Identification of a potent benzoxaborole drug candidate for treating cryptosporidiosis.

Cryptosporidiosis is a leading cause of life-threatening diarrhea in young children and causes chronic diarrhea in AIDS patients, but the only approved treatment is ineffective in malnourished children and immunocompromised people. We here use a drug repositioning strategy and identify a promising anticryptosporidial drug candidate. Screening a library of benzoxaboroles comprised of analogs to four antiprotozoal chemical scaffolds under pre-clinical development for neglected tropical diseases for Cryptosporidium growth inhibitors identifies the 6-carboxamide benzoxaborole AN7973. AN7973 blocks intracellular parasite development, appears to be parasiticidal, and potently inhibits the two Cryptosporidium species most relevant to human health, C. parvum and C. hominis. It is efficacious in murine models of both acute and established infection, and in a neonatal dairy calf model of cryptosporidiosis. AN7973 also possesses favorable safety, stability, and PK parameters, and therefore, is an exciting drug candidate for treating cryptosporidiosis.

Endoperoxide-8-aminoquinoline hybrids as dual-stage antimalarial agents with enhanced metabolic stability.

Hybrid compounds may play a critical role in the context of the malaria eradication agenda, which will benefit from therapeutic tools active against the symptomatic erythrocytic stage of Plasmodium infection, and also capable of eliminating liver stage parasites. To address the need for efficient multistage antiplasmodial compounds, a small library of 1,2,4,5-tetraoxane-8- aminoquinoline hybrids, with the metabolically labile C-5 position of the 8-aminoquinoline moiety blocked with aryl groups, was synthesized and screened for antiplasmodial activity and metabolic stability. The hybrid compounds inhibited development of intra-erythrocytic forms of the multidrug-resistant Plasmodium falciparum W2 strain, with EC50 values in the nM range, and with low cytotoxicity against mammalian cells. The compounds also inhibited the development of P. berghei liver stage parasites, with the most potent compounds displaying EC50 values in the low µM range. SAR analysis revealed that unbranched linkers between the endoperoxide and 8-aminoquinoline pharmacophores are most beneficial for dual antiplasmodial activity. Importantly, hybrids were significantly more potent than a 1:1 mixture of 8-aminoquinoline-tetraoxane, highlighting the superiority of the hybrid approach over the combination therapy. Furthermore, aryl substituents at C-5 of the 8-aminoquinoline moiety improve the compounds' metabolic stability when compared with their primaquine (i.e. C-5 unsubstituted) counterparts. Overall, this study reveals that blocking the quinoline C-5 position does not result in loss of dual-stage antimalarial activity, and that tetraoxane-8- aminoquinoline hybrids are an attractive approach to achieve elimination of exo- and intraerythrocytic parasites, thus with the potential to be used in malaria eradication campaigns.

Cryptosporidium and Toxoplasma Parasites Are Inhibited by a Benzoxaborole Targeting Leucyl-tRNA Synthetase.

The apicomplexan parasites Cryptosporidium and Toxoplasma are serious threats to human health. Cryptosporidiosis is a severe diarrheal disease in malnourished children and immunocompromised individuals, with the only FDA-approved drug treatment currently being nitazoxanide. The existing therapies for toxoplasmosis, an important pathology in immunocompromised individuals and pregnant women, also have serious limitations. With the aim of developing alternative therapeutic options to address these health problems, we tested a number of benzoxaboroles, boron-containing compounds shown to be active against various infectious agents, for inhibition of the growth of Cryptosporidium parasites in mammalian cells. A 3-aminomethyl benzoxaborole, AN6426, with activity in the micromolar range and with activity comparable to that of nitazoxanide, was identified and further characterized using biophysical measurements of affinity and crystal structures of complexes with the editing domain of Cryptosporidium leucyl-tRNA synthetase (LeuRS). The same compound was shown to be active against Toxoplasma parasites, with the activity being enhanced in the presence of norvaline, an amino acid that can be mischarged by LeuRS. Our observations are consistent with AN6426 inhibiting protein synthesis in both Cryptosporidium and Toxoplasma by forming a covalent adduct with tRNA(Leu) in the LeuRS editing active site and suggest that further exploitation of the benzoxaborole scaffold is a valid strategy to develop novel, much needed antiparasitic agents.

