A novel diazaspiro[3.4]octane series was identified from a Plasmodium falciparum whole-cell high-throughput screening campaign. Hits displayed activity against multiple stages of the parasite lifecycle, which together with a novel sp3-rich scaffold provided an attractive starting point for a hit-to-lead medicinal chemistry optimization and biological profiling program. Structure-activity-relationship studies led to the identification of compounds that showed low nanomolar asexual blood-stage activity (<50 nM) together with strong gametocyte sterilizing properties that translated to transmission-blocking activity in the standard membrane feeding assay. Mechanistic studies through resistance selection with one of the analogues followed by whole-genome sequencing implicated the P. falciparum cyclic amine resistance locus in the mode of resistance.

Chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium . Here, we report the parallel de novo screening of the Medicines for Malaria Venture (MMV) Pandemic Response Box against Plasmodium asexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. Unique chemotypes were identified with both multistage activity or stage-specific activity, including structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity, such as the JmjC inhibitor ML324 and the antitubercular clinical candidate SQ109. Mechanistic investigations prove that ML324 prevents histone demethylation, resulting in aberrant gene expression and death in gametocytes. Moreover, the selection of parasites resistant to SQ109 implicates the druggable V-type H + -ATPase for the reduced sensitivity. Our data therefore provides an expansive dataset of compounds that could be redirected for antimalarial development and also point towards proteins that can be targeted in multiple parasite life cycle stages.

Met die toenemende voorkoms van weerstandige Plasmodium stamme het die beheer van malaria-voorkoms en -mortaliteit weer op die voorgrond getree. Nuwe teikens en antimalariamiddels wat effektief is teen weerstandige malaria-parasiete word dus dringend benodig. Kalsium-afhanklike proteïenkinases (calcium dependent protein kinases – CDPKs) is betrokke by die beheer van ’n aantal biologiese prosesse in die malaria-parasiet, Plasmodium falciparum, met CDPK4 die belangrikste ensiem in hierdie klas. In hierdie studie is die struktuur van PfCDPK4 gebruik as templaat vir die soeke na nuwe malariamiddels. Die PfCDPK4 modelstruktuur is deur middel van homologiemodellering gegenereer en die stereochemiese kwaliteit gevalideer. Die molekulêre modelleringbenadering deur middel van in silico sifting teen die teiken-molekuul PfCDPK4 het ’n beskeie biblioteek van 20 000 chemiese verbindings ingesluit, asook ’n aantal aktiewe natuurprodukte en kliniesgoedgekeurde kinase-inhibeerders. In silico sifting van die Biofocus biblioteek teen PfCDPK4 het 26 verbindings opgelewer; in vitro sifting het bevestig dat drie van hierdie verbindings matig aktief is teen Plasmodium falciparum NF54, met persentasie inhibisie tussen 42% en 47%.

Background African populations are characterised by high genetic diversity, which provides opportunities for discovering and elucidating novel variants of clinical importance, especially those affecting therapeutic outcome. Significantly more knowledge is however needed before such populations can take full advantage of the advances in precision medicine. Coupled with the need to concisely map and better understand the pharmacological implications of genetic diversity in populations of sub-Sharan African ancestry, the aim of this study was to identify and characterize known and novel variants present within 65 important absorption, distribution, metabolism and excretion genes. Patients and methods Targeted ultra-deep next-generation sequencing was used to screen a cohort of 40 South African individuals of Bantu ancestry. Results We identified a total of 1662 variants of which 129 are novel. Moreover, out of the 1662 variants 22 represent potential loss-of-function variants. A high level of allele frequency differentiation was observed for variants identified in this study when compared with other populations. Notably, on the basis of prior studies, many appear to be pharmacologically important in the pharmacokinetics of a broad range of drugs, including antiretrovirals, chemotherapeutic drugs, antiepileptics, antidepressants, and anticoagulants. An in-depth analysis was undertaken to interrogate the pharmacogenetic implications of this genetic diversity. Conclusion Despite the new insights gained from this study, the work illustrates that a more comprehensive understanding of population-specific differences is needed to facilitate the development of pharmacogenetic-based interventions for optimal drug therapy in patients of African ancestry.

