falciparum.

Artemisinin resistance constitutes a major threat to the continued success of control programs for malaria, particularly in light of developing resistance to partner drugs. Improving our understanding of how artemisinin-based drugs act and how resistance manifests is essential for the optimization of dosing regimens and the development of strategies to prolong the life span of current first-line treatment options. Recent short-drug-pulse experiments have shown that the parasite killing rate depends not only on drug concentration but also the exposure time, challenging the standard pharmacokinetic-pharmacodynamic (PK-PD) paradigm in which the killing rate depends only on drug concentration.

Structural Determinants Defining the Allosteric Inhibition of an Essential Antibiotic Target.

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the lysine biosynthesis pathway of bacteria. The pathway can be regulated by feedback inhibition of DHDPS through the allosteric binding of the end product, lysine. The current dogma states that DHDPS from Gram-negative bacteria are inhibited by lysine but orthologs from Gram-positive species are not.

i-bodies, Human Single Domain Antibodies That Antagonize Chemokine Receptor CXCR4.

CXCR4 is a G protein-coupled receptor with excellent potential as a therapeutic target for a range of clinical conditions, including stem cell mobilization, cancer prognosis and treatment, fibrosis therapy, and HIV infection. We report here the development of a fully human single-domain antibody-like scaffold termed an "i-body," the engineering of which produces an i-body library possessing a long complementarity determining region binding loop, and the isolation and characterization of a panel of i-bodies with activity against human CXCR4. The CXCR4-specific i-bodies show antagonistic activity in a range of in vitro and in vivo assays, including inhibition of HIV infection, cell migration, and leukocyte recruitment but, importantly, not the mobilization of hematopoietic stem cells.

Haemoglobin degradation underpins the sensitivity of early ring stage Plasmodium falciparum to artemisinins.

Current first-line artemisinin antimalarials are threatened by the emergence of resistant Plasmodium falciparum. Decreased sensitivity is evident in the initial (early ring) stage of intraerythrocytic development, meaning that it is crucial to understand the action of artemisinins at this stage. Here, we examined the roles of iron (Fe) ions and haem in artemisinin activation in early rings using Fe ion chelators and a specific haemoglobinase inhibitor (E64d).

Targeting the cell stress response of Plasmodium falciparum to overcome artemisinin resistance.

Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein.

Optimal assay design for determining the in vitro sensitivity of ring stage Plasmodium falciparum to artemisinins.

Recent reports demonstrate that failure of artemisinin-based antimalarial therapies is associated with an altered response of early blood stage Plasmodium falciparum. This has led to increased interest in the use of pulse assays that mimic clinical drug exposure for analysing artemisinin sensitivity of highly synchronised ring stage parasites. We report a methodology for the reliable execution of drug pulse assays and detail a synchronisation strategy that produces well-defined tightly synchronised ring stage cultures in a convenient time-frame.

Identification of the bona fide DHDPS from a common plant pathogen.

Agrobacterium tumefaciens is a Gram-negative soil-borne bacterium that causes Crown Gall disease in many economically important crops. The absence of a suitable chemical treatment means there is a need to discover new anti-Crown Gall agents and also characterize bona fide drug targets. One such target is dihydrodipicolinate synthase (DHDPS), a homo-tetrameric enzyme that catalyzes the committed step in the metabolic pathway yielding meso-diaminopimelate and lysine.

From knock-out phenotype to three-dimensional structure of a promising antibiotic target from Streptococcus pneumoniae.

Given the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobials targeting this pathogen and an equally urgent need to characterize new drug targets. A promising antibiotic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the rate-limiting step in lysine biosynthesis. In this study, we firstly show by gene knock out studies that S.

Cloning to crystallization of dihydrodipicolinate synthase from the intracellular pathogen Legionella pneumophila.

Dihydrodipicolinate synthase (DHDPS) catalyses the rate-limiting step in the biosynthesis of meso-diaminopimelate and lysine. Here, the cloning, expression, purification and crystallization of DHDPS from the intracellular pathogen Legionella pneumophila are described. Crystals grown in the presence of high-molecular-weight PEG precipitant and magnesium chloride were found to diffract beyond 1.

Defining the interaction of perforin with calcium and the phospholipid membrane.

Following its secretion from cytotoxic lymphocytes into the immune synapse, perforin binds to target cell membranes through its Ca(2+)-dependent C2 domain. Membrane-bound perforin then forms pores that allow passage of pro-apoptopic granzymes into the target cell. In the present study, structural and biochemical studies reveal that Ca(2+) binding triggers a conformational change in the C2 domain that permits four key hydrophobic residues to interact with the plasma membrane.

Disarming bacterial virulence through chemical inhibition of the DNA binding domain of an AraC-like transcriptional activator protein.

The misuse of antibiotics during past decades has led to pervasive antibiotic resistance in bacteria. Hence, there is an urgent need for the development of new and alternative approaches to combat bacterial infections. In most bacterial pathogens the expression of virulence is tightly regulated at the transcriptional level.

Structural, kinetic and computational investigation of Vitis vinifera DHDPS reveals new insight into the mechanism of lysine-mediated allosteric inhibition.

Lysine is one of the most limiting amino acids in plants and its biosynthesis is carefully regulated through inhibition of the first committed step in the pathway catalyzed by dihydrodipicolinate synthase (DHDPS). This is mediated via a feedback mechanism involving the binding of lysine to the allosteric cleft of DHDPS. However, the precise allosteric mechanism is yet to be defined.

Cloning, expression, purification and crystallization of dihydrodipicolinate synthase from Agrobacterium tumefaciens.

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of the lysine-biosynthesis pathway in bacteria, plants and some fungi. This study describes the cloning, expression, purification and crystallization of DHDPS (NP_354047.1) from the plant pathogen Agrobacterium tumefaciens (AgT-DHDPS).

Comparative structure and function analyses of native and his-tagged forms of dihydrodipicolinate reductase from methicillin-resistant Staphylococcus aureus.

Given the rise of multi drug resistant bacterial strains, such as methicillin-resistant Staphylococcus aureus (MRSA), there is an urgent need to discover new antimicrobial agents. A validated but as yet unexplored target for new antibiotics is dihydrodipicolinate reductase (DHDPR), an enzyme that catalyzes the second step of the lysine biosynthesis pathway in bacteria. We report here the cloning, expression and purification of N-terminally his-tagged recombinant DHDPR from MRSA (6H-MRSA-DHDPR) and compare its secondary and quaternary structure with the wild type (MRSA-DHDPR) enzyme.

Crystal, solution and in silico structural studies of dihydrodipicolinate synthase from the common grapevine.

Dihydrodipicolinate synthase (DHDPS) catalyzes the rate limiting step in lysine biosynthesis in bacteria and plants. The structure of DHDPS has been determined from several bacterial species and shown in most cases to form a homotetramer or dimer of dimers. However, only one plant DHDPS structure has been determined to date from the wild tobacco species, Nicotiana sylvestris (Blickling et al.