Friday, September 03, 2010

'huge black box'

Science Daily (September 2, 2010)

New Type of Anti-Malarial Compound Discovered

An international team led by scientists from The Scripps Research Institute, the Swiss Tropical Institute, the Genomics Institute of the Novartis Research Foundation and the Novartis Institute for Tropical Diseases has discovered a promising new drug candidate that represents a new class of drug to treat malaria. Clinical trials for the compound are planned for later this year.

The research is published on September 3, 2010, in the journal Science.

In Pursuit of a New Drug

The impetus for the new study began in the Scripps Research Winzeler laboratory about seven years ago when Winzeler received funding from the Keck Foundation to develop new antimalarial drugs by pursuing target-based drug discovery methods (designing a drug based on known molecular interactions). The approach was not yielding many interesting compounds, so Winzeler and her collaborators at GNF decided to take a different tack.

Noting that serendipity and observation played a role in all previous breakthrough antimalarials (for example, the drug artemisinin was derived from an herb used in traditional Chinese medicine), the team decided to pursue cell-based screening. The Winzeler lab at GNF then developed a high-throughput screen to look for compounds active against the malaria parasite Plasmodium falciparum. Scientists at Novartis, which had compiled a library of 12,000 purified natural products, then offered their library for the screen.

The first screen returned a set of 275 compounds with anti-malarial activity. Subsequent screens weeded out those with little activity against multi-drug resistant parasites and those toxic for mammalian cells. Seventeen compounds remained in the running.

An evaluation of the remaining compounds' toxicity and pharmacokinetic profiles provided additional information to evaluate their potential drug candidates. One compound -- belonging to a chemical class of molecules called spiroindolones, which had never before been associated with anti-malarial activity -- stood out as particularly promising.

Novartis Institute for Tropical Medicine's project team head Bryan Yeung noted, "Of the remaining compound classes, the spirotetrahydro-beta-carbolines or spiroindolones displayed the desired physicochemical properties for drug development, as well as a mechanism of action distinct from the currently used therapies based on aminoquinolines and artemisinin derivatives."

In an effort based at the Novartis Institute of Tropical Diseases in Singapore, the chemistry team synthesized and evaluated some 200 derivatives of this molecule to optimize its safety profile and pharmacokinetic properties. At the end of several hundred rounds of medicinal chemistry and efficacy testing at the Swiss Tropical and Public Health Institute, the team advanced NITD609 as the best candidate for proceeding to clinical trials.

Shining Light in the Black Box

The new study, however, doesn't stop there. To gain insight into how NITD609 worked, Winzeler applied a distinctive and elegant evolutionary approach.

Winzeler noted, "One of the disadvantages of doing cellular screening has been chemists will say, 'You don't know what the target is. You don't know if the parasites are going to become resistant to it. It's a huge black box.' It has been extremely difficult to find the genes involved in malarial drug resistance using traditional methods. So what we've been doing in my lab is developing ways to find single-base changes in drug-exposed genomes."

In this case, Case McNamara at GNF, a lead author, took a parasite and cloned it to create two identical organisms. One was allowed to reproduce in regular culture. The other was placed in a culture with a sub-lethal dose of the anti-malarial drug candidate. After three to four months and many generations, the parasites in the culture with NITD609 started to display low-level drug resistance.

At that point, the team used an advanced tiling array to compare the 26 million base pairs of coding sequence in the genome of the drug-exposed organisms to the genome of the control organisms.

"We were expecting hundreds or thousands of mutations because we grew the parasites for many generations," Winzeler said. "We got only a handful."

When McNamara analyzed the genomes of the six resistant clones, it turned out that all of the mutant strains had at least one mutation mapping to a single gene, pfatp4. This suggests that the protein PfATP4 is either the target for the new drug candidate or is involved in the parasite's resistance to it in some other way.

"PfATP4 is a cation transporting ATPase, so it is a very well validated drug target," said Winzeler. "That class of proteins, for example, is the target of antacids. It hasn't really been explored in malaria. This is one of the first cases where an evolution study has been used to identify the action of a compound in a parasite cell."


Spiroindolones, a new and potent chemotype for the treatment of malaria.
Matthias Rottmann, Case McNamara, Bryan K. S. Yeung, Marcus C. S. Lee, Bin Zou, Bruce Russell, Patrick Seitz, David M. Plouffe, Neekesh V. Dharia, Jocelyn Tan, Steven B. Cohen, Kathryn R. Spencer, Gonzalo E. González-Páez, Suresh B. Lakshminarayana, Anne Goh, Rossarin Suwanarusk, Timothy Jegla, Esther K. Schmitt, Hans-Peter Beck, Reto Brun, Francois Nosten, Laurent Renia, Veronique Dartois, Thomas H. Keller, David A. Fidock, Elizabeth A. Winzeler, Thierry T. Diagana
Science, 3 September 2010: Vol. 329. no. 5996, pp. 1175 – 1180 DOI: 10.1126/science.1193225

Link to Science abstract

Link to Science Daily article

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