Christel Brunschwig,a Nina Lawrence,a Dale Taylor,a Efrem Abay,a Mathew Njoroge,a Gregory S. Basarab,a Claire Le Manach,b Tanya Paquet,b Diego Gonzàlez Cabrera,b Aloysius T. Nchinda,b Carmen de Kock,a Lubbe Wiesner,c Paolo Denti,c David Waterson,d Benjamin Blasco,d Didier Leroy,d Michael J. Witty,d Cristina Donini,d James Duffy,d Sergio Wittlin,e,f Karen L. White,g Susan A. Charman,g Maria Belén Jiménez-Díaz,h Iñigo Angulo-Barturen,h Esperanza Herreros,h Francisco Javier Gamo,h Rosemary Rochford,i Dalu Mancama,j Theresa L. Coetzer,k Mariëtte E. van der Watt,l Janette Reader,l Lyn-Marie Birkholtz,l Kennan C. Marsh,m Suresh M. Solapure,n† John E. Burke,z Jacob A. McPhail,z Manu Vanaerschot,o David A. Fidock,o,p Paul V. Fish,q Peter Siegl,r Dennis A. Smith,s Grennady Wirjanata,t Rintis Noviyanti,u Ric N. Price,t,v Jutta Marfurt,t Kigbafori D. Silue,w Leslie J. Street,b Kelly Chibaleb,x,y

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.

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 <100 nM (8 were IMPs and 4 were 2-APs) and were also active against early stage gametocytes and asexual parasites, with >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.

Background: Discovery of novel gametocytocidal molecules is a major pharmacological strategy in the elimination and eradication of malaria. The high patronage of the aqueous root extract of the popular West African anti-malarial plant Cryptolepis sanguinolenta (Periplocaceae) in traditional and hospital settings in Ghana has directed this study investigating the gametocytocidal activity of the plant and its major alkaloid, cryptolepine. This study also investigates the anti-malarial interaction of cryptolepine with standard anti-malarials, as the search for new anti-malarial combinations continues. Methods: The resazurin-based assay was employed in evaluating the gametocytocidal properties of C. sanguinolenta and cryptolepine against the late stage (IV/V) gametocytes of Plasmodium falciparum (NF54). A fixed ratio method based on the SYBR Green I fluorescence-based assay was used to build isobolograms from a combination of cryptolepine with four standard anti-malarial drugs in vitro using the chloroquine sensitive strain 3D7. Results: Cryptolepis sanguinolenta (IC50 = 49.65 nM) and its major alkaloid, cryptolepine (IC50 = 1965 nM), showed high inhibitory activity against the late stage gametocytes of P. falciparum (NF54). In the interaction assays in asexual stage, cryptolepine showed an additive effect with both lumefantrine and chloroquine with mean ΣFIC50s of 1.017 ± 0.06 and 1.465 ± 0.17, respectively. Cryptolepine combination with amodiaquine at therapeutically relevant concentration ratios showed a synergistic effect (mean ΣFIC50 = 0.287 ± 0.10) whereas an antagonistic activity (mean ΣFIC50 = 4.182 ± 0.99) was seen with mefloquine. Conclusions: The findings of this study shed light on the high gametocytocidal properties of C. sanguinolenta and cryptolepine attributing their potent anti-malarial activity mainly to their effect on both the sexual and asexual stages of the parasite. Amodiaquine is a potential drug partner for cryptolepine in the development of novel fixed dose combinations.

There are limited data describing acute kidney injury (AKI) in HIV-infected adult patients in resource-limited settings where tenofovir disoproxil fumarate (TDF), which is potentially nephrotoxic, is increasingly prescribed. We describe risk factors for and prognosis of AKI in HIV-infected individuals, stratified by those receiving and those naive to TDF. A prospective case cohort study of hospitalized HIV-infected adults with AKI stratified by TDF exposure. Adults (>= 18 years) were recruited: clinical and biochemical data were collected at admission; their renal recovery, discharge, or mortality was ascertained as an in-patient and, subsequently, to a scheduled 3-month follow-up. Among this predominantly female (61%), almost exclusively black African cohort of 175 patients with AKI, 93 (53%) were TDF exposed; median age was 41 years (interquartile range 35-50). Median CD4 count and viral load and creatinine at baseline were 116 cells/mm(3) and 110,159 copies/ml, respectively. A greater proportion of the TDF group had severe AKI on admission (61% vs. 43%, p =.014); however, both groups had similar rates of newly diagnosed tuberculosis (TB; 52%) and nonsteroidal anti-inflammatory drug (NSAID; 32%) use. Intravenous fluid was the therapeutic mainstay; only seven were dialyzed. Discharge median serumcreatinine (SCr) was higher in the TDF group (p=.032) and fewer in the TDF group recovered renal function after 3 months (p=.043). Three-month mortality was 27% in both groups, but 55% of deaths occurred in hospital. Those that died had a higher SCr and more severe AKI than survivors; TB was diagnosed in 33 (70%) of those who died. AKI was more severe and renal recovery slower in the TDF group; comorbidities, risk factors, and prognosis were similar regardless of TDF exposure. Because TB is linked to higher mortality, TB coinfection inHIV-infected patients with AKI warrants more intensive monitoring. In all those with poor renal recovery, our data suggest that a lower threshold for dialysis is needed.

