The essential malaria protein PfCyRPA targets glycans to invade erythrocytes.

Plasmodium falciparum is a human-adapted apicomplexan parasite that causes the most dangerous form of malaria. P. falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. The precise role of PfCyRPA in this process has not been resolved. Here, we show that PfCyRPA is a lectin targeting glycans terminating with α2-6-linked N-acetylneuraminic acid (Neu5Ac). PfCyRPA has a >50-fold binding preference for human, α2-6-linked Neu5Ac over non-human, α2-6-linked N-glycolylneuraminic acid. PfCyRPA lectin sites were predicted by molecular modeling and validated by mutagenesis studies. Transgenic parasite lines expressing endogenous PfCyRPA with single amino acid exchange mutants indicated that the lectin activity of PfCyRPA has an important role in parasite invasion. Blocking PfCyRPA lectin activity with small molecules or with lectin-site-specific monoclonal antibodies can inhibit blood-stage parasite multiplication. Therefore, targeting PfCyRPA lectin activity with drugs, immunotherapy, or a vaccine-primed immune response is a promising strategy to prevent and treat malaria.

The malaria blood stage antigen PfCyRPA formulated with the TLR-4 agonist adjuvant GLA-SE elicits parasite growth inhibitory antibodies in experimental animals.

BACKGROUND: Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. In contrast to several previously clinically tested merozoite vaccine candidate antigens, PfCyRPA is not polymorphic, making it a promising candidate antigen for blood stage vaccine development. METHODS: Mice and rabbits were immunized with vaccine formulations of recombinantly expressed PfCyRPA adjuvanted either with the glucopyranosyl lipid A (GLA) containing adjuvants GLA-LSQ, GLA-SE, GLA-Alum or with Nanoalum. ELISA and indirect immunofluorescence assays (IFA) were used to analyse elicited IgG titers and the P. falciparum growth inhibitory activity was determined with a standardized in vitro [3H]-hypoxanthine incorporation assay. RESULTS: In the mouse experiments, the GLA adjuvanted formulations were superior to the Nanoalum formulation with respect to antibody titer development, IFA sero-conversion rates and in vitro parasite growth-inhibitory activity. In rabbits, the highest titers of parasite growth inhibitory antibodies were obtained with the GLA-SE formulation. Comparable mean ELISA IgG endpoint titers were reached in rabbits after three immunizations with GLA-SE adjuvanted PfCyRPA doses of 5, 25 and 100 µg, but with 100 µg of antigen, only two immunizations were required to reach this titer. CONCLUSION: PfCyRPA formulated with the human-compatible adjuvant GLA-SE represents an attractive vaccine candidate for early clinical testing in a controlled P. falciparum blood stage challenge trial.

Scalable Process for High-Yield Production of PfCyRPA Using Insect Cells for Inclusion in a Malaria Virosome-Based Vaccine Candidate.

Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) has been identified as a promising blood-stage candidate antigen to include in a broadly cross-reactive malaria vaccine. In the last couple of decades, substantial effort has been committed to the development of scalable cost-effective, robust, and high-yield PfCyRPA production processes. Despite insect cells being a suitable expression system due to their track record for protein production (including vaccine antigens), these are yet to be explored to produce this antigen. In this study, different insect cell lines, culture conditions (baculovirus infection strategy, supplementation schemes, culture temperature modulation), and purification strategies (affinity tags) were explored aiming to develop a scalable, high-yield, and high-quality PfCyRPA for inclusion in a virosome-based malaria vaccine candidate. Supplements with antioxidants improved PfCyRPA volumetric titers by 50% when added at the time of infection. In addition, from three different affinity tags (6x-His, 4x-His, and C-tag) evaluated, the 4x-His affinity tag was the one leading to the highest PfCyRPA purification recovery yields (61%) and production yield (26 mg/L vs. 21 mg/L and 13 mg/L for 6x-His and C-tag, respectively). Noteworthy, PfCyRPA expressed using High Five cells did not show differences in protein quality or stability when compared to its human HEK293 cell counterpart. When formulated in a lipid-based virosome nanoparticle, immunized rabbits developed functional anti-PfCyRPA antibodies that impeded the multiplication of P. falciparum in vitro. This work demonstrates the potential of using IC-BEVS as a qualified platform to produce functional recombinant PfCyRPA protein with the added benefit of being a non-human expression system with short bioprocessing times and high expression levels.

Development of an ELISA for the quantification of mycolactone, the cytotoxic macrolide toxin of Mycobacterium ulcerans.

