In vitro inhibition of Plasmodium falciparum early and late stage gametocyte viability by extracts from eight traditionally used South African plant species

ETHNOPHARMACOLOGICAL RELEVANCE : Extracts of plant species, used traditionally to treat malaria, have been extensively investigated for their activity against Plasmodium intraerythrocytic asexual parasites in search of new antimalarial drugs. However, less effort has been directed towards examining their efficacy in blocking transmission. Here, we report the results of the in vitro screening of extracts from eight selected plant species used traditionally to treat malaria in South Africa for activity against P. falciparum NF54 early and late stage gametocytes. The species used were Khaya anthotheca, Trichilia emetica, Turraea floribunda, Leonotis leonurus, Leonotis leonurus ex Hort, Olea europaea subsp. Africana, Catha edulis and Artemisia afra. AIM OF STUDY : To investigate the activities of extracts from plant species traditionally used for malaria treatment against P. falciparum gametocytes. MATERIAL AND METHODS : Air-dried and ground plant leaves were extracted using acetone. Primary two point in vitro phenotypic screens against both early and late stage gametocytes were done at 10 and 20 μg/ml followed by full IC50 determination of the most active extracts. Inhibition of gametocyte viability in vitro was assessed using the parasite lactate dehydrogenase (pLDH) assay. RESULTS : Of the eight crude acetone extracts from plant species screened in vitro, four had good activity with over 50-70% inhibition of early and late stage gametocytes’ viability at 10 and 20 μg/ml, respectively. Artemisia afra (Asteraceae), Trichilia emetica (Meliaceae) and Turraea floribunda (Meliaceae) were additionally highly active against both gametocyte stages with IC50 values of less than 10 μg/ml while Leonotis leonurus ex Hort (Lamiaceae) was moderately active (IC50<20 μg/ml). The activity of these three highly active plant species was significantly more pronounced on late stage gametocytes compared to early stages. CONCLUSION : This study shows the potential transmission blocking activity of extracts from selected South African medicinal plants and substantiates their traditional use in malaria control that broadly encompasses prevention, treatment and transmission blocking. Further studies are needed to isolate and identify the active principles from the crude extracts of A. afra, T. emetica and T. floribunda, as well as to examine their efficacy towards blocking parasite transmission to mosquitoes.A research grant from the University of Pretoria Centre for Sustainable Malaria Control (UP CSMC), the South African National Research Foundation (UID:84627), and the Medical Research Council Strategic Health Innovation Partnership.http://www.elsevier.com/locate/jethpharm2017-06-30hb2016BiochemistryChemistryParaclinical Science

Activities of 11‐azaartemisinin and N‐sulfonyl derivatives against asexual and transmissible malaria parasites

Dihydroartemisinin (DHA), either used in its own right or as the active drug generated in vivo from the other artemisinins in current clinical use—artemether and artesunate—induces quiescence in ring‐stage parasites of Plasmodium falciparum (Pf). This induction of quiescence is linked to artemisinin resistance. Thus, we have turned to structurally disparate artemisinins that are incapable of providing DHA on metabolism. Accordingly, 11‐azaartemisinin 5 and selected N‐sulfonyl derivatives were screened against intraerythrocytic asexual stages of drug‐sensitive Pf NF54 and drug‐resistant K1 and W2 parasites. Most displayed appreciable activities against all three strains, with IC50 values 2000 toward asexual parasites. Overall, the readily accessible 11‐azaartemisinin 5 and the sulfonyl derivatives 11 and 16 represent potential candidates for further development, in particular for transmission blocking of artemisinin‐resistant parasites.http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1860-71872018-12-19hj2018Biochemistr

Artemisone and artemiside - potent pan-reactive antimalarial agents that also synergize redox imbalance in P. falciparum transmissible gametocyte stages

