Characterization of AMA1-RON2L complex with native gel electrophoresis and capillary isoelectric focusing

Rhoptry neck protein 2 (RON2) binds to the hydrophobic groove of apical membrane antigen 1 (AMA1), an interaction essential for invasion of red blood cells (RBCs) by Plasmodium falciparum (Pf) parasites. Vaccination with AMA1 alone has been shown to be immunogenic, but unprotective even against homologous challenge in human trials. However, the AMA1-RON2L (L is referred to as the loop region of RON2 peptide) complex is a promising candidate, as preclinical studies with Freund's adjuvant have indicated complete protection against lethal challenge in mice and superior protection against virulent infection in Aotus monkeys. To prepare for clinical trials of the AMA1-RON2L complex, identity and integrity of the candidate vaccine must be assessed, and characterization methods must be carefully designed to not dissociate the delicate complex during evaluation. In this study, we developed a native Tris-glycine gel method to separate and identify the AMA1-RON2L complex, which was further identified and confirmed by Western blotting using anti-AMA1 monoclonal antibodies (mAbs 4G2 and 2C2) and anti-RON2L polyclonal Ab coupled with mass spectrometry. The formation of complex was also confirmed by Capillary Isoelectric Focusing (cIEF). A short-term (48 h and 72 h at 4°C) stability study of AMA1-RON2L complex was also performed. The results indicate that the complex was stable for 72 h at 4°C. Our research demonstrates that the native Tris-glycine gel separation/Western blotting coupled with mass spectrometry and cIEF can fully characterize the identity and integrity of the AMA1–RON2L complex and provide useful quality control data for the subsequent clinical trials.     

Simultaneous Determination of Artemether and Azithromycin in Suppositories by Reversed Phase HPLC

Chromatographic parameter assessments for RP-HPLC-UV method development for the simultaneous analysis of artemether and azithromycin for the pharmaceutical analysis of a rectal coformulation currently under development for the treatment of malaria infected children. Using methanol based mobile phase for the analysis of both artemether and azithromycin provided a more robust method in terms of resolution and peak symmetry. The method validated for suppository used 80% methanol and 20% phosphate buffer 15 mM at pH 9. The UV detection was at 210 nm. The accuracy profiles indicated a method validation between 80–120% for both active pharmaceutical ingredients. The preparation process of the suppository was validated based on theoretical values of artemether and azithromycin present in the formulation; active pharmaceutical ingredients were homogenously distributed within the suppository.     

Highly efficient one-pot synthesis of D-ring chloro-substituted neocryptolepines via a condensation—Pd-catalyzed intramolecular direct arylation strategy

D-ring chloro-substituted neocryptolepines have been synthesized in excellent yield starting from 3-bromo-2-chloro-1-methylquinolinium triflate via a one-pot condensation—Pd-catalyzed intramolecular direct arylation strategy involving chloroanilines. The 3-bromo-2-chloro-1-methylquinolinium triflate was obtained via methylation of commercial 3-bromo-2-chloroquinoline with methyl triflate.     

Disulfide-Reductase Inhibitors as Chemotherapeutic Agents: The Design of Drugs for Trypanosomiasis and Malaria

Viewed globally, parasitic diseases such as malaria and Chagas' cardiopathy pose an increasing threat to human health and welfare. Recognition of this problem and the challenge of synthesizing a quinine-like antimalarial agent sparked off the development of the chemical industry about 100 years ago. Our contribution deals with aspects of drug design, a young branch of pharmaceutical chemistry. As drug targets the flavoenzyme, glutathione reductase, and the recently discovered parasite enzyme, trypanothione reductase, were chosen. Based on the knowledge of the structure of these molecules, the modeling of enzyme inhibitors as potential chemotherapeutic agents against parasites has become possible. In addition, biochemical and clinical observations are considered since chemical principles of biological evolution can serve as guidelines for the pharmaceutical chemists. The picture shows two erythrocytes destroyed by malaria parasites. In the center of the photograph a parasite is just leaving its host cell through the ruptured cell membrane. Its target could be a neighboring healthy erythrocyte.     

