Several new fluorescence malaria in vitro drug susceptibility microtiter plate assays that detect the presence of malarial DNA in infected erythrocytes have recently been reported, in contrast to traditional isotopic screens that involve radioactive substrate incorporation to measure in vitro malaria growth inhibition. We have assessed and further characterized the malaria SYBR Green I-based fluorescence (MSF) assay for its ability to monitor drug resistance. In order to use the MSF assay as a drug screen, all assay conditions must be thoroughly examined. In this study we expanded upon the capabilities of this assay by including antibiotics and antifolates in the drug panel and testing folic acid-free growth conditions. To do this, we evaluated a more expansive panel of antimalarials in combination with various drug assay culture conditions commonly used in drug sensitivity screening for their activity against Plasmodium falciparum strains D6 and W2. The detection and quantitation limits of the MSF assay were 0.04 to 0.08% and ϳ0.5% parasitemia, respectively. The MSF assay quality was significantly robust, displaying a Z range of 0.73 to 0.95. The 50% inhibitory concentrations for each drug and culture condition combination were determined by using the MSF assay. Compared to the standard [ 3 H]hypoxanthine assay, the MSF assay displayed the expected parasite drug resistance patterns with a high degree of global and phenotypic correlation (r 2 > 0.9238), regardless of which culture condition combination was used. In conclusion, the MSF assay allows for reliable one-plate high-throughput, automated malaria in vitro susceptibility testing without the expense, time consumption, and hazard of other screening assays.

The discovery of the rules governing the inhibition of the various HDAC isoforms is likely to be key to identifying improved therapeutics that act as epigenetic modulators of gene transcription. Herein we present results on the modification of the CAP region of a set of triazolylphenyl-based HDACIs, and show that the nature of substitution on the phenyl ring plays a role in their selectivity for HDAC1 versus HDAC6, with low to moderate selectivity (2-51-fold) being achieved. In light of the valuable selectivity and potency that were identified for the triazolylphenyl ligand 6b in the inhibition of HDAC6 (IC50 = 1.9 nM), this compound represents a valuable research tool and a candidate for further chemical modifications. Lastly, these new HDACIs were studied for both their anticancer and antimalarial activity, which serve to validate the superior activity of the HDACI 10c.

Cyclin dependent protein kinases (CDKs) have become attractive drug targets in an effort to identify effective inhibitors of the parasite Plasmodium falciparum, the causative agent of the most severe form of human malaria. We tested known CDK inhibitors for their ability to inhibit two malarial CDKs: Pfmrk and PfPK5. Many broad spectrum CDK inhibitors failed to inhibit Pfmrk suggesting that the active site differs from other CDKs in important ways. By screening compounds in the Walter Reed chemical database, we identified oxindole-based compounds as effective inhibitors of Pfmrk (IC(50) = 1.5 microM). These compounds have low cross-reactivity against PfPK5 and human CDK1 demonstrating selectivity for Pfmrk. Amino acid comparison of the active sites of Pfmrk and PfPK5 identified unique amino acid differences that may explain this selectivity and be exploited for further drug development efforts.

The clinical potential of mefloquine has been compromised by reports of adverse neurological effects. A series of 4-quinolinecarbinolamines were compared in terms of neurotoxicity and antimalarial activity in an attempt to identify replacement drugs. Neurotoxicity (MTT [thiazolyl blue reduction] assay) was assessed by exposure of cultured embryonic rat neurons to graded concentrations of the drugs for 20 min. The 50% inhibitory concentration (IC 50 ) of mefloquine was 25 M, while those of the analogs were 19 to 200 M. The relative (to mefloquine) therapeutic indices of the analogs were determined after using the tritiated hypoxanthine assay for assessment of the antimalarial activity of the analogs against mefloquine-sensitive (W2) and -resistant (D6 and TM91C235) Plasmodium falciparum strains. Five analogs, WR157801, WR073892, WR007930, WR007333, and WR226253, were less neurotoxic than mefloquine and exhibited higher relative therapeutic indices (RTIs) against TM91C235 (2.9 to 12.2). Conventional quinoline antimalarials were generally less neurotoxic (IC 50 s of 400, 600, and 900 for amodiaquine, chloroquine, and quinine) or had higher RTIs (e.g., 30 for halofantrine against TM91C235). The neurotoxicity data for the 4-quinolinecarbinolamines were used to develop a three-dimensional (3D), function-based pharmacophore. The crucial molecular features correlated with neurotoxicity were a hydrogen bond acceptor (lipid) function, an aliphatic hydrophobic function, and a ring aromatic function specifically distributed in the 3D surface of the molecule. Mapping of the 3D structures of a series of structurally diverse quinolines to the pharmacophore allowed accurate qualitative predictions of neurotoxicity (or not) to be made. Extension of this in silico screening approach may aid in the identification of less-neurotoxic quinoline analogs.

The importance of fatty acids to the human malaria parasite, Plasmodium falciparum, and differences due to a type I fatty acid synthesis (FAS) pathway in the parasite, make it an attractive drug target. In the present study, we developed and a utilized a pharmacophore to select compounds for testing against PfKASIII, the initiating enzyme of FAS. This effort identified several PfKASIII inhibitors that grouped into various chemical classes of sulfides, sulfonamides, and sulfonyls. Approximately 60% of the submicromolar inhibitors of PfKASIII inhibited in vitro growth of the malaria parasite. These compounds inhibited both drug sensitive and resistant parasites and testing against a mammalian cell line revealed an encouraging in vitro therapeutic index for the most active compounds. Docking studies into the active site of PfKASIII suggest a potential binding mode that exploits amino acid residues at the mouth of the substrate tunnel.

Sophisen, a new ophthalmic drug carrier, was characterized using physicochemical and morphological criteria. Diclofenac belongs to a nonsteroidal anti-inflammatory molecule group and its ophthalmic use avoids side effects produced by steroid drugs. Cyclosporine-A is a cyclic peptide used as an immunosuppressive when administrated systemically. Its application in ophthalmology has been reported, but it is a very poor soluble drug. Diclofenac sodium and Cyclosporine-A were mixed with Sophisen to render two new ophthalmic solutions that were named 3A Ofteno and Modusik-A Ofteno, respectively. Based on transmission electron microscopy and dynamic light scattering studies, we concluded that Sophisen is a polydisperse solution with a molecular weight of 413 +/-122 kDa, whereas 3A Ofteno and Modusik-A Ofteno are monodisperse solutions with molecular weights of 169 +/- 44 and 153 +/- 10, respectively. Sophisen was shown to be a good carrier for diclofenac sodium as evaluated by passive diffusion through the cornea. A comparative study suggests that diclofenac applied as eye drops was better tolerated when associated with Sophisen. In addition, Modusik-A Ofteno, a new aqueous solution of Cyclosporine-A, improved tear production in patients with moderate or severe dry eye condition.

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