Mycolactones, macrolide cytotoxins, are key virulence factors of Mycobacterium ulcerans, the etiological agent of the chronic necrotizing skin disease Buruli ulcer. There is urgent need for a simple point-of-care laboratory test for Buruli ulcer and mycolactone represents a promising target for the development of an immunological assay. However, for a long time, all efforts to generate mycolactone-specific antibodies have failed. By using a protein conjugate of a truncated non-toxic synthetic mycolactone derivative, we recently described generation of a set of mycolactone-specific monoclonal antibodies. Using the first mycolactone-specific monoclonal antibodies that we have described before, we were able to develop an antigen competition assay that detects mycolactones. By the systematic selection of a capturing antibody and a reporter molecule, and the optimization of assay conditions, we developed an ELISA that detects common natural variants of mycolactone with a limit of detection in the low nanomolar range. The mycolactone-specific ELISA described here will be a very useful tool for research on the biology of this macrolide toxin. After conversion into a simple point-of-care test format, the competition assay may have great potential as laboratory assay for both the diagnosis of Buruli ulcer and for the monitoring of treatment efficacy.

Vaccination with virosomally formulated recombinant CyRPA elicits protective antibodies against Plasmodium falciparum parasites in preclinical in vitro and in vivo models.

The Plasmodium falciparum (Pf) cysteine-rich protective antigen (PfCyRPA) has emerged as a promising blood-stage candidate antigen for inclusion into a broadly cross-reactive malaria vaccine. This highly conserved protein among various geographical strains plays a key role in the red blood cell invasion process by P. falciparum merozoites, and antibodies against PfCyRPA can efficiently prevent the entry of the malaria parasites into red blood cells. The aim of the present study was to develop a human-compatible formulation of the PfCyRPA vaccine candidate and confirming its activity in preclinical studies. Recombinant PfCyRPA expressed in HEK 293 cells was chemically coupled to phosphoethanolamine and then incorporated into the membrane of unadjuvanted influenza virosomes approved as antigen delivery system for humans. Laboratory animals were immunised with the virosome-based PfCyRPA vaccine to determine its immunogenic properties and in particular, its capacity to elicit parasite binding and growth-inhibitory antibodies. The vaccine elicited in mice and rabbits high titers of PfCyRPA-specific antibodies that bound to the blood-stage parasites. At a concentration of 10 mg/mL, purified total serum IgG from immunised rabbits inhibited parasite growth in vitro by about 80%. Furthermore, in a P. falciparum infection mouse model, passive transfer of 10 mg of purified total IgG from PfCyRPA vaccinated rabbits reduced the in vivo parasite load by 77%. Influenza virosomes thus represent a suitable antigen delivery system for the induction of protective antibodies against the recombinant PfCyRPA, designating it as a highly suitable component for inclusion into a multivalent and multi-stage virosomal malaria vaccine.

Potent Inhibitors of Plasmodial Serine Hydroxymethyltransferase (SHMT) Featuring a Spirocyclic Scaffold.

With the discovery that serine hydroxymethyltransferase (SHMT) is a druggable target for antimalarials, the aim of this study was to design novel inhibitors of this key enzyme in the folate biosynthesis cycle. Herein, 19 novel spirocyclic ligands based on either 2-indolinone or dihydroindene scaffolds and featuring a pyrazolopyran core are reported. Strong target affinities for Plasmodium falciparum (Pf) SHMT (14-76 nm) and cellular potencies in the low nanomolar range (165-334 nm) were measured together with interesting selectivity against human cytosolic SHMT1 (hSHMT1). Four co-crystal structures with Plasmodium vivax (Pv) SHMT solved at 2.2-2.4 Šresolution revealed the key role of the vinylogous cyanamide for anchoring ligands within the active site. The spirocyclic motif in the molecules enforces the pyrazolopyran core to adopt a substantially more curved conformation than that of previous non-spirocyclic analogues. Finally, solvation of the spirocyclic lactam ring of the receptor-bound ligands is discussed.

Conformational Aspects in the Design of Inhibitors for Serine Hydroxymethyltransferase (SHMT): Biphenyl, Aryl Sulfonamide, and Aryl Sulfone Motifs.