Structural insights into the binding mechanism of Plasmodium falciparum exported Hsp40-Hsp70 chaperone pair

Expression of heat shock proteins in Plasmodium falciparum (Pf) increases during febrile episodes to play key roles in several necessary cellular processes. ‘PFA0660w-PfHsp70-x’, an exported chaperone pair is known to co-localize to specialized intracellular structures termed J-dots, and has been implicated in trafficking of the major virulence factor, PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) across the host cell. This article highlights for the first time detailed structural analysis of PFA0660w-PfHsp70-x chaperone pair to better understand their binding mechanism. Here, we have modeled reliable molecular structures for the complete conserved region of PFA0660w and PfHsp70-x. These structures were evaluated by different structure verification tools followed by molecular dynamics (MD) simulations. The model of PFA0660w was subjected to docking with PfHsp70-x using Haddock to reveal a number of residues crucial for their bipartite interaction, and also performed MD simulations on the complex. The peptide binding clefts of PFA0660w and its other Plasmodium species homologs were found to be bigger than their counterparts in higher eukaryotes like yeast, humans and C. parvum. Based on our results, we propose a model for PFA0660w-PfHsp70-x interaction and a mechanism of substrate binding, and compare it with its dimeric human counterparts. Owing to these striking structural differences between the host and parasite chaperones, such information on the essential Hsp40 and its partner Hsp70 may form the basis for rational drug design against fatal malaria.     

Raman spectroscopic investigation of the antimalarial agent mefloquine

The antimalarial agent mefloquine was investigated using Fourier transform near-infrared (FT NIR) Raman and FT IR spectroscopy. The IR and Raman spectra were calculated with the help of density functional theory (DFT) and a very good agreement with the experimental spectra was achieved. These DFT calculations were applied to unambiguously assign the prominent features in the experimental vibrational spectra. The calculation of the potential energy distribution (PED) and the atomic displacements provide further valuable insight into the molecular vibrations. The most prominent NIR Raman bands at 1,363 cm⁻¹ and 1,434 cm⁻¹ are due to C=C stretching (in the quinoline part of mefloquine) and CH₂ wagging vibrations, while the most intense IR peaks at 1,314 cm⁻¹; 1,147 cm⁻¹; and 1,109 cm⁻¹ mainly consist of ring breathings and δCH (quinoline); C–F stretchings; and asymmetric ring breathings, C–O stretching as well as CH₂ twisting/rockings located at the piperidine moiety. Since the active agent (mefloquine) is usually present in very low concentrations within the biological samples, UV resonance Raman spectra of physiological solutions of mefloquine were recorded. By employing the detailed non-resonant mode assignment it was also possible to unambiguously identify the resonantly enhanced modes at 1,619 cm⁻¹, 1,603 cm⁻¹ and 1,586 cm⁻¹ in the UV Raman spectra as high symmetric C=C stretching vibrations in the quinoline part of mefloquine. These spectroscopic results are important for the interpretation of upcoming in vitro and in vivo mefloquine target interaction experiments.     

Synthesis of imidazolidin-4-one and 1H-imidazo[2,1-a]isoindole-2,5(3H,9bH)-dione derivatives of primaquine: scope and limitations

The synthesis of imidazolidin-4-one derivatives of primaquine as potential antimalarial agents is described. The target compounds were synthesized in three steps: (i) condensation of (±)-primaquine with N^α-protected amino acids, (ii) removal of the N^α-protecting group, and (iii) reaction of the N-acylprimaquine with a carbonyl compound: acetone, three cyclic ketones and veratraldehyde. Using 2-formylbenzoic acid in the third step afforded 1H-imidazo[2,1-a]isoindole-2,5(3H,9bH)-diones. All products were isolated in good to excellent yields. Whereas imidazolidin-4-ones were formed as mixtures of all possible diastereomers in equal amounts, 1H-imidazo[2,1-a]isoindole-2,5(3H,9bH)-diones were produced in a stereoselective fashion. The compounds hydrolyse very slowly (t_1/2 5-30 d) in pH 7.4 buffer to release primaquine. These primaquine derivatives are being submitted to biological assays, and preliminary results of their antimalarial activity are quite encouraging.