SEARCHING FOR NEW EFFECTIVE DRUGS AGAINST PARASITIC DISEASES

(Funding from the NIH SCORE Program Grant # SC1GM089558)

MALARIA
Malaria is one of the most prevalent and deadly parasitic diseases in the world. Up to 289 million cases of malaria may have occurred in 2010, causing between 660,000 and 1.25 million deaths, mainly in Africa and mostly of children younger than 5 years.
(WHO: http://www.who.int/malaria/publications/world_malaria_report_2012/en/index.html; Fidock, D. A. Eliminating Malaria. Science 2013, 340, 1531-1533.)




The most serious problem in malaria treatment is that the parasites causing the disease, particularly the deadly Plasmodium falciparum, have developed resistance to widely used drugs, particularly chloroquine (CQ). Currently, the most efficacious therapies are combinations of an artemisinin-type compound with a long-lasting partner drug like lumefantrine, amodiaquine or mefloquine.


Unfortunately, signs of resistance to artemisinin drugs are beginning to emerge in parts of Asia. Therefore new chemotherapeutic agents to treat drug-resistant malaria are urgently needed, and a good understanding of the mechanisms of therapeutic action, and of parasite drug resistance is essential for the development of new drugs. 

Our long-term goal is to discover new leads for anti-malarial agents, to elucidate their mechanisms of action, and to better understand the mechanism of resistance to CQ and related drugs. Our specific aims are:

•    To synthesize and evaluate the antimalarial potency of new compounds specifically designed to be active against resistant parasites

•    To understand the mechanisms of action of the new compounds

•  To understand the mechanism of resistance to chloroquine by computational modeling of the plasmodial resistance protein PfCRT, and its interactions with known and new drugs

RECENT ACCOMPLISHMENTS
 

Organometallic antimalarials:

We demonstrated that binding CQ to organo-ruthenium fragments selectively enhances the activity against CQ-resistant P. falciparum. The principal mechanism of action is heme aggregation inhibition, and the increased efficacy is due to a combination of structural and physicochemical effects that results in a marked lowering of the resistance.

New N-benzyl-4-aminoquinolines:

Amodiaquine (AQ) is effective against CQ-resistant parasites, but serious toxicity issues linked to a quinone-imine metabolite limit its clinical use. We synthesized the new N-benzyl-4-aminoquinolines 1-5, which are incapable of forming quinone-type metabolites. The new compounds display activity markedly superior to CQ and comparable to AQ against CQ-resistant P. falciparum, with very low toxicity to normal L6 mammalian cells
     
Biological data from Prof. J. Schrevel and coworkers at the National Museum of Natural History, Paris (France)

We have developed synthetic methods for a variety of new compounds with antimalarial potential, including

The antimalarial potency and cytotoxicity of the new compounds is currently being evaluated.
Mechanistic studies include the determination of physicochemical properties, heme aggregation inhibition ability, PfDHODH inhibition activity measurements, and metabolic stability assays.

Computational studies of chloroquine resistance:






A computational model of the PfCRT wild type and mutant proteins responsible for chloroquine resistance has been developed. Molecular docking methods are being applied to known drugs and to our lead compounds in order to locate binding pocket(s) in both wild type and mutant proteins. This modeling approach will lead us to a better understanding of the resistance mechanism and it will serve a predictive tool in the search for new drugs able to overcome malaria resistance. The interactions of different drug and drug-like aminoquinolines with heme are also studied using molecular docking and DFT methods.


CANCER
Chloroquine and related molecules have shown promise against certain types of cancer. Very recently, hydroxychloroquine has entered Phase II clinical trials as a possible effective treatment against metastatic pancreatic cancer (http://clinicaltrials.gov/ct2/show/NCT01273805).
In collaboration with Dr. Gary Schwartz, from Memorial Sloan Kettering Cancer Center, we have found that our compound (p-cymene)RuCl2(CQ) inhibits the in vitro growth of colon cancer cells, independently of p53 status, and more notably of dedifferentiated liposarcoma tumor cells lines (LS 141, IC50 8 μM), for which there is currently no chemotherapy available. In work with Prof. Renato Aguilera from the University of Texas at El Paso, we further observed interesting in vitro activities of Ru-complexes of chloroquine, clotrimazole, and ketokonazole against several cancer cell lines, predominantly by induction of apoptosis, and low toxicity against normal mouse and human cells.
        