Discovery of novel gametocytocidal molecules is a major pharmacological strategy in the elimination and eradication of malaria. The high patronage of the aqueous root extract of the popular West African anti-malarial plant Cryptolepis sanguinolenta (Periplocaceae) in traditional and hospital settings in Ghana has directed this study investigating the gametocytocidal activity of the plant and its major alkaloid, cryptolepine. This study also investigates the anti-malarial interaction of cryptolepine with standard anti-malarials, as the search for new anti-malarial combinations continues.

Cryptolepine is the main alkaloid in the root of the popular West African plant Cryptolepis sanguinolenta, frequently used as an antimalarial, antidysentery and febrifuge remedy [1]. The high patronage of the aqueous root extract of the popular West African antimalarial plant Cryptolepis sanguinolenta (Lindl.) Schlechter (Periplocaceae) in traditional and hospital settings in Ghana has directed this studies investigating the gametocytocidal potential as well as the antimalarial interaction of its major alkaloid, cryptolepine with standard antimalarial agents as the search for new antimalarial combinations continue. A fixed ratio method based on the SYBR Green I fluorescent-based assay [2] was used to build isobolograms from a combination of cryptolepine with four standard antimalarial drugs in vitro using the chloroquine sensitive strain 3D7. The ProtoBlue® assay [3] was employed in evaluating the transmission-blocking properties of Cryptolepis sanguinolenta and cryptolepine against the late stage gametocytes of Plasmodium falciparum (NF54). In the interaction assays, cryptolepine showed an additive effect with both lumefantrine and chloroquine with mean ΣFIC50s of 1.017 ± 0.45 and 1.342 ± 0.34, respectively. Cryptolepine combination with amodiaquine at therapeutically relevant concentration ratios showed a synergistic effect (mean ΣFIC50= 0.235 ± 0.15) whereas an antagonistic activity (mean ΣFIC50= 4.182 ± 0.68) was seen with mefloquine. Cryptolepis sanguinolenta and its major alkaloid showed minimal inhibitory activity against the late stage gametocytes of P. falciparum (NF54). The findings of this study shed light on the low transmission- blocking properties of Cryptolepis sanguinolenta and cryptolepine attributing their antimalarial activity mainly to their effect on the asexual stages of the parasite. Amodiaquine is a potential drug partner for cryptolepine in the development of novel fixed dose combinations.

The ability to target human-mosquito parasite transmission challenges global malaria elimination. However, it is not obvious what a transmission-blocking drug will look like; should it target only parasite transmission stages; be combined with a partner drug killing the pathogenic asexual stages; or kill both the sexual and asexual blood stages, preferably displaying polypharmacology? The development of transmission-blocking antimalarials requires objective analyses of the current strategies. Here, pertinent issues and questions regarding the target candidate profile of a transmission-blocking compound, and its role in malaria elimination strategies, are highlighted and novel perspectives proposed. The essential role of a test cascade that integrates screening and validation strategies to identify next-generation transmission-blocking antimalarials is emphasised.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

The objective of this review was to evaluate the experimental approaches used in clinical trials to support the benefit claims of plant food supplements (PFS) with reported activity on infectious diseases. A literature search was conducted on a list of 309 plant species currently used in food supplements in Europe using the National Centre for Biotechnology Information (NCBI) PubMed Database to identify all the clinical trials for evaluating the benefit claim for preventing or treating infectious diseases in humans. The searches included a combination of terms related to the name of plants, class of infectious agents and therapeutic activities against infectious disease. By limiting the searches to clinical studies, only 27 articles representing 19 plant species were identified. From this list, 13 papers from the 10 plants most extensively researched were critically evaluated for assessing methods used to assess the benefits of PFS. Different study designs were used ranging from an open trial with no placebo or control and no randomization to double-blind randomized placebo controlled trials including a crossover design. Although the experimental approaches described in this review were found to be suitable for evaluating the benefit claims of PFS for treating infectious diseases caused by bacteria, viruses, fungi and parasites, the clinical study design should be more standardised as many studies lacked a control group and sufficient population size to be statistically acceptable taking into consideration patient variability. The reporting of the results varied and should also be standardised to include all the study parameters and data collected.