Lead identification to clinical candidate selection: drugs for Chagas disease.

Chagas disease affects 8 million people worldwide and remains a main cause of death due to heart failure in Latin America. The number of cases in the United States is now estimated to be 300,000, but there are currently no Food and Drug Administration (FDA)-approved drugs available for patients with Chagas disease. To fill this gap, we have established a public-private partnership between the University of California, San Francisco and the Genomics Institute of the Novartis Research Foundation (GNF) with the goal of delivering clinical candidates to treat Chagas disease. The discovery phase, based on the screening of more than 160,000 compounds from the GNF Academic Collaboration Library, led to the identification of new anti-Chagas scaffolds. Part of the screening campaign used and compared two screening methods, including a colorimetric-based assay using Trypanosoma cruzi expressing ß-galactosidase and an image-based, high-content screening (HCS) assay using the CA-I/72 strain of T. cruzi. Comparing molecules tested in both assays, we found that ergosterol biosynthesis inhibitors had greater potency in the colorimetric assay than in the HCS assay. Both assays were used to inform structure-activity relationships for antiparasitic efficacy and pharmacokinetics. A new anti-T. cruzi scaffold derived from xanthine was identified, and we describe its development as lead series.

4-Aminoquinoline-ß-lactam conjugates: synthesis, antimalarial, and antitubercular evaluation.

A library of quinoline-ß-lactam-based hybrids was synthesized and tested for their antimalarial and antitubercular activities. The present antimalarial data showed the dependence of activity on the nature of linker, N-1 substituent of the ß-lactam ring as well as the length of alkyl chain. Most of the compounds are not as efficient as chloroquine in inhibiting the culture growth of Plasmodium falciparum W2 strain. Nevertheless, the synthesized hybrids showed better antitubercular activities (up to five times) compared with cephalexin (up to three times) and ethionamide.

Compounds from Sorindeia juglandifolia (Anacardiaceae) exhibit potent anti-plasmodial activities in vitro and in vivo.

BACKGROUND: Discovering new lead compounds against malaria parasites is a crucial step to ensuring a sustainable global pipeline for effective anti-malarial drugs. As far as we know, no previous phytochemical or pharmacological investigations have been carried out on Sorindeia juglandifolia. This paper describes the results of an anti-malarial activity-driven investigation of the fruits of this Cameroonian plant. METHODS: Air-dried fruits were extracted by maceration using methanol. The extract was fractionated by flash chromatography followed by column chromatography over silica gel, eluting with gradients of hexane-ethyl acetate mixtures. Resulting fractions and compounds were tested in vitro against the Plasmodium falciparum chloroquine-resistant strain W2, against field isolates of P. falciparum, and against the P. falciparum recombinant cysteine protease falcipain-2. Promising fractions were assessed for acute toxicity after oral administration in mice. One of the promising isolated compounds was assessed in vivo against the rodent malaria parasite Plasmodium berghei. RESULTS: The main end-products of the activity-guided fractionation were 2,3,6-trihydroxy benzoic acid (1) and 2,3,6-trihydroxy methyl benzoate (2). Overall, nine fractions tested against P. falciparum W2 and falcipain-2 were active, with IC50 values of 2.3-11.6 µg/ml for W2, and 1.1-21.9 µg/ml for falcipain-2. Purified compounds (1) and (2) also showed inhibitory effects against P. falciparum W2 (IC50s 16.5 µM and 13.0 µM) and falcipain-2 (IC50s 35.4 and 6.1 µM). In studies of P. falciparum isolates from Cameroon, the plant fractions demonstrated IC50 values of 0.14-19.4 µg/ml and compounds (1) and (2) values of 6.3 and 36.1 µM. In vivo assessment of compound (1) showed activity against P. berghei strain B, with mean parasitaemia suppressive dose and curative dose of 44.9 mg/kg and 42.2 mg/kg, respectively. Active fractions were found to be safe in mice after oral administration of 7 g/kg body weight. CONCLUSIONS: Fractions of Sorindeia juglandifolia and two compounds isolated from these fractions were active against cultured malaria parasites, the P. falciparum protease falcipain-2, and in a rodent malaria model. These results suggest that further investigation of the anti-malarial activities of natural products from S. juglandifolia will be appropriate.

