Chemical synthesis was used to prepare the HIV-1 protease specifically 13C-labelled in the catalytically essential Asp 25 in each monomer. The NMR chemical shift of the 13C-enriched homodimeric enzyme was measured in the presence of the inhibitor pepstatin, a mimic of the tetrahedral intermediate formed in enzyme catalysis. In this complex, the catalytic carboxyls do not titrate in the pH range where the enzyme is active; throughout the range pH 2.5-6.5, one Asp 25 side chain is protonated and the other deprotonated. By contrast, in the absence of inhibitor the two Asp side chains are chemically equivalent and both deprotonated at pH6, the optimum for enzymatic activity. These direct observations of the chemical properties of the catalytic apparatus of the enzyme provide concrete information on which to base the design of improved HIV-1 protease inhibitors.
The malarial parasite Plasmodium falciparum depends on the purine salvage enzyme hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) to convert purine bases from the host to nucleotides needed for DNA and RNA synthesis. An approach to developing antimalarial drugs is to use HGXPRT to convert introduced purine base analogs to nucleotides that are toxic to the parasite. This strategy requires that these compounds be good substrates for the parasite enzyme but poor substrates for the human counterpart, HGPRT. Bases with a chlorine atom in the 6-position or a nitrogen in the 8-position exhibited strong discrimination between P. falciparum HGXPRT and human HGPRT. The k(cat)/K(m) values for the Plasmodium enzyme using 6-chloroguanine and 8-azaguanine as substrates were 50 - 80-fold and 336-fold higher than for the human enzyme, respectively. These and other bases were effective in inhibiting the growth of the parasite in vitro, giving IC(50) values as low as 1 microM.
Caribbean ciguatoxins (C-CTXs) are responsible for the
widespread occurrence of ciguatera in the
Caribbean Sea. The structure and configuration of C-CTX-1
(1), the major ciguatoxin isolated from the
horse-eye jack (Caranx latus), has been determined from DQF-COSY,
E-COSY, TOCSY, NOESY, ROESY, ge-HSQC, and HMQC experiments performed at 750 MHz and 500 MHz on a 0.13
μmol sample. C-CTX-1 ([M
+ H]+
m/z 1141.6 Da, molecular
formula C62H92O19) has a
ciguatoxin/brevetoxin ladder structure comprising
14 trans-fused, ether-linked rings (7/6/6/7/8/9/7/6/8/6/7/6/7/6)
assembled from 6 protonated fragments. The
relative stereochemistry and ring configuration of 1 was
determined from an analysis of coupling constant and
NOE data. Like ciguatoxins in the Pacific Ocean (P-CTX), C-CTX-1
possesses a flexible nine-membered
ring which may be a conserved feature among ciguatoxins. However,
C-CTX-1 has a longer contiguous carbon
backbone (57 vs 55 carbons for P-CTX-1), one extra ring, and a
hemiketal in ring N but no spiroketal as
found in P-CTX. C-CTX-1 possesses a primary hydroxyl which may
allow selective derivatization. A minor
analogue, C-CTX-2, was also isolated from fish and assigned the
structure 56 epi-C-CTX-1 (2), since it slowly
rearranged to C-CTX-1 in solution. Given the structural
similarities between Caribbean and Pacific ciguatoxins,
we propose that C-CTX-1 and C-CTX-2 arise from a Caribbean strain of
the benthic dinoflagellate,
Gambierdiscus toxicus.
Seven cysteine-rich repeats form the ligand-binding region of the low-density lipoprotein (LDL) receptor. Each of these repeats is assumed to bind a calcium ion, which is needed for association of the receptor with its ligands, LDL and beta-VLDL. The effects of metal ions on the folding of the reduced N-terminal cysteine-rich repeat have been examined by using reverse-phase high-performance liquid chromatography to follow the formation of fully oxidized isomers with different disulfide connectivities. In the absence of calcium many of the 15 possible isomers formed on oxidation, whereas in its presence the predominant product at equilibrium had the native disulfide bond connectivities. Other metals were far less effective at directing disulfide bond formation: Mn2+ partly mimicked the action of Ca2+, but Ba2+, Sr2+, and Mg2+ had little effect. This metal-ion specificity was also observed in two-dimensional 1H NMR spectral studies; only Ca2+ induced the native three-dimensional fold. The two paramagnetic ions, Gd3+ and Mn2+, and Cd2+ did not promote adoption of a well-defined structure, and the two paramagnetic ions did not displace calcium ions. The location of calcium ion binding sites in the repeat was also explored by NMR spectroscopy. The absence of chemical shift changes for the side chain proton resonances of Asp26, Asp36, and Glu37 from pH 3.9 to 6.8 in the presence of calcium ions and their proximal location in the NMR structures implicated these side chains as calcium ligands. Deuterium exchange NMR experiments also revealed a network of hydrogen bonds that stabilizes the putative calcium-binding loop.
Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) are the foremost causative agents of malaria. Due to the development of resistance to current antimalarial medications, new drugs for this parasitic disease need to be discovered. The activity of hypoxanthine-guanine-[xanthine]-phosphoribosyltransferase, HG[X]PRT, is reported to be essential for the growth of both of these parasites, making it an excellent target for antimalarial drug discovery. Here, we have used rational structure-based methods to design an inhibitor, [3R,4R]-4-guanin-9-yl-3-((S)-2-hydroxy-2-phosphonoethyl)oxy-1-N-(phosphonopropionyl)pyrrolidine, of PvHGPRT and PfHGXPRT that has K values of 8 and 7 nM, respectively, for these two enzymes. The crystal structure of PvHGPRT in complex with this compound has been determined to 2.85 Å resolution. The corresponding complex with human HGPRT was also obtained to allow a direct comparison of the binding modes of this compound with the two enzymes. The tetra-(ethyl l-phenylalanine) tetraamide prodrug of this compound was synthesized, and it has an IC of 11.7 ± 3.2 μM against Pf lines grown in culture and a CC in human A549 cell lines of 102 ± 11 μM, thus giving it a ∼10-fold selectivity index.
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