Biological and Medicinal Chemistry in Africa.

The young, fast-growing population of Africa means that harnessing the economic benefits of scientific research is critical to sustained and equitable growth in the continent. Moreover, the whole world would benefit from the added intellectual contribution that would come from nurturing African science. The high burden of neglected diseases in Africa makes chemical biology a particularly important field. In this editorial, the reconvergence of science conducted at the interface of chemistry and biology is placed in the context of African participation, its importance to global science and the unique blend of supporting and hindering factors that influence African scientific contributions. The new Biological and Medicinal Chemistry in Africa special collection showcases a broad spectrum of African chemical biology.

Native Mass Spectrometry-Guided Screening Identifies Hit Fragments for HOP-HSP90 PPI Inhibition.

Contemporary medicinal chemistry considers fragment-based drug discovery (FBDD) and inhibition of protein-protein interactions (PPI) as important means of expanding the volume of druggable chemical space. However, the ability to robustly identify valid fragments and PPI inhibitors is an enormous challenge, requiring the application of sensitive biophysical methodology. Accordingly, in this study, we exploited the speed and sensitivity of nanoelectrospray (nano-ESI) native mass spectrometry to identify a small collection of fragments which bind to the TPR2AB domain of HOP. Follow-up biophysical assessment of a small selection of binding fragments confirmed binding to the single TPR2A domain, and that this binding translated into PPI inhibitory activity between TPR2A and the HSP90 C-terminal domain. An in-silico assessment of binding fragments at the PPI interfacial region, provided valuable structural insight for future fragment elaboration strategies, including the identification of losartan as a weak, albeit dose-dependent inhibitor of the target PPI.

Native ion mobility mass spectrometry reveals that small organic acid fragments impart gas-phase stability to carbonic anhydrase II.

RATIONALE: A key element of studies that utilise ion mobility mass spectrometry (IM-MS) under native electrospray conditions for the analysis of protein-ligand binding is the maintenance of the native conformation of a protein during the removal of bulk solvent. Ruotolo and co-workers have demonstrated that the binding and subsequent dissociation of the anionic component of inorganic salts stabilise native protein conformations in the gas phase. In this study, we investigated the effect that organic acid fragments identified from a fragment-based drug discovery (FBDD) campaign might have on the gas-phase stability of carbonic anhydrase II (CA II). METHODS: We utilised native IM-MS to monitor changes in the conformation of CA II in the absence and presence of four acidic fragments. By performing a series of collision-induced unfolding (CIU) experiments we determined the effect of fragment binding on the gas-phase stability of CA II. RESULTS: Binding and dissociation of acidic fragments result in increased gas-phase stability of CA II. CFU experiments revealed that the native-like compact gas-phase conformation of the protein is stable with higher degree of pre-activation when bound to a series of acidic fragments. Importantly, although acetate was present in high concentrations, the stabilising effect was not observed without the addition of the acidic fragments. CONCLUSIONS: Binding and subsequent dissociation of acidic fragments from CA II significantly delayed CIU in a manner which is probably analogous to the effect of inorganic anions. Furthermore, we saw a slightly altered stabilising effect between the different fragments investigated in this study. This suggests that the prevention of CIU by organic acids may be tuneable to specific properties of a bound ligand. These observations may open avenues to exploit IM-MS as a screening platform in FBDD.

Use of a non-hepatic cell line highlights limitations associated with cell-based assessment of metabolically induced toxicity.

Metabolically induced drug-toxicity is a major cause of drug failure late in drug optimization phases. Accordingly, in vitro metabolic profiling of compounds is being introduced at earlier stages of the drug discovery pipeline. An increasingly common method to obtain these profiles is through overexpression of key CYP450 metabolic enzymes in immortalized liver cells, to generate competent hepatocyte surrogates. Enhanced cytotoxicity is presumed to be due to toxic metabolite production via the overexpressed enzyme. However, metabolically induced toxicity is a complex multi-parameter phenomenon and the potential background contribution to metabolism arising from the use of liver cells which endogenously express CYP450 isoforms is consistently overlooked. In this study, we sought to reduce the potential background interference by applying this methodology in kidney-derived HEK293 cells which lack endogenous CYP450 expression. Overexpression of CYP3A4 resulted in increased HEK293 proliferation, while exposure to four compounds with reported metabolically induced cytotoxicity in liver-derived cells overexpressing CYP3A4 resulted in no increase in cytotoxicity. Our results indicate that overexpression of a single CYP450 isoform in hepatic cell lines may not be a reliable method to discriminate which enzymes are responsible for metabolic induced cytotoxicity.

