High throughput screening (hts) of fundación Medina´s microbial extracts collection to identify and characterize anti-protozoal natural products

Resumen

Malaria and Neglected Tropical Diseases such as Human African Trypanosomiasis (Sleeping sickness), American Trypanosomiasis (Chagas Disease) and Leishmaniasis are four major protozoan diseases that still plague the world´s poorest populations causing hundreds of thousands of deaths each year. Current drugs used in combating these four protozoan diseases are woefully inadequate, have problems with parasite resistance and some even have toxic side effects causing as high as 20 % death in patients. There is an urgent need for innovation in finding lead compounds against these diseases. Natural products from microbial and plant sources offer a highly underexplored pool of unique chemical diversity for the discovering novel compounds against current unmet medical challenges. Chemical entities from this source can serve as excellent templates for the synthesis of novel bioactive compounds. With respect to the first and second specific objectives of this thesis, which involved setting up and validating an HTS platform for screening kinetoplastid parasites (chapter 1 section 1.6), this has been realized, as a new HTS platform has been set up in this thesis. The methodology for this platform has been described in full details for the pilot screening of a subset (5,976) of the microbial extracts collection of Fundación MEDINA against three neglected tropical disease parasites, namely, Trypanosoma cruzi, Leishmania donovani and Trypanosoma brucei brucei. From the primary screening through to the confirmatory and then dose response stages of the screening process, a total of eighty extracts (80) were selected in all the three parasites screened and further processed by early LC/MS de-replication and cytotoxicity test in a secondary intracellular L. donovani amastigote assay. At this stage, twenty six (26) extracts were excluded since they contained metabolites already known and four (4) additional extracts were excluded for their cytotoxicity in the L. donovani secondary assay. Hence from the eighty (80) extracts that were progressed from the dose response stage, a total of thirty (30) extracts were excluded at this early LC/MS de-replication and cytotoxicity stage, leaving fifty (50) extracts which were classified as potentially containing novel and active metabolites for further processing. Of this number only twenty six (26) extracts have so far been prioritized for fermentation in 100 mL medium scale volumes and followed up, hence twenty four (24) extracts remain, pending their fermentation and follow up in 100 mL volumes. Of the twenty six (26) extracts already fermented and followed up in 100 mL volumes, there were a total of four (4) extracts (1T. cruzi-active, 2 L. donovani-active and 1 T. b. brucei-active) in which the identity of the active components could not be confirmed by LC-HRMS and NMR due to extremely low concentrations problems. Hence, these four (4) extracts require fermentation in larger volumes (≥1 L) for further investigation, especially the T. cruzi-active extract in which additional preliminary work has already confirmed the interesting nature of the metabiolites that could be isolated here. Apart from the four (4) extracts mentioned above, the identity of the metabolites responsible for the activities observed in fifteen (15) extracts, have been identified and dereplicated. Additionally seven (7) extracts from this set have pending issues regarding the chemistry of the possible active metabolites, and are yet to be resolved. Regarding the known compounds dereplicated, the data from this HTS platform provides useful information for drug repositioning programs as an alternative path of providing useful therapeutic compounds in the fight against neglected tropical diseases. Since the screening platform has already been set up and validated in this thesis, it is currently available and ready to be used for the possible screening of the entire Fundación MEDINA´s microbial extract collection and other externally available collections against the three neglected tropical disease parasites used in setting up the platform. The third, fourth and fifth specific objectives of this thesis involved bioassay-guided compound isolation, structure elucidation and preclinical profiling of novel anti-protozoal compounds (chapter 1 section 1.6). For this purpose, there were two extracts (one from a fungus and the other from an actinomycete) previously identified from an HTS of Fundación MEDINA´s 22,000 microbial extracts against P.falciparum 3D7 parasites, which provided the starting materials this work. Bioassay-guided purification of the fungal extract produced by Stresseria sp., led to the isolation of a novel family of four nanomolar-active P. falciparum macrolides, namely, MDN-109, MDN-0110, MDN-0111 and MDN-0112. The planar structures of these four compounds have been elucidated and the novelty of their chemistry described. In a comprehensive preclinical study, these novel macrolides were tested against P. falciparum chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) parasites by LDH and SYBR Green fluorescence assays. The results from these experiments have shown that MDN-0109, MDN-0110 and MDN-0111, consistently exhibit potent nanomolar activities in both parasite strains. Further characterization of these novel macrolides have been performed in other protozoan parasites, namely, T. b. brucei, L. donovani and T. cruzi. Other tests performed with these compounds involved, cytotoxicity and cardiotoxicity studies in HepG2 (or THLE-2) hepatocytes and in a hERG channel-expressing HEK cell line. Metabolic stability and drug-drug interation studies in human liver microsomes (HLM), and also in vivo toxicity studies in mice have also been performed with these compounds. Respecting their potencies and issues surrounding their saftey and suitability for further development, the comprehensive preclinical study has cummulatively shown that MDN-0109 and MDN-0110 are the most readily suitable and promising compounds for further tests in efficacy studies. Although MDN-0111 was the most potent compound, it presented inherent problems of toxicity at the current state, whereas the least potent compound, MDN-0112, presented no safety issues at all. Although MDN-0111 and MDN-0112 could not be readily progressed into efficacy stuides in their current states, they will provide critical information with respect to SAR and mode of action stuides in further development of this novel family of macrolides for possible use in malaria chemotherapy. In addition to the above, bioassay-guided purification of the bacterial extract produced by the actinomycete Micromonospora sp., led to the isolation of a polycyclic xanthone (MDN-0185) with an IC50 of 9 nM against P. falciuparum 3D7 parasites. Structural elucidation of this compound identified it to be composed of a fusion between the ring systems of two known polycyclic xanthones (a fusion between the ABCD ring system of xantholipin and a derivative of the EFG ring system of the compound known as Sch 54445). As at the time of the writing of this thesis, simaomicin α was the only other polycyclic xanthone reported in the literature to posses antiplasmodial activity but in this case against P. falciuparum K1 parasites, hence highlighling the potential for further development of the newly discovered member of the xanthone family described in this thesis. This newly discovered polycyclic xanthone is yet to undergo preclinical profiling, just as has already been done for the novel macrolides discovered in this thesis.