Diverse inhibitor chemotypes targeting Trypanosoma cruzi CYP51.

BACKGROUND: Chagas Disease, a WHO- and NIH-designated neglected tropical disease, is endemic in Latin America and an emerging infection in North America and Europe as a result of population moves. Although a major cause of morbidity and mortality due to heart failure, as well as inflicting a heavy economic burden in affected regions, Chagas Disease elicits scant notice from the pharmaceutical industry because of adverse economic incentives. The discovery and development of new routes to chemotherapy for Chagas Disease is a clear priority. METHODOLOGY/PRINCIPAL FINDINGS: The similarity between the membrane sterol requirements of pathogenic fungi and those of the parasitic protozoon Trypanosoma cruzi, the causative agent of Chagas human cardiopathy, has led to repurposing anti-fungal azole inhibitors of sterol 14α-demethylase (CYP51) for the treatment of Chagas Disease. To diversify the therapeutic pipeline of anti-Chagasic drug candidates we exploited an approach that included directly probing the T. cruzi CYP51 active site with a library of synthetic small molecules. Target-based high-throughput screening reduced the library of ∼104,000 small molecules to 185 hits with estimated nanomolar K(D) values, while cross-validation against T. cruzi-infected skeletal myoblast cells yielded 57 active hits with EC(50) <10 µM. Two pools of hits partially overlapped. The top hit inhibited T. cruzi with EC(50) of 17 nM and was trypanocidal at 40 nM. CONCLUSIONS/SIGNIFICANCE: The hits are structurally diverse, demonstrating that CYP51 is a rather permissive enzyme target for small molecules. Cheminformatic analysis of the hits suggests that CYP51 pharmacology is similar to that of other cytochromes P450 therapeutic targets, including thromboxane synthase (CYP5), fatty acid ω-hydroxylases (CYP4), 17α-hydroxylase/17,20-lyase (CYP17) and aromatase (CYP19). Surprisingly, strong similarity is suggested to glutaminyl-peptide cyclotransferase, which is unrelated to CYP51 by sequence or structure. Lead compounds developed by pharmaceutical companies against these targets could also be explored for efficacy against T. cruzi.

WormAssay: a novel computer application for whole-plate motion-based screening of macroscopic parasites.

Lymphatic filariasis is caused by filarial nematode parasites, including Brugia malayi. Adult worms live in the lymphatic system and cause a strong immune reaction that leads to the obstruction of lymph vessels and swelling of the extremities. Chronic disease leads to the painful and disfiguring condition known as elephantiasis. Current drug therapy is effective against the microfilariae (larval stage) of the parasite, but no drugs are effective against the adult worms. One of the major stumbling blocks toward developing effective macrofilaricides to kill the adult worms is the lack of a high throughput screening method for candidate drugs. Current methods utilize systems that measure one well at a time and are time consuming and often expensive. We have developed a low-cost and simple visual imaging system to automate and quantify screening entire plates based on parasite movement. This system can be applied to the study of many macroparasites as well as other macroscopic organisms.

Computer-aided drug design of falcipain inhibitors: virtual screening, structure-activity relationships, hydration site thermodynamics, and reactivity analysis.