Malaria remains a major threat to mankind due to the perpetual emergence of resistance against marketed drugs. Twenty-one pyrazolopyran-based inhibitors bearing terminal biphenyl, aryl sulfonamide, or aryl sulfone motifs were synthesized and tested towards serine hydroxymethyltransferase (SHMT), a key enzyme of the folate cycle. The best ligands inhibited Plasmodium falciparum (Pf) and Arabidopsis thaliana (At) SHMT in target, as well as PfNF54 strains in cell-based assays in the low nanomolar range (18-56 nm). Seven co-crystal structures with P. vivax (Pv) SHMT were solved at 2.2-2.6 Šresolution. We observed an unprecedented influence of the torsion angle of ortho-substituted biphenyl moieties on cell-based efficacy. The peculiar lipophilic character of the sulfonyl moiety was highlighted in the complexes with aryl sulfonamide analogues, which bind in their preferred staggered orientation. The results are discussed within the context of conformational preferences in the ligands.

Antimalarial Inhibitors Targeting Serine Hydroxymethyltransferase (SHMT) with in Vivo Efficacy and Analysis of their Binding Mode Based on X-ray Cocrystal Structures.

Target-based approaches toward new antimalarial treatments are highly valuable to prevent resistance development. We report several series of pyrazolopyran-based inhibitors targeting the enzyme serine hydroxymethyltransferase (SHMT), designed to improve microsomal metabolic stability and to identify suitable candidates for in vivo efficacy evaluation. The best ligands inhibited Plasmodium falciparum (Pf) and Arabidopsis thaliana (At) SHMT in target assays and PfNF54 strains in cell-based assays with values in the low nanomolar range (3.2-55 nM). A set of carboxylate derivatives demonstrated markedly improved in vitro metabolic stability (t1/2 > 2 h). A selected ligand showed significant in vivo efficacy with 73% of parasitemia reduction in a mouse model. Five new cocrystal structures with PvSHMT were solved at 2.3-2.6 Å resolution, revealing a unique water-mediated interaction with Tyr63 at the end of the para-aminobenzoate channel. They also displayed the high degree of conformational flexibility of the Cys364-loop lining this channel.

An amphiphilic graft copolymer-based nanoparticle platform for reduction-responsive anticancer and antimalarial drug delivery.

Medical applications of anticancer and antimalarial drugs often suffer from low aqueous solubility, high systemic toxicity, and metabolic instability. Smart nanocarrier-based drug delivery systems provide means of solving these problems at once. Herein, we present such a smart nanoparticle platform based on self-assembled, reduction-responsive amphiphilic graft copolymers, which were successfully synthesized through thiol-disulfide exchange reaction between thiolated hydrophilic block and pyridyl disulfide functionalized hydrophobic block. These amphiphilic graft copolymers self-assembled into nanoparticles with mean diameters of about 30-50 nm and readily incorporated hydrophobic guest molecules. Fluorescence correlation spectroscopy (FCS) was used to study nanoparticle stability and triggered release of a model compound in detail. Long-term colloidal stability and model compound retention within the nanoparticles was found when analyzed in cell media at body temperature. In contrast, rapid, complete reduction-triggered disassembly and model compound release was achieved within a physiological reducing environment. The synthesized copolymers revealed no intrinsic cellular toxicity up to 1 mg mL(-1). Drug-loaded reduction-sensitive nanoparticles delivered a hydrophobic model anticancer drug (doxorubicin, DOX) to cancer cells (HeLa cells) and an experimental, metabolically unstable antimalarial drug (the serine hydroxymethyltransferase (SHMT) inhibitor (±)-1) to Plasmodium falciparum-infected red blood cells (iRBCs), with higher efficacy compared to similar, non-sensitive drug-loaded nanoparticles. These responsive copolymer-based nanoparticles represent a promising candidate as smart nanocarrier platform for various drugs to be applied to different diseases, due to the biocompatibility and biodegradability of the hydrophobic block, and the protein-repellent hydrophilic block.

Chiral Dihydrobenzofuran Acids Show Potent Retinoid X Receptor-Nuclear Receptor Related 1 Protein Dimer Activation.

The nuclear receptor Nurr1 can be activated by RXR via heterodimerization (RXR-Nurr1) and is a promising target for treating neurodegenerative diseases. We herein report the enantioselective synthesis and SAR of sterically constricted benzofurans at RXR. The established SAR, using whole cell functional assays, lead to the full agonist 9a at RXR (pEC50 of 8.2) and RXR-Nurr1. The X-ray structure shows enantiomeric discrimination where 9a optimally addresses the ligand binding pocket of RXR.

Stabilization of protein-protein interactions by small molecules.