Synthesis, Structure and In Vitro Anti-Trypanosomal Activity of Non-Toxic Arylpyrrole-Based Chalcone Derivatives.

With an intention of identifying chalcone derivatives exhibiting anti-protozoal activity, a cohort of relatively unexplored arylpyrrole-based chalcone derivatives were synthesized in moderate to good yields. The resultant compounds were evaluated in vitro for their potential activity against a cultured Trypanosoma brucei brucei 427 strain. Several compounds displayed mostly modest in vitro anti-trypanosomal activity with compounds 10e and 10h emerging as active candidates with IC50 values of 4.09 and 5.11 µM, respectively. More importantly, a concomitant assessment of their activity against a human cervix adenocarcinoma (HeLa) cell line revealed that these compounds are non-toxic.

Identification, Isolation and Determination of Biomarkers for Quality Control of Bush Tea (Athrixia phyllicoides).

Athrixia phylicoides, known as "bush tea", grows abundantly in South Africa. An infusion of the leaves is used as a beverage and to treat a multitude of health conditions. The aim of this study was to investigate the chemical variation within A. phylicoides and to identify characteristic compounds for quality control. Samples from 12 locations in South Africa were analysed using ultra-performance liquid chromatography. Hierarchical cluster analysis of the aligned ultra-performance liquid chromatography-time-of-flight mass spectrometry data indicated two groups on the resulting dendrogram, representing 48 samples. Five marker compounds, identified through visual inspection and the construction of a discriminant analysis model, were evident on the ultra-performance liquid chromatography-MS profiles. Four of these compounds were isolated and identified, three as hydroxy methoxyflavones and the fourth as a coumarate, using nuclear magnetic resonance spectroscopy. An ultra-performance liquid chromatography-photodiode array method was developed and validated for the determination of the marker compounds using the isolates as standards. The limits of detection for the four compounds ranged from 0.92 - 2.50 µg/mL. Their recoveries at three concentration levels (1.00, 10.0, and 100 µg/mL) were between 97.0 and 101%, while acceptable intra- and inter-day precision was obtained as reflected by percentage relative standard deviation values below 2.24%. The concentrations of all the marker compounds were found to be higher in samples corresponding to Group 1 of the dendrogram than in those from Group 2. This may be attributable to differences in altitude, climate, and some edaphic factors. Identification of these marker compounds will make a valuable contribution towards the quality control and sustainable commercialisation of bush tea.

Application of the Huisgen cycloaddition and 'click' reaction toward various 1,2,3-triazoles as HIV non-nucleoside reverse transcriptase inhibitors.

The development of novel anti-HIV agents remains an important medicinal chemistry challenge given that no cure for the disease is imminent, and the continued use of current NNRTIs inevitably leads to problems associated with resistance. Inspired by the pyrazole-containing NNRTI lersivirine (LSV), we embarked upon a study to establish whether 1,2,3-triazole heterocycles could be used as a new scaffold for the creation of novel NNRTIs. An especially attractive feature of triazoles used for this purpose is the versatility in accessing variously functionalised systems using either the thermally regulated Huisgen cycloaddition, or the related 'click' reaction. Employing three alternative forms of these reactions, we were able to synthesise a range of triazole compounds and evaluate their efficacy in a phenotypic HIV assay. To our astonishment, even compounds closely mimicking LSV were only moderately effective against HIV.

Synthetic analogues of the marine bisindole deoxytopsentin: potent selective inhibitors of MRSA pyruvate kinase.