Falcipains (FPs) are hemoglobinases of Plasmodium falciparum that are validated targets for the development of antimalarial chemotherapy. A combined ligand- and structure-based virtual screening of commercial databases was performed to identify structural analogs of virtual screening hits previously discovered in our laboratory. A total of 28 low micromolar inhibitors of FP-2 and FP-3 were identified and the structure-activity relationship (SAR) in each series was elaborated. The SAR of the compounds was unusually steep in some cases and could not be explained by a traditional analysis of the ligand-protein interactions (van der Waals, electrostatics, and hydrogen bonds). To gain further insights, a statistical thermodynamic analysis of explicit solvent in the ligand binding domains of FP-2 and FP-3 was carried out to understand the roles played by water molecules in binding of these inhibitors. Indeed, the energetics associated with the displacement of water molecules upon ligand binding explained some of the complex trends in the SAR. Furthermore, low potency of a subset of FP-2 inhibitors that could not be understood by the water energetics was explained in the context of poor chemical reactivity of the reactive centers of these compounds. The present study highlights the importance of considering energetic contributors to binding beyond traditional ligand-protein interactions.

Mining a cathepsin inhibitor library for new antiparasitic drug leads.

The targeting of parasite cysteine proteases with small molecules is emerging as a possible approach to treat tropical parasitic diseases such as sleeping sickness, Chagas' disease, and malaria. The homology of parasite cysteine proteases to the human cathepsins suggests that inhibitors originally developed for the latter may be a source of promising lead compounds for the former. We describe here the screening of a unique ∼ 2,100-member cathepsin inhibitor library against five parasite cysteine proteases thought to be relevant in tropical parasitic diseases. Compounds active against parasite enzymes were subsequently screened against cultured Plasmodium falciparum, Trypanosoma brucei brucei and/or Trypanosoma cruzi parasites and evaluated for cytotoxicity to mammalian cells. The end products of this effort include the identification of sub-micromolar cell-active leads as well as the elucidation of structure-activity trends that can guide further optimization efforts.

Enone- and chalcone-chloroquinoline hybrid analogues: in silico guided design, synthesis, antiplasmodial activity, in vitro metabolism, and mechanistic studies.

Analogues of the previously reported antimalarial hybrid compounds 8b and 12 were proposed with the aim of identifying compounds with improved solubility and retained antimalarial potency. In silico characterization predicted improved solubilities of the analogues, particularly at low pH; they retained acceptable predicted permeability properties but were predicted to be susceptible to hepatic metabolism. These analogues were synthesized and found to exhibit notable in vitro antimalarial activity. Compounds 25 and 27 were the most active of the analogues. In vitro metabolism studies indicated susceptibility of the analogues to hepatic metabolism. There was also evidence of primary glucuronidation for analogues 24-27. Presumed cis-trans isomerism of 12, 22, and 23 under in vitro metabolism assay conditions was also observed, with differences in the nature and rates of metabolism observed between isomers. Biochemical studies strongly suggested that inhibition of hemozoin formation is the primary mechanism of action of these analogues.

An image-based assay for high throughput screening of Giardia lamblia.

Giardia lamblia is a protozoan parasite that causes widespread gastrointestinal illness. Drugs to treat giardiasis are limited, but efforts to discover new anti-giardial compounds are constrained by the lack of a facile system for cell culture and inhibitor testing. We achieved robust and reproducible growth of G. lamblia in 384-well tissue culture plates in a modified TYI-S-33 medium. A high throughput assay for the screening of potential anti-giardial compounds was developed utilizing the WB strain of G. lamblia and automated optical detection of parasites after growth with tested inhibitors. We screened a library of 1600 known bioactive molecules and identified 12 compounds that inhibited growth of G. lamblia at low- or sub-micromolar concentrations. Our high throughput assay should facilitate evaluation of available chemical libraries for novel drugs to treat giardiasis.

Potent antiplasmodial extracts from Cameroonian Annonaceae.