CHAGAS DISEASE AND LEISHMANIASIS
Leishmaniasis is among the most neglected diseases, affecting the poorest populations of developing countries. This disease, caused by hemoflagellate protozoa of the genus Leishmania, is endemic in 88 countries; some 2 million new cases occur yearly and 350 million people are considered at risk of contracting leishmaniasis. American Trypanosomiasis (Chagas' disease), caused by Trypanosoma cruzi, is also largely neglected; it affects over 20 million people in Central and South America, of which up to 5 million develop severe digestive and cardiac impairments, causing 10-15,000 deaths per year and a heavy burden of persons unable to work. Available treatments for these ailments suffer, in most of cases, from toxicity problems, limited efficacy, and emerging resistance; thus, the search for novel chemotherapeutic agents requires urgent attention.
Over the last several years we have been searching for new metal-based therapies against Trypanosoma cruzi and Leishmania major, by combining a compound of known anti-parasitic activity like clotrimazole (CTZ) or ketoconazole (KTZ) and a metal (Ru, Rh, Ir, Au, Cu) in a single molecule. Our strategy makes use of the fact that (1) azole compounds cause a depletion of normal sterols and an accumulation of abnormal amounts of sterol precursors with cytostatic or cytotoxic consequences; and (2) Metals are known to bind to DNA, causing cellular disruption. The combination results in dual-target compounds with enhanced antiparasitic potency and reduced toxicity to mammalian cells.

We have synthesized a series of organo-Ru complexes of clotrimazole (CTZ) and ketoconazole (KTZ) with high potential as possible dual-target chemotherapeutic agents against Trypanosoma cruzi (Chagas) and Leishmania major (leishmaniasis). With Prof. Rosa A. Maldonado (U. Texas at El paso), we have demonstrated very high in vitro and in vivo activities against these pathogens by complexes like (p-cymene)RuCl2(Azole), [(p-cymene)RuCl(acac)(Azole)]BF4 (Azole is CTZ or KTZ) and related species. The metal-drugs also display high selectivity indexes with respect to normal mammalian cells in vitro and no acute toxicity in vivo.

 
The biological activity of these compounds is due to a dual mechanism, still under study, that involves intracellular dissociation of the azole ligand, which promotes sterol biosynthesis inhibition, plus selective binding of the organo-Ru fragment to the DNA of the parasites. The metal-azole drugs also inhibit the activity of protein kinases, particularly the human serine/threonine CAMKII (Ca2+-calmodulin) family, which has further mechanistic implications.


Recent Publications
  • E. Robles-Escajeda, A. Martínez, A. Varela-Ramirez, R. A. Sánchez-Delgado, and R. J. Aguilera,* “Analysis of the cytotoxic effects of ruthenium-ketoconazole and ruthenium-clotrimazole complexes on cancer cells” Cell Biol. Toxicol. 2013, 29, 431-443.

  • E. Iniguez, A. Sánchez, M. A. Vasquez, A. Martínez, J. Olivas, A. Sattler, R. A. Sánchez-Delgado*, and R. A. Maldonado*, “The Metal-Drug Synergy: New RutheniumII Complexes of Ketoconazole are Highly Active against Leishmania major and Trypanosoma cruzi and Non-toxic to Human or Murine Normal Cells” J. Biol. Inorg. Chem. 2013, 18, 779-790.

  • J. G. Estrada and R. A. Sánchez-Delgado,* “Spectroscopic Study of the Interactions of Ruthenium-Ketoconazole Complexes of Known Antiparasitic Activity with Human Serum Albumin and Apotransferrin”, J. Mex. Chem. Soc., 2013, 57, 169-174.

  • Martínez, T. Carreon, E. Iniguez, A. Anzellotti, A. Sánchez, M. Tyan, A. Sattler, L. Herrera, R. A. Maldonado* and R. A. Sánchez-Delgado*, “Searching for new chemotherapies for tropical diseases: Ruthenium-clotrimazole complexes display high in vitro activity against Leishmania major and Trypanosoma cruzi and low toxicity toward normal mammalian cells.” J, Med. Chem. 2012, 55, 3867-3877.

  • L. Glans, A. Ehnbom, C. de Kock, A. Martínez, J. Estrada, P. J. Smith, M. Haukka, R. A. Sánchez-Delgado* and E. Nordlander*, “Ruthenium(II) arene complexes with chelating chloroquine analogue ligands: Synthesis, characterization and in vitro antimalarial activity.” Dalton Trans. 2012, 41, 2764-2773.

  • M. Navarro,* W. Castro, A. R, Higuera-Padilla, A. Sierraalta, M. J. Abad, P. Taylor and R. A. Sánchez-Delgado, “Synthesis, characterization and biological activity of trans-platinum (II) complexes with chloroquine”, J. Inorg. Biochem. 2011, 105, 1684-1691.

  • Martínez, J. Suárez, T. Shand, R. S. Magliozzo and R. A. Sánchez-Delgado*, “Interactions of Arene-Ru(II)-Chloroquine Complexes of Known Antimalarial and Antitumor Activity with Human Serum Albumin (HSA) and Transferrin”, J. Inorg. Biochem. 2011, 105, 39-45.

  • A. Martínez, C. S. K. Rajapakse, A. Varela-Ramirez, C. Lema, R. Aguilera and R. A. Sánchez-Delgado*, “Arene-Ru(II)-Chloroquine Complexes Interact With DNA, Induce Apoptosis Against Human Lymphoid Cell Lines and Are Less Toxic to Normal Mammalian Cells than Chloroquine”, J. Inorg. Biochem. 2010, 104, 967-977.