A novel Plasmodium falciparum expression system for assessing antifolate resistance caused by mutant P. vivax dihydrofolate reductase-thymidylate synthase.

With the emergence of drug-resistant vivax malaria, in vitro studies are urgently needed to examine resistance mechanisms and for drug development. Currently, Plasmodium vivax culturing is inadequate for addressing these needs; therefore, surrogate biological systems have been developed. Although these systems are informative, they do not address Plasmodium species-specific mechanisms, such as drug delivery through erythrocytes and parasite membranes. Here, we demonstrate that P. falciparum is an excellent biological system for expression of P. vivax dhfr-ts alleles to assess dihydrofolate reductase (DHFR)-thymidylate synthase interactions with antifolates. Our results show that the P. vivax dhfr-ts quadruple-mutant allele AMRU1, expressed in P. falciparum, provides significant protection against pyrimethamine, cycloguanil, and clocicguanil. Moreover, the PvDHFR quadruple mutant confers greater resistance to cycloguanil, clociguanil, and WR99210 than the PfDHFR quadruple mutant. Modeling of both P. vivax and P. falciparum DHFR quadruple mutants suggests that mutations unique to P. vivax DHFR are responsible for differences seen in parasite susceptibility to antifolates.

Discovery of an MIT-like atracotoxin family: spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins.

This project identified a novel family of six 66-68 residue peptides from the venom of two Australian funnel-web spiders, Hadronyche sp. 20 and H. infensa: Orchid Beach (Hexathelidae: Atracinae), that appear to undergo N- and/or C-terminal post-translational modifications and conform to an ancestral protein fold. These peptides all show significant amino acid sequence homology to atracotoxin-Hvf17 (ACTX-Hvf17), a non-toxic peptide isolated from the venom of H. versuta, and a variety of AVIT family proteins including mamba intestinal toxin 1 (MIT1) and its mammalian and piscine orthologs prokineticin 1 (PK1) and prokineticin 2 (PK2). These AVIT family proteins target prokineticin receptors involved in the sensitization of nociceptors and gastrointestinal smooth muscle activation. Given their sequence homology to MIT1, we have named these spider venom peptides the MIT-like atracotoxin (ACTX) family. Using isolated rat stomach fundus or guinea-pig ileum organ bath preparations we have shown that the prototypical ACTX-Hvf17, at concentrations up to 1muM, did not stimulate smooth muscle contractility, nor did it inhibit contractions induced by human PK1 (hPK1). The peptide also lacked activity on other isolated smooth muscle preparations including rat aorta. Furthermore, a FLIPR Ca2+ flux assay using HEK293 cells expressing prokineticin receptors showed that ACTX-Hvf17 fails to activate or block hPK1 or hPK2 receptors. Therefore, while the MIT-like ACTX family appears to adopt the ancestral disulfide-directed beta-hairpin protein fold of MIT1, a motif believed to be shared by other AVIT family peptides, variations in the amino acid sequence and surface charge result in a loss of activity on prokineticin receptors.

Disulfide bond mutagenesis and the structure and function of the head-to-tail macrocyclic trypsin inhibitor SFTI-1.

SFTI-1 is a novel 14 amino acid peptide comprised of a circular backbone constrained by three proline residues, a hydrogen-bond network, and a single disulfide bond. It is the smallest and most potent known Bowman-Birk trypsin inhibitor and the only one with a cyclic peptidic backbone. The solution structure of [ABA(3,11)]SFTI-1, a disulfide-deficient analogue of SFTI-1, has been determined by (1)H NMR spectroscopy. The lowest energy structures of native SFTI-1 and [ABA(3,11)]SFTI-1 are similar and superimpose with a root-mean-square deviation over the backbone and heavy atoms of 0.26 +/- 0.09 and 1.10 +/- 0.22 A, respectively. The disulfide bridge in SFTI-1 was found to be a minor determinant for the overall structure, but its removal resulted in a slightly weakened hydrogen-bonding network. To further investigate the role of the disulfide bridge, NMR chemical shifts for the backbone H(alpha) protons of two disulfide-deficient linear analogues of SFTI-1, [ABA(3,11)]SFTI-1[6,5] and [ABA(3,11)]SFTI-1[1,14] were measured. These correspond to analogues of the cleavage product of SFTI-1 and a putative biosynthetic precursor, respectively. In contrast with the cyclic peptide, it was found that the disulfide bridge is essential for maintaining the structure of these open-chain analogues. Overall, the hydrogen-bond network appears to be a crucial determinant of the structure of SFTI-1 analogues.

Sunflower trypsin inhibitor-1.

SFTI-1 is a bicyclic 14 amino acid peptide that was originally isolated from the seeds of the sunflower Helianthus annuus. It is a potent inhibitor of trypsin, with a sub-nanomolar K(i) value and is homologous to the active site region of the well-known family of serine protease inhibitors known as the Bowman-Birk trypsin inhibitors. It has a cyclic backbone that is cross-braced by a single disulfide bridge and a network of hydrogen bonds that result in a well-defined structure. SFTI