Hands-On Introduction to Synthetic Biology for Security Professionals.

The rapid pace of life sciences innovations and a growing list of nontraditional actors engaging in biological research make it challenging to develop appropriate policies to protect sensitive infrastructures. To address this challenge, we developed a five-day awareness program for security professionals, including laboratory work, site visits, and lectures.

Point mutations in the dihydrofolate reductase and dihydropteroate synthase genes of Plasmodium falciparum and resistance to sulfadoxine-pyrimethamine in Sri Lanka.

Sulfadoxine-pyrimethamine (SP) is the second-line treatment for Plasmodium falciparum malaria in Sri Lanka. Resistance to SP is caused by point mutations in the dihydrofolate reductase (Pf-dhfr) and dihydropteroate synthase (Pf-dhps) genes of P. falciparum. We determined the genotype of Pf-dhfr and Pf-dhps and the clinical response to SP in 30 field isolates of P. falciparum from Sri Lanka. All patients treated with SP had an adequate clinical response. Eighty-five percent (23 of 27) of pure field isolates carried parasites with double mutant alleles of Pf-dhfr (C59R + S108N) and showed about 200-fold higher levels of resistance to pyrimethamine than the wild type in a yeast system. None of the isolates had either known or novel mutations at other positions in the dhfr domain. In contrast, 67% (20 of 30) of the isolates carried parasites that were wild type for Pf-dhps. In Sri Lanka, detection of the triple mutant allele of Pf-dhfr will require tracking mutations at codon 51.

Identification of the optimal third generation antifolate against P. falciparum and P. vivax.

Inhibitors of dihydrofolate reductase (DHFR) have been mainstays in the treatment of falciparum malaria. Resistance to one of these antifolates, pyrimethamine, is now common in Plasmodium falciparum populations. Antifolates have not traditionally been recommended for treatment of vivax malaria. However, recent studies have suggested that a third-generation antifolate, WR99210, is remarkably effective even against highly pyrimethamine-resistant parasites from both species. Two methods were used to identify a compound that is effective against quadruple mutant alleles from P. falciparum (N51I/C59R/S108N/I164L) and from Plasmodium vivax (57L/111L/117T/173F). The first was simple yeast system used to screen a panel of WR99210 analogs. The biguanide prodrug, JPC-2056, of the 2-chloro-4-trifluoromethoxy analog of WR99210 was effective against both the P. falciparum and P. vivax enzymes, and has been selected for further development. The second method compared the analogs in silico by docking them in the known structure of the P. falciparum DHFR-thymidylate synthase. The program reproduced well the position of the triazine ring, but the calculated energies of ligand binding were very similar for different compounds and therefore did not reproduce the observed trends in biological activity. The WR99210 family of molecules is flexible due to a long bridge between the triazine ring and the substituted benzene. During docking, multiple conformations were observed for the benzene ring part of the molecules in the DHFR active site, making computer-based predictions of binding energy less informative than for more rigid ligands. This flexibility is a key factor in their effectiveness against the highly mutant forms of DHFR.

Novel alleles of Plasmodium falciparum dhfr that confer resistance to chlorcycloguanil.

In Plasmodium falciparum, resistance to folate inhibitors like pyrimethamine is mediated by point mutations in the target gene dihydrofolate reductase (dhfr). The resistance to pyrimethamine increases with the accumulation of particular point mutations. These mutations also confer increased resistance to chlorcycloguanil, the active metabolite of chlorproguanil and one component of a newly introduced DHFR inhibitor, LapDap. One genotype (16V/108T) has been previously identified that confers resistance to cycloguanil but not to pyrimethamine. This study was designed to identify novel alleles that might confer resistance to chlorcycloguanil, but escape the surveillance methods currently in place for common pyrimethamine-resistant alleles. Directed mutagenesis was performed using the wild type and the common pyrimethamine-resistant allele, 51I/59R/108N, to determine the effect of the 16V and 108T mutations on enzyme activity and drug resistance. In addition, we randomly mutagenized the 51I/59R/108N allele and identified nine novel alleles that could confer resistance to chlorcycloguanil. These yeast strains were also resistant to pyrimethamine, but retained sensitivity to the experimental DHFR inhibitor, WR99210. None of the alleles generated in this study was as resistant to chlorcycloguanil as the common quadruple mutant, 51I/59R/108N/164L. In addition, selection of high levels of chlorcycloguanil resistance in parasites that carry the 51I/59R/108N allele will require two directed steps, a change from 108N to 108T followed by a mutation from A16 to 16V. The resulting allele, 16V/51I/59R/108T is highly resistant to chlorcycloguanil, but 200-fold more sensitive to pyrimethamine than the 51I/59R/108N allele.