The emergence of resistance towards artemisinin combination therapies (ACTs) by the malaria parasite Plasmodium falciparum has the potential to severely compromise malaria control. Therefore, development of new artemisinins in combination with new drugs that impart activities towards both intraerythrocytic proliferative asexual and transmissible gametocyte stages, in particular those of resistant parasites, are urgently required. We define artemisinins as oxidant drugs through their ability to oxidize reduced flavin cofactors of flavin disulfide reductases critical for maintaining redox-homeostasis in the malaria parasite. Here we compare the activities of 10-amino artemisinin derivatives towards the asexual and gametocyte stages of P. falciparum parasites. Of these, artemisone and artemiside inhibited asexual and gametocyte stages, particularly stage V gametocytes in the low nM range. Further, treatment of both early and late gametocyte stages with artemisone or artemiside combined with the pro-oxidant redox partner methylene blue displays notable synergism. These data suggest that modulation of redox-homeostasis likely is an important druggable process, particularly in gametocytes, and thereby enhances the prospect of using combinations of oxidant and redox drugs for malaria control.The South African Medical Research Council (MRC) Flagship Project MALTB-Redox with funds from the National Treasury under its Economic Competitiveness and Support Package to Richard K. Haynes; a South African MRC Strategic Health Innovation Partnership (SHIP) grant, a South African MRC Collaborative Centre for Malaria Research grant, and a South African National Research Foundation grant (UID 84627) to Lyn-Marie Birkholtz; and South African National Research Foundation grants to Richard K. Haynes (UIDs 90682 and 98934). Donatella Taramelli and Sarah D'Alessandro acknowledge the support from the Global Health Program of the Bill & Melinda Gates Foundation (grant OPP1040394 to Donatella Taramelli, Pietro Alano coordinator, and COST Action CM1307).http://aac.asm.org2019-02-01hj2018Biochemistr

Potent Plasmodium falciparum gametocytocidal compounds identified by exploring the kinase inhibitor chemical space for dual active antimalarials

OBJECTIVES : Novel chemical tools to eliminate malaria should ideally target both the asexual parasites and transmissible gametocytes. Several imidazopyridazines (IMPs) and 2-aminopyridines (2-APs) have been described as potent antimalarial candidates targeting lipid kinases. However, these have not been extensively explored for stage-specific inhibition of gametocytes in Plasmodium falciparum parasites. Here we provide an in-depth evaluation of the gametocytocidal activity of compounds from these chemotypes and identify novel starting points for dual-acting antimalarials. METHODS : We evaluated compounds against P. falciparum gametocytes using several assay platforms for cross-validation and stringently identified hits that were further profiled for stage specificity, speed of action and ex vivo efficacy. Physicochemical feature extraction and chemogenomic fingerprinting were applied to explore the kinase inhibition susceptibility profile. RESULTS : We identified 34 compounds with submicromolar activity against late stage gametocytes, validated across several assay platforms. Of these, 12 were potent at 1000-fold selectivity towards the parasite over mammalian cells. Front-runner compounds targeted mature gametocytes within 48 h and blocked transmission to mosquitoes. The resultant chemogenomic fingerprint of parasites treated with the lead compounds revealed the importance of targeting kinases in asexual parasites and gametocytes. CONCLUSIONS : This study encompasses an in-depth evaluation of the kinase inhibitor space for gametocytocidal activity. Potent lead compounds have enticing dual activities and highlight the importance of targeting the kinase superfamily in malaria elimination strategies.The South African Medical Research Council (SAMRC) Self-initiated Research (to JN) and Strategic Health Initiatives Partnerships (MRC-SHIP) programmes to L.B., T.C., D.M. K.C. further acknowledges the SAMRC for funding of the extramural Drug Discovery and Development Research Unit at UCT. The SAMRC is acknowledged for funding of the UP ISMC (LMB) and WRIM (TLC) as Collaborating Centres for Malaria Research. The Council for Scientific and Industrial Research and the 3R Foundation (project 118–10) to D.M. We thank the Medicines for Malaria Venture and South African Technology Innovation Agency (TIA) for funding to K.C. (Project MMV09/0002). The University of Cape Town, University of Pretoria, and South African Research Chairs Initiative of the Department of Science and Technology, administered through the South African National Research Foundation are gratefully acknowledged for support to K.C. and L.B. (UID84627). JN was supported through an International Society for Infectious Diseases grant.https://academic.oup.com/jac2019-05-01hj2018Biochemistr

Defining the clinical and cognitive phenotype of child savants with autism spectrum disorder