Protein-protein interactions (PPIs) are implicated in every disease and mastering the ability to influence PPIs with small molecules would considerably enlarge the druggable genome. Whereas inhibition of PPIs has repeatedly been shown to work successfully, targeted stabilization of PPIs is underrepresented in the literature. This is all the more surprising because natural products like FK506, rapamycin, brefeldin, forskolin and fusicoccin confer their physiological activity by stabilizing specific PPIs. However, recently a number of very interesting synthetic molecules have been reported from drug discovery projects that indeed achieve their desired activities by stabilizing either homo- or hetero-oligomeric complexes of their target proteins.

Small molecules, peptides and natural products: getting a grip on 14-3-3 protein-protein modulation.

One of the proteins that is found in a diverse range of eukaryotic protein-protein interactions is the adaptor protein 14-3-3. As 14-3-3 is a hub protein with very diverse interactions, it is a good model to study various protein-protein interactions. A wide range of classes of molecules, peptides, small molecules or natural products, has been used to modify the protein interactions, providing both stabilization or inhibition of the interactions of 14-3-3 with its binding partners. The first protein crystal structures were solved in 1995 and gave molecular insights for further research. The plant analog of 14-3-3 binds to a plant plasma membrane H(+)-ATPase and this protein complex is stabilized by the fungal phytotoxin fusicoccin A. The knowledge gained from the process in plants was transferred to and applied in human models to find stabilizers or inhibitors of 14-3-3 interaction in human cellular pathways.

Bexarotene prodrugs: targeting through cleavage by NQO1 (DT-diaphorase).

Bexarotene, a retinoid X receptor (RXR) agonist, is being tested as a potential disease modifying treatment for neurodegenerative conditions. To limit the peripheral exposure of bexarotene and release it only in the affected areas of the brain, we designed a prodrug strategy based on the enzyme NAD(P)H/quinone oxidoreductase (NQO1) that is elevated in neurodegenerative diseases. A series of indolequinones (known substrates of NQO1) was synthesized and coupled to bexarotene. Bexarotene-3-(hydroxymethyl)-5-methoxy-1,2-dimethyl-1H-indole-4,7-dione ester 7a was cleaved best by NQO1. The prodrugs are not cleaved by esterase.

Influence of chlorine or fluorine substitution on the estrogenic properties of 1-alkyl-2,3,5-tris(4-hydroxyphenyl)-1H-pyrroles.

In continuation of our previous work, several 1-alkyl-2,3,5-tris(4-hydroxyphenyl)aryl-1H-pyrroles with chlorine or fluorine substituents in the aryl residues were synthesized and tested for estrogen receptor (ER) binding at isolated ERα/ERß receptors (HAP assay) and in transactivation assays using ERα-positive MCF-7/2a as well as U2-OS/ERα and U2-OS/ERß cells. In the competition experiment at ERα the compounds displayed very high relative binding affinities of up to 37% (determined for 8m) but with restricted subtype selectivity (e.g., ERα/ERß (8m) = 9). The highest estrogenic potency in ERα-positive MCF-7/2a cells was determined for 2,3,5-tris(2-fluoro-4-hydroxyphenyl)-1-propyl-1H-pyrrole 8m (EC(50) = 23 nM), while in U2-OS/ERα cells 2-(2-fluoro-4-hydroxyphenyl)-3,5-bis(4-hydroxyphenyl)-1-propyl-1H-pyrrole 8b (EC(50) = 0.12 nM) was the most potent agonist, only 30-fold less active than estradiol (E2, EC(50) = 0.004 nM). In U2-OS/ERß cells for all pyrroles no transactivation could be observed, which indicates that they are selective ERα agonists in cellular systems.

Development of 2,3,5-triaryl-1H-pyrroles as estrogen receptor†α selective ligands.

1-Alkyl-2,3,5-triaryl-1H-pyrroles (for which alkyl=methyl, ethyl, n-propyl, or 2-methylpropyl) were tested for stability, estrogen receptor (ER) binding, and inhibition of tumor cell growth. These pyrroles (type B) showed higher stability in aqueous solution than their 1,2,4-triaryl-1H-pyrrole congeners (type A pyrroles), exclusive ERα binding (no ERß interaction), and a hormonal profile of partial agonists at ERα. The most potent compound, 1-(2-methylpropyl)-2,3,5-tris(4-hydroxyphenyl)-1H-pyrrole (5 d), was less active than the lead structure 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) in MCF-7 cells stably transfected with the plasmid EREwtcluc (MCF-7/2a), but more potent in U2-OS/α cells. Furthermore, 5 d showed weak anti-estrogenic properties (IC50=310 nM). An additional propyl chain at C4 decreased the stability and pharmacological effects.