As part of an ongoing study to elucidate the SAR of bisindole alkaloid inhibitors against the evolutionary conserved MRSA pyruvate kinase (PK), we present here the synthesis and biological activity of six dihalogenated analogues of the naturally occurring sponge metabolite deoxytopsentin, including the naturally occurring dibromodeoxytopsentin. The most active compounds displayed potent low nanomolar inhibitory activity against MRSA PK with concomitant significant selectivity for MRSA PK over human PK orthologues. Computational studies suggest that these potent MRSA PK inhibitors occupy a region of the small interface of the enzyme tetramer where amino acid sequence divergence from common human PK orthologues may contribute to the observed selectivity.

Recent Advances in Drug Discovery from South African Marine Invertebrates.

Recent developments in marine drug discovery from three South African marine invertebrates, the tube worm Cephalodiscus gilchristi, the ascidian Lissoclinum sp. and the sponge Topsentia pachastrelloides, are presented. Recent reports of the bioactivity and synthesis of the anti-cancer secondary metabolites cephalostatin and mandelalides (from C. gilchristi and Lissoclinum sp., respectively) and various analogues are presented. The threat of drug-resistant pathogens, e.g., methicillin-resistant Staphylococcus aureus (MRSA), is assuming greater global significance, and medicinal chemistry strategies to exploit the potent MRSA PK inhibition, first revealed by two marine secondary metabolites, cis-3,4-dihydrohamacanthin B and bromodeoxytopsentin from T. pachastrelloides, are compared.

A native mass spectrometry approach to qualitatively elucidate interfacial epitopes of transient protein-protein interactions.

Native mass spectrometric analysis of TPR2A and GrpE with unpurified peptides derived from limited proteolysis of their respective PPI partners (HSP90 C-terminus and DnaK) facilitated efficient, qualitative identification of interfacial epitopes involved in transient PPI formation. Application of this approach can assist in elucidating interfaces of currently uncharacterised transient PPIs.

Methionine-Containing Peptides: Avoiding Secondary Reactions in the Final Global Deprotection.

The solid-phase synthesis of Met-containing peptides using a fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu) protection scheme is inevitably accompanied by two stubborn side reactions, namely, oxidation and S-alkylation (tert-butylation), which result in the formation of Met(O) and sulfonium salt impurities of the target peptide, respectively. These two reactions are acid-catalyzed, and they occur during the final trifluoroacetic (TFA)-based acidolytic cleavage step. Herein, we developed two new cleavage solutions that eradicate the oxidation and reduce S-alkylation. TFA-anisole-trimethylsilyl chloride (TMSCl)-Me2S-triisopropylsilane (TIS) containing 1 mg of triphenyl phosphine per mL of solution was the optimal mixture for Cys-containing peptides, while for the remaining peptides, TIS was not required. Both cleavage solutions proved to be excellent when sensitive amino acids such as Cys and Trp were involved. TMSCl did not affect either of these sensitive amino acids. Reversing the sulfonium salt to free Met-containing peptide was achieved by heating the peptide at 40 °C for 24 h using 5% acetic acid.

Into the Fray! A Beginner's Guide to Medicinal Chemistry.

Modern medicinal chemistry is a complex, multidimensional discipline that operates at the interface of the chemical and biological sciences. The medicinal chemistry contribution to drug discovery is typically described in the context of the well-recited linear progression of the drug discovery pipeline. However, compound optimization is idiosyncratic to each project, and clear definitions of hit and lead molecules and the subsequent progress along the pipeline becomes easily blurred. In addition, this description lacks insight into the entangled relationship between chemical and pharmacological properties, and thus provides limited guidance on how innovative medicinal chemistry strategies can be applied to solve optimization problems, regardless of the stage in the pipeline. Through discussion and illustrative examples, this article seeks to provide insights into the finesse of medicinal chemistry and the subtlety of balancing chemical properties pharmacology. In so doing, it aims to serve as an accessible and simple-to-digest guide for anyone who wishes to learn about the underlying principles of medicinal chemistry, in a context that has been decoupled from the pipeline description.

Virtual screening and in vitro validation identifies the first reported inhibitors of Salmonella enterica HPPK.

HPPK, which directly precedes DHPS in the folate biosynthetic pathway, is a promising but chronically under-exploited anti-microbial target. Here we report the identification of new S. enterica HPPK inhibitors, offering potential for new resistance circumventing S. enterica therapies as well as avenues for diversifying the current HPPK inhibitor space.