AIM OF THE STUDY: In a search for new antimalarial leads, we have carried out a preliminary ethnopharmacological study with the aim of evaluating the in vitro antiplasmodial activity of extracts from thirteen Annonaceae species growing in Cameroon, and of assessing the acute toxicity of promising fractions in Swiss albino mice. MATERIALS AND METHODS: Plants were selected on the basis of an ethnobotanical survey carried out in four sites in centre and south regions of Cameroon (Yaoundé neighbourhoods, Kon-Yambetta, Ngobayang and Mbalmayo) on Annonaceae plants locally used to treat malaria and related symptoms. The choice of the sites was mainly based on environmental factors enabling mosquito breeding, cosmopolitan areas regrouping people from different cultural origins, areas with limited access to health centers, and areas with people relying exclusively on traditional medical practices. Collected materials were extracted by maceration in 95% ethanol. The crude extract was partitioned using organic solvents and the fractions afforded were evaluated for antiplasmodial activity in culture against the W2 strain of Plasmodium falciparum. Promising fractions (methanol fractions) were assessed for their acute toxicity in Swiss albino mice. RESULTS: From the results achieved, 37 (31.3%) out of 118 extracts tested exhibited antiplasmodial activity, with IC(50) values ranging from 1.07 µg/ml to 9.03 µg/ml. Of the active extracts, 29 (78.4%) were methanol fractions, 21 (72.4%) of which inhibited the parasites with IC(50)<5 µg/ml. The promising fractions proved to be safe through oral administration in mice. CONCLUSIONS: The activities and toxicity profiles of methanol fractions indicate that they deserve to be further investigated in detail for antimalarial lead discovery.

Antiplasmodial volatile extracts from Cleistopholis patens Engler & Diels and Uvariastrum pierreanum Engl. (Engl. & Diels) (Annonaceae) growing in Cameroon.

In a search for alternative treatment for malaria, plant-derived essential oils extracted from the stem barks and leaves of Cleistopholis patens and Uvariastrum pierreanum (Annonaceae) were evaluated in vitro for antiplasmodial activity against the W2 strain of Plasmodium falciparum. The oils were obtained from 500 g each of stem barks and leaves, respectively, by hydrodistillation, using a Clevenger-type apparatus with the following yields: 0.23% and 0.19% for C. patens and 0.1% and 0.3% for U. pierreanum (w/w relative to dried material weight). Analysis of 10% (v/v) oil in hexane by gas chromatography and mass spectrometry identified only terpenoids in the oils, with over 81% sesquiterpene hydrocarbons in C. patens extracts and U. pierreanum stem bark oil, while the leaf oil from the latter species was found to contain a majority of monoterpenes. For C. patens, the major components were α-copaene, δ-cadinene, and germacrene D for the stem bark oil and ß-caryophyllene, germacrene D, and germacrene B for the leaf oil. The stem bark oil of U. pierreanum was found to contain mainly ß-bisabolene and α-bisabolol, while α- and ß-pinenes were more abundant in the leaf extract. Concentrations of oils obtained by diluting 1-mg/mL stock solutions were tested against P. falciparum in culture. The oils were active, with IC(50) values of 9.19 and 15.19 µg/mL for the stem bark and leaf oils, respectively, of C. patens and 6.08 and 13.96 µg/mL, respectively, for those from U. pierreanum. These results indicate that essential oils may offer a promising alternative for the development of new antimalarials.

Chemical genetics of Plasmodium falciparum.

Malaria caused by Plasmodium falciparum is a disease that is responsible for 880,000 deaths per year worldwide. Vaccine development has proved difficult and resistance has emerged for most antimalarial drugs. To discover new antimalarial chemotypes, we have used a phenotypic forward chemical genetic approach to assay 309,474 chemicals. Here we disclose structures and biological activity of the entire library-many of which showed potent in vitro activity against drug-resistant P. falciparum strains-and detailed profiling of 172 representative candidates. A reverse chemical genetic study identified 19 new inhibitors of 4 validated drug targets and 15 novel binders among 61 malarial proteins. Phylochemogenetic profiling in several organisms revealed similarities between Toxoplasma gondii and mammalian cell lines and dissimilarities between P. falciparum and related protozoans. One exemplar compound displayed efficacy in a murine model. Our findings provide the scientific community with new starting points for malaria drug discovery.