Synthesis and investigations on the oxidative degradation of C3/C5-alkyl-1,2,4-triarylpyrroles as ligands for the estrogen receptor.

In this study, we synthesized 1,2,4-triarylpyrroles as ligands for the estrogen receptor (ER). Two pyrrole series were prepared with either C3-alkyl or C3/C5-dialkyl residues. Compounds from both series were susceptible to oxidative degradation-dialkylated compounds (t(1/2) =33-66 h) to a higher extent than their monoalkylated congeners (t(1/2) =140-211 h). Nevertheless, stability was sufficient for determination of in vitro ER binding affinity. The most active agonist in hormone-dependent, ERα-positive MCF-7/2a and U2-OS/α cells was 1,2,4-tris(4-hydroxyphenyl)-3-propyl-1H-pyrrole (6 d) (MCF-7/2a: EC(50) =70 nM; U2-OS/α: EC(50) =1.6 nM). A corresponding inactivity in U2-OS/ß cells demonstrated the high ERα selectivity. This trend was confirmed in a competition experiment using estradiol (E2) and purified hERα and hERß proteins (relative binding affinity (RBA) calculated for 6 d: RBA(ERα)=1.85 %; RBA(ERß) <0.01 %). Generally, C3/C5-dialkyl substitution led to reduction of activity, possibly due to lower stability.

Diffusion tensor magnetic resonance imaging of the breast: a pilot study.

OBJECTIVES: Diffusion-weighted MR imaging has shown diagnostic value for differential diagnosis of breast lesions. Diffusion tensor imaging (DTI) adds information about tissue microstructure by addressing diffusion direction. We have examined the diagnostic application of DTI of the breast. METHODS: A total of 59 patients (71 lesions: 54 malignant, 17 benign) successfully underwent prospective echo planar imaging-DTI (EPI-DTI) (1.5 T). First, diffusion direction both of parenchyma as well as lesions was assessed on parametric maps. Subsequently, apparent diffusion coefficient (ADC) and fractional anisotropy (FA) values were measured. Statistics included univariate (Mann-Whitney U test, receiver operating analysis) and multivariate (logistic regression analysis, LRA) tests. RESULTS: Main diffusion direction of parenchyma was anterior-posterior in the majority of cases (66.1%), whereas lesions (benign, malignant) showed no predominant diffusion direction in the majority of cases (23.9%). ADC values showed highest differences between benign and malignant lesions (P<0.001) with resulting area under the curve (AUC) of 0.899. FA values were lower in benign (interquartile range, IR, 0.14-0.24) compared to malignant lesions (IR 0.21-0.35, P<0.002) with an AUC of 0.751-0.770. Following LRA, FA did not prove to have incremental value for differential diagnosis over ADC values. CONCLUSIONS: Microanatomical differences between benign and malignant breast lesions as well as breast parenchyma can be visualized by using DTI.

Intramembrane proteolysis of Mgm1 by the mitochondrial rhomboid protease is highly promiscuous regarding the sequence of the cleaved hydrophobic segment.

Rhomboids are a family of intramembrane serine proteases that are conserved in bacteria, archaea, and eukaryotes. They are required for numerous fundamental cellular functions such as quorum sensing, cell signaling, and mitochondrial dynamics. Mitochondrial rhomboids form an evolutionarily distinct class of rhomboids. It is largely unclear how their activity is controlled and which substrate determinants are responsible for recognition and cleavage. We investigated these requirements for the mitochondrial rhomboid protease Pcp1 and its substrate Mgm1. In contrast to several other rhomboid proteases, Pcp1 does not require helix-breaking amino acids in the cleaved hydrophobic region of Mgm1, termed 'rhomboid cleavage region' (RCR). Even transmembrane segments of inner membrane proteins that are normally not processed by Pcp1 become cleavable when put in place of the authentic RCR of Mgm1. We further show that mutational alterations of a highly negatively charged region located C-terminally to the RCR led to a strong processing defect. Moreover, we show that the determinants required for Mgm1 processing by mitochondrial rhomboid protease are conserved during evolution, as PARL (the human ortholog of Pcp1) showed similar substrate requirements. These results suggest a surprising promiscuity of the mitochondrial rhomboid protease regarding the sequence requirements of the cleaved hydrophobic segment. We propose a working hypothesis on how the mitochondrial rhomboid protease can, despite this promiscuity, achieve a high specificity in recognizing Mgm1. This hypothesis relates to the exceptional biogenesis pathway of Mgm1.