Objective: Whilst savant syndrome is most commonly observed in individuals with Autism Spectrum Disorder (ASD), it has historically been associated with intellectual impairment, and little is known about the clinical and cognitive characteristics of intellectually able individuals with ASD and savant skills. Methods: Participants with ASD and validated savant skills were compared with age and intelligence matched non-savants with ASD using a range of diagnostic and standardised tests. Results: Although the analysis of the clinical data revealed few differences between the groups, striking differences emerged during cognitive testing. Children with savant skills exhibited highly superior working memory and their scores on tests of analytic skills were also superior to those of non-savants. Conclusion: We propose that obsessionality, focused attention, superior working memory and analytic skills facilitate veridical mapping and pattern perception abilities characteristic in savant syndrome

The use of HIV-1 envelope epitopes for characterizing humoral immune responses

Background: HIV envelope (env) proteins are highly antigenic making them useful for detecting the immune response to natural infection. Envelope protein epitopes are not just virus derived but can be acquired from the host during budding. Some epitopes of host protein beta 2 microglobulin (?2M) become exposed only during incorporation by the virus. These epitopes are defined as cryptic (newly exposed) and are being investigated here, alongside virus-derived envelope protein epitopes for the ability to detect HIV-induced immune responses in vitro. Data obtained in this manner provides information on the potential use of these epitopes as vaccine components or antigens in prognostic/diagnostic assays. Methods: Synthetic peptides based on epitopes of ?2M (designated as R7V, F7E, S7K, ?2Mp) or env (designated as 2F5, DC1, DV3, and MPER) were synthesized and used in an indirect ELISA to detect antibodies in the serum of HIV infected patients who were treatment-naïve or receiving highly active antiretroviral treatment (HAART). The effect of the peptides on peripheral blood momonuclear cells (PBMCs) was assessed through measuring cytokine secretion (using Cytometric beads) and proliferation (tetrazolium dye uptake, real time cell electronic sensing (RTCES) and flow cytometry using carboxyfluorescein diacetate succimidyl ester (CFSE)). New Zealand white rabbits were immunized with the peptides and affinity purified antibodies prepared and tested for the ability to neutralize viral constructs (pseudo-virus designed to undergo one replication cycle). Results and Discussion: In individuals infected with HIV-1 subtype C the seroprevalence of antibodies against epitopes of ?2m was variable (between and within experimental groups). ?2M antibodies were prominent (up to 2 fold) in newly infected individuals who were not on treatment or had recently started HAART. One ?2M epitope (R7V) sparked interest in the literature when it repeatedly served as an indicator of non-progression to disease. In the current study the strongest response was observed for antibodies against V3 loop peptides while antibodies against gp41 epitopes were generally low. At times a notable response was seen against a peptide based on the membrane proximal external region (MPER) of gp41. Proliferation data confirmed that the peptide concentrations used were non-toxic to cells. However, PBMC proliferation was minimal (stimulation index ±1) on all accounts. Tetrazolium dyes are notoriously weak detectors of peptide-induced proliferation, which is why RTCES and flow cytometry were employed as methods with improved levels of sensitivity. Flow cytometry and a fluorescent dye, CFSE, were better detectors of proliferation, allowing the calculation of stimulation indices between 4 and 7. DV3, B2Mp and R7V stimulated proliferation of infected cells. IL-6 (P1:6400 and up to 1:152000 were observed. These Polyclonal antibodies (raised against DV3 and MPER) neutralized HIV-1 pseudo-virus ZM53 at >50%. Conclusion: Antibodies against host-derived beta-2 microglobulin epitopes were reportedly ideal prognostic indicators (prominent in long term non-progressors (LTNP) according to Galea et al, 1996) in infection with HIV-1 subtypes A and B. Margolick et al. (2010) qualified these findings by retrospectively demonstrating that the presence of ?2M epitope antibodies in early infection indicated a tendency toward LTNP. In the current study, using sera collected from HIV-1 subtype C infected individuals; peptides based on ?2M were able to detect antibodies in recently infected individuals and also stimulated cytokine production in vitro. When compared to the viral-derived antigens, the responses detected using host-derived peptides were lower. Although immunogenic, host-derived epitopes do not appear to have value in diagnostic use. The responses to host-derived peptides distinguished recent from later infection, suggesting prognostic potential. The antigenicity of the viral peptides confirmed, what is well reported in the literature, potential use in vaccine development, while the peptides based on host derived protein epitopes showed less ability in this regard.Dissertation (MSc)--University of Pretoria, 2016.BiochemistryMScUnrestricte