Emilio Alvarez

Thousands of chemical starting points for antimalarial lead identification

Malaria is a devastating infection caused by protozoa of the genus Plasmodium. Drug resistance is widespread, no new chemical class of antimalarials has been introduced into clinical practice since 1996 and there is a recent rise of parasite strains with reduced sensitivity to the newest drugs. We screened nearly 2 million compounds in GlaxoSmithKline’s chemical library for inhibitors of P. falciparum, of which 13,533 were confirmed to inhibit parasite growth by at least 80% at 2 µM concentration. More than 8,000 also showed potent activity against the multidrug resistant strain Dd2. Most (82%) compounds originate from internal company projects and are new to the malaria community. Analyses using historic assay data suggest several novel mechanisms of antimalarial action, such as inhibition of protein kinases and host–pathogen interaction related targets. Chemical structures and associated data are hereby made public to encourage additional drug lead identification efforts and further research into this disease.

Screening and catalytic activity in organic synthesis of novel fungal and yeast lipases

A total of 969 microbial strains were isolated from soil samples and tested to determine their lipolytic activity by employing screening techniques on solid and in liquid media. Ten lipase-producing microorganisms were selected and their taxonomic identification was carried out. From these strains Achremonium murorum, Monascus mucoroides, Arthroderma ciferri, Fusarium poae, Ovadendron sulphureo-ochraceum and Rhodotorula araucariae are described as lipase-producers for the first time. Hydrolysis activity of the crude lipases against both tributyrin and olive oil was measured. Heptyl oleate synthesis was carried out to test the activity of the selected lipases as biocatalysts in organic medium. All the selected lipases were tested as biocatalysts in several organic reactions using unnatural substrates. Lipases from the fungi Fusarium. oxysporum and O. sulphureo-ochraceum gave the best yields and enantioselectivities in the esterification of carboxylic acids. F. oxysporum and Penicillium chrysogenum lipases were the most active ones for the acylation of alcohols without steric hindrance. A. murorum lipase is very useful for the esterification of menthol. F. oxysporum and Fusarium. solani lipases were very stereoselective in the synthesis of carbamates.

General characterization of noncommercial microbial lipases in hydrolytic and synthetic reactions

Fourteen noncommercial preparations of microbial lipases were investigated with respect to their catalytic activity for hydrolysis and synthesis of ester bonds. Six of the lipases were derived from microorganisms that have not previously been described as lipase producers, and another four were characterized for the first time. The synthetic reactions were carried out in two solvents of different polarities (n-heptane and acetone) using a series of fatty acids and primary and secondary alcohols with different chain lengths. Under the culture conditions employed, Pseudomonas cepacia produced more active enzyme than the other microorganisms. The lipase preparations produced using Ovadendron sulphureo-ochraceum, Monascus mucoroides, Monascus sp., Fusarium oxysporum, Penicillium chrysogenum, Rhodotorula araucariae, Pseudomonas cepacia, Streptomyces halstedii, and Streptomyces sp. were the most efficient catalysts for hydrolysis at lipid-water interfaces. Enzyme preparations from P. cepacia, Streptomyces sp., S. halstedii, and R. araucariae were good biocatalysts for esterification in the polar medium (acetone). When the lipase preparations with the greatest activity for hydrolytic, reactions were excluded, regression analysis of the data for the hydrolytic and synthetic activities of the remaining lipase preparations yielded high multiple correllation coefficients for these reactions in both n-heptane and acetone (R=0.82 and 0.91, respectively).

Antitubercular drugs for an old target: GSK693 as a promising InhA direct inhibitor

Despite being one of the first antitubercular agents identified, isoniazid (INH) is still the most prescribed drug for prophylaxis and tuberculosis (TB) treatment and, together with rifampicin, the pillars of current chemotherapy. A high percentage of isoniazid resistance is linked to mutations in the pro-drug activating enzyme KatG, so the discovery of direct inhibitors (DI) of the enoyl-ACP reductase (InhA) has been pursued by many groups leading to the identification of different enzyme inhibitors, active against Mycobacterium tuberculosis (Mtb), but with poor physicochemical properties to be considered as preclinical candidates. Here, we present a series of InhA DI active against multidrug (MDR) and extensively (XDR) drug-resistant clinical isolates as well as in TB murine models when orally dosed that can be a promising foundation for a future treatment.