A native mass spectrometry platform identifies HOP inhibitors that modulate the HSP90-HOP protein-protein interaction.

Herein we describe a native mass spectromery protein-peptide model as a competent surrogate for the HOP-HSP90 protein-protein interaction (PPI), application of which led to the qualititive identification of two new peptides capable of in vitro PPI disruption. This proof of concept study offers a viable alternative for PPI inhibitor screening.

Anti-seizure activity of African medicinal plants: The identification of bioactive alkaloids from the stem bark of Rauvolfia caffra using an in vivo zebrafish model.

ETHNOPHARMACOLOGICAL RELEVANCE: Epilepsy is one of the major chronic diseases that does not have a cure to date. Adverse drug reactions have been reported from the use of available anti-epileptic drugs (AEDs) which are also effective in only two-thirds of the patients. Accordingly, the identification of scaffolds with promising anti-seizure activity remains an important first step towards the development of new anti-epileptic therapies, with improved efficacy and reduced adverse effects. Herbal medicines are widely used in developing countries, including in the treatment of epilepsy but with little scientific evidence to validate this use. In the search for new epilepsy treatment options, the zebrafish has emerged as a chemoconvulsant-based model for epilepsy, mainly because of the many advantages that zebrafish larvae offer making them highly suitable for high-throughput drug screening. AIM OF THE STUDY: In this study, 20 medicinal plants traditionally used in South Africa to treat epilepsy were screened for anti-epileptic activity using a zebrafish larvae model. MATERIALS AND METHODS: Toxicity triaging was conducted on 120 crude extracts, 44 fractions and three isolated compounds to determine the maximum tolerated concentration (MTC) of each extract, fraction or compound. MTC values were used to guide the concentration range selection in bioactivity studies. The effectiveness of crude extracts, fractions and isolated compounds from Rauvolfia caffra Sond. in suppression of pentylenetetrazole (PTZ) induced seizure-like behaviour in a 6-dpf zebrafish larvae model was measured using the PTZ assay. RESULTS: Following a preliminary toxicity triage and bioactivity screen of crude extracts from 20 African plants used traditionally for the treatment and management of epilepsy, the methanolic extract of Rauvolfia caffra Sond. was identified as the most promising at suppressing PTZ induced seizure-like behaviour in a zebrafish larvae model. Subsequent bioactivity-guided fractionation and spectroscopic structural elucidation resulted in the isolation and identification of two tryptoline derivatives; a previously unreported alkaloid to which we assigned the trivial name rauverine H (1) and the known alkaloid pleiocarpamine (2). Pleiocarpamine was found to reduce PTZ-induced seizures in a dose-dependent manner. CONCLUSIONS: Accordingly, pleiocarpamine represents a promising scaffold for the development of new anti-seizure therapeutic compounds. Furthermore, the results of this study provide preliminary evidence to support the traditional use of Rauvolfia caffra Sond. in the treatment and management of epilepsy. These findings warrant further studies on the anti-epileptic potential of Rauvolfia caffra Sond. using other models.

Recent Highlights in Anti-infective Medicinal Chemistry from South Africa.

Global advancements in biological technologies have vastly increased the variety of and accessibility to bioassay platforms, while simultaneously improving our understanding of druggable chemical space. In the South African context, this has resulted in a rapid expansion in the number of medicinal chemistry programmes currently operating, particularly on university campuses. Furthermore, the modern medicinal chemist has the advantage of being able to incorporate data from numerous related disciplines into the medicinal chemistry process, allowing for informed molecular design to play a far greater role than previously possible. Accordingly, this review focusses on recent highlights in drug-discovery programmes, in which South African medicinal chemistry groups have played a substantive role in the design and optimisation of biologically active compounds which contribute to the search for promising agents for infectious disease.

Insights into structural and physicochemical properties required for ß-hematin inhibition of privileged triarylimidazoles.

In this study, we investigated a series of triarylimidazoles, in an effort to elucidate critical SAR information pertaining to their anti-plasmodial and ß-hematin inhibitory activity. Our results showed that in addition to the positional effects of ring substitution, subtle changes to lipophilicity and imidazole ionisability were important factors in SAR interpretation. Finally, in silico adsorption analysis indicated that these compounds exert their effect by inhibiting ß-hematin crystal growth at the fast growing 001 face.