On-bead screening of a combinatorial fumaric acid derived peptide library yields antiplasmodial cysteine protease inhibitors with unusual peptide sequences.

A new class of cysteine protease inhibitors based on fumaric acid derived oligopeptides was successfully identified from a high-throughput screening of a solid-phase bound combinatorial library. As target enzymes falcipain and rhodesain were used, which play important roles in the life cycles of the parasites which cause malaria (Plasmodium falciparum) and African sleeping sickness (Trypanosoma brucei rhodesiense). The best inhibitors with unusual amino acid sequences not reported before for this type of enzyme were also fully analyzed in detail in solution. K(i) values in the lower micromolar and even nanomolar region were found. Some inhibitors are even active against plasmodia and show good selectivity relative to other enzymes. Also the mechanism of action was studied and could be shown to be irreversible inhibition.

Antiplasmodial activity of extracts from seven medicinal plants used in malaria treatment in Cameroon.

AIM OF THE STUDY: In a search for new plant-derived biologically active compounds against malaria parasites, we have carried out an ethnopharmacological study to evaluate the susceptibility of cultured Plasmodium falciparum to extracts and fractions from seven Cameroonian medicinal plants used in malaria treatment. We have also explored the inhibition of the Plasmodium falciparum cysteine protease Falcipain-2. MATERIALS AND METHODS: Plant materials were extracted by maceration in organic solvents, and subsequently partitioned or fractionated to afford test fractions. The susceptibility of erythrocytes and the W2 strain of Plasmodium falciparum to plant extracts was evaluated in culture. In addition, the ability of annonaceous extracts to inhibit recombinant cysteine protease Falcipain-2 was also assessed. RESULTS AND DISCUSSION: The extracts showed no toxicity against erythrocytes. The majority of plant extracts were highly active against Plasmodium falciparumin vitro, with IC(50) values lower than 5 microg/ml. Annonaceous extracts (acetogenin-rich fractions and interface precipitates) exhibited the highest potency. Some of these extracts exhibited modest inhibition of Falcipain-2. CONCLUSION: These results support continued investigation of components of traditional medicines as potential new antimalarial agents.

Composition and anti-plasmodial activities of essential oils from some Cameroonian medicinal plants.

In a search for new plant-derived biologically active compounds against malaria parasites, five essential oils extracted from the Cameroonian plants Xylopia phloiodora, Pachypodanthium confine, Antidesma laciniatum, Xylopia aethiopica, and Hexalobus crispiflorus were evaluated in regard to their anti-plasmodial activity against the W2 strain of Plasmodium falciparum. The oils were obtained from the plants with 0.12, 0.13, 0.18, 0.6 and 0.1% yields (relatively to dried material weight) respectively. Analysis by gas chromatography and mass spectrometry identified mainly terpenoids, among which alpha-copaene, gamma-cadinene, delta-cadinene, alpha-cadinol, spathulenol and caryophyllene oxide were most commonly found. The five oils were active against Plasmodium falciparum in culture. The most effective was the oil of Hexalobus crispiflorus, with an IC50 of 2 microg/ml.

Synthesis and evaluation of isatins and thiosemicarbazone derivatives against cruzain, falcipain-2 and rhodesain.

While commercial isatins were practically inactive against the target proteases, thiosemicarbazone derivatives were found to be active. The most active compound from the series displayed an inhibitory IC(50) value of 1 microM against rhodesain. One thiosemicarbazone was found to be active against all three proteases with inhibitory IC(50) values of 10 microM or less. A combination of N-benzylation and appropriate substitution on the aromatic portion of the isatin scaffold was generally found to be beneficial especially against cruzain for ketone inhibitors.