Identifying inhibitors of the Leishmania inositol phosphorylceramide synthase with antiprotozoal activity using a yeast-based assay and ultra-high throughput screening platform

Leishmaniasis is a Neglected Tropical Disease caused by the insect-vector borne protozoan parasite, Leishmania species. Infection affects millions of the world’s poorest, however vaccines are absent and drug therapy limited. Recently, public-private partnerships have developed to identify new modes of controlling leishmaniasis. Drug discovery is a significant part of these efforts and here we describe the development and utilization of a novel assay to identify antiprotozoal inhibitors of the Leishmania enzyme, inositol phosphorylceramide (IPC) synthase. IPC synthase is a membrane-bound protein with multiple transmembrane domains, meaning that a conventional in vitro assay using purified protein in solution is highly challenging. Therefore, we utilized Saccharomyces cerevisiae as a vehicle to facilitate ultra-high throughput screening of 1.8 million compounds. Antileishmanial benzazepanes were identified and shown to inhibit the enzyme at nanomolar concentrations. Further chemistry produced a benzazepane that demonstrated potent and specific inhibition of IPC synthase in the Leishmania cell.

Three New Lipases from Actinomycetes and Their Use in Organic Reactions

Three novel lipase-producing microorganisms have been isolated from 526 actinomycete strains by employing screening techniques on solid media. Time-course and scale-up of enzyme production were analyzed. The lipases, produced by microorganisms belonging to the Streptomyces genus, were tested in several reactions in organic medium using unnatural substrates. The lyophilized crude lipases are stable at least for 1 month at 4°C (100% recovered activity). The lipase activity per milliliter of cell culture broth was higher than described in the literature for other lipases from actinomycetes. The three selected lipases displayed better activity than commercial lipase from Candida rugosa in the resolution of chiral secondary alcohols. The lipase from S. halstedii also displayed very good activity in the synthesis of carbamates.

Production of Tricarboxylic Acid Anhydrides from Fatty Acids by a Lipase-producing Strain of Pseudomonas cepacia

A lipase-producing strain of Pseudomonas cepacia isolated from a soil sample was found to produce five compounds when oleic acid was added to the culture medium as lipase inducer. The five compounds were isolated by solvent extraction, silicagel column chromatography and preparative HPLC, and their structural elucidation was performed by mass spectrometry, and infrared and nuclear magnetic resonance spectroscopies. The products were identified as dec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 1 ), undec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 2 ), dodec-3-ene-I,3,4-tricarboxylic acid 3,4-anhydride (product 3 ), dodec-3,8-diene-1,3,4-tricarboxylic acid 3,4-anhydride (product 4 ) and dodec-3,6-diene-I,3,4-tricarboxylic acid 3,4-anhydride (product 5 ). Accumulation of these compounds in the culture medium started after oleic acid consumption and followed a pattern similar to that found for cell growth and for lipase production. The five compounds were radioactively labeled when [U- 14 C]oleic acid was supplied to the culture medium, thus showing that they were produced by transformation of the acid. When isolated from cultures containing [1,2- 13 C]acetic acid and oleic acid as the sole sources of carbon, the compounds showed to contain the 13 C isotope only in the first five atoms of carbon of the molecule. Several long chain fatty acids also acted as precursors of these compounds, with maximal yields for chain lengths between 11 and 18 atoms of carbon. None of the five compounds acted as lipase inducer when added to the culture medium instead of oleic acid. The compounds showed moderate antibacterial and antifungal activities when tested in solid media bioassays.