Detection of the in vitro modulation of Plasmodium falciparum Arf1 by Sec7 and ArfGAP domains using a colorimetric plate-based assay.

The regulation of human Arf1 GTPase activity by ArfGEFs that stimulate GDP/GTP exchange and ArfGAPs that mediate GTP hydrolysis has attracted attention for the discovery of Arf1 inhibitors as potential anti-cancer agents. The malaria parasite Plasmodium falciparum encodes a Sec7 domain-containing protein - presumably an ArfGEF - and two putative ArfGAPs, as well as an Arf1 homologue (PfArf1) that is essential for blood-stage parasite viability. However, ArfGEF and ArfGAP-mediated activation/deactivation of PfArf1 has not been demonstrated. In this study, we established an in vitro colorimetric microtiter plate-based assay to detect the activation status of truncated human and P. falciparum Arf1 and used it to demonstrate the activation of both proteins by the Sec7 domain of ARNO, their deactivation by the GAP domain of human ArfGAP1 and the inhibition of the respective reactions by the compounds SecinH3 and QS11. In addition, we found that the GAP domains of both P. falciparum ArfGAPs have activities equivalent to that of human ArfGAP1, but are insensitive to QS11. Library screening identified a novel inhibitor which selectively inhibits one of the P. falciparum GAP domains (IC50 4.7 µM), suggesting that the assay format is suitable for screening compound collections for inhibitors of Arf1 regulatory proteins.

Chemotypic variation of non-volatile constituents of Artemisia afra (African wormwood) from South Africa.

Artemisia afra (African wormwood) is a popular medicinal plant of southern Africa and is an excellent candidate for commercialisation. This current study was aimed at exploring the phytochemistry and chemical variation of non-volatile compounds within wild populations of A. afra, and developing chromatographic quality control protocols for raw materials based on the identification of marker compounds. Chromatographic data, from samples representing 12 distinct populations, were obtained using liquid chromatography-mass spectrometry. An untargeted chemometric approach revealed three clusters. Marker compounds for each cluster, revealed through discriminant analysis, were isolated and identified using NMR spectroscopy, as acacetin (1) (Group 1), chrysoeriol (2) (Group 2), and 3,5-di-O-caffeoylquinic acid (3) and scopoletin (4) (Group 3). In addition, (3) and rutin (5), (both reported for the first time from A. afra), and (1), (2), (4) and 4-caffeoylquinic acid (6) were established as reliable markers for species identification, since they were abundant in most samples. Quantitative analysis using a validated method established (4) as the dominant compound in the samples (1080-19,600 µg/g dry weight (d.w.)), followed by (5) (49.5-2490 µg/g d.w.). A high performance thin layer chromatography (HPTLC) method was developed. The Rf values and colours of the bands corresponding to the marker compounds were recorded so that these compounds could be easily identified for quality control purposes. Multivariate analysis of the data using the rTLC online application confirmed the presence of different chemical groupings within the samples. It was deduced that quantitative, rather than qualitative differences, characterised the samples. Future research should focus on comparing the efficacy of the various chemical clusters in multi-target biological assays aligned to the traditional use of the plant.

Unpacking the Pathogen Box-An Open Source Tool for Fighting Neglected Tropical Disease.

The Pathogen Box is a 400-strong collection of drug-like compounds, selected for their potential against several of the world's most important neglected tropical diseases, including trypanosomiasis, leishmaniasis, cryptosporidiosis, toxoplasmosis, filariasis, schistosomiasis, dengue virus and trichuriasis, in addition to malaria and tuberculosis. This library represents an ensemble of numerous successful drug discovery programmes from around the globe, aimed at providing a powerful resource to stimulate open source drug discovery for diseases threatening the most vulnerable communities in the world. This review seeks to provide an in-depth analysis of the literature pertaining to the compounds in the Pathogen Box, including structure-activity relationship highlights, mechanisms of action, related compounds with reported activity against different diseases, and, where appropriate, discussion on the known and putative targets of compounds, thereby providing context and increasing the accessibility of the Pathogen Box to the drug discovery community.