The CSIR, in collaboration with several partner institutions, has begun screening vast libraries of synthetic compounds that could be used in a new generation of antimalarial drugs. These drugs will disrupt the life cycle of the parasite (that causes malaria), paving the way for the eradication of the disease. Compounds have already been identified that meet the criteria and exhibit the ideal characteristics required for further development.

BACKGROUND:The discovery of malaria transmission-blocking compounds is seen as key to malaria elimination strategies and gametocyte-screening platforms are critical filters to identify active molecules. However, unlike asexual parasite assays measuring parasite proliferation, greater variability in end-point readout exists between different gametocytocidal assays. This is compounded by difficulties in routinely producing viable, functional and stage-specific gametocyte populations. Here, a parallel evaluation of four assay platforms on the same gametocyte populations was performed for the first time. This allowed the direct comparison of the ability of different assay platforms to detect compounds with gametocytocidal activity and revealed caveats in some assay readouts that interrogate different parasite biological functions.METHODS:Gametocytogenesis from Plasmodium falciparum (NF54) was optimized with a robust and standardized protocol. ATP, pLDH, luciferase reporter and PrestoBlue® assays were compared in context of a set of 10 reference compounds. The assays were performed in parallel on the same gametocyte preparation (except for luciferase reporter lines) using the same drug preparations (48 h). The remaining parameters for each assay were all comparable.RESULTS:A highly robust method for generating viable and functional gametocytes was developed and comprehensively validated resulting in an average gametocytaemia of 4%. Subsequent parallel assays for gametocytocidal activity indicated that different assay platforms were not able to screen compounds with variant chemical scaffolds similarly. Luciferase reporter assays revealed that synchronized stage-specific gametocyte production is essential for drug discovery, as differential susceptibility in various gametocyte developmental populations is evident.CONCLUSIONS:With this study, the key parameters for assays aiming at testing the gametocytocidal activity of potential transmission blocking molecules against Plasmodium gametocytes were accurately dissected. This first and uniquely comparative study emphasizes differential effects seen with the use of different assay platforms interrogating variant biological systems. Whilst this data is informative from a biological perspective and may provide indications of the drug mode of action, it does highlight the care that must be taken when screening broad-diversity chemotypes with a single assay platform against gametocytes for which the biology is not clearly understood.

As per the 2007 Global Malaria Eradication Plan, malaria transmission blocking is seen as key to malaria elimination strategies. A highlight that has emerged from gametocytocidal assays to identify novel compounds with malaria transmission blocking ability is that, unlike asexual-based assays, greater variability in end-point readout may exist between these assays that interrogate different parasite biological functions. Drug mode-of-action is likely to be an important factor on this outcome. Such variability may be mitigated by screening compounds based on similar pharmacophores in series. One of the major concerns with the current assay platforms is their inability to be robustly used to screen variant pharmacophores accurately as the different assay principles may interrogate different biological functions. As such, compounds targeting a specific biological pathway may in extreme cases either fail in a certain assay, or by contrast, may be flagged as false positives. Taking assay platform differences into account, and relying on good intra-assay variability for each assay optimized in our laboratories, the ATP, pLDH, luciferase reporter and PrestoBlue™ assays were compared in context of a blinded MMV 10-compound set. All the assays were performed in parallel on the same gametocyte population (except for the luciferase reporter lines). The remaining parameters for each assay were all comparable. In each case, the assay was performed for 48 h of continuous drug pressure for at least three replicates. Although direct comparison of absolute inhibition values are difficult between assay platforms, similar trends were observed including comparative performance of the luciferase marker assay and the PrestoBlue™ assay for e.g. DHA and Methylene blue. Interestingly, the ATP assay could not detect any inhibitory activity for some quinoline family members and may therefore be more sensitive in indicating the inability of these compounds to inhibit gametocytes. Data from the luciferase reporter assays for these compounds indicate that the compounds are indeed more active against early stage gametocytes. The signal obtained for these compounds in the PrestoBlue™ and pLDH assays may therefore rather reflect the inability of these assays in discriminating activity of compounds against earlier stages of gametocytes, whereas the ATP assay more accurately reflect these compounds’ activity. Whilst this data is informative from a biological perspective and may provide indications of the drug mode of action, it does highlight the care that has to be taken in screening platforms where compounds may be falsely assigned activity (or lack thereof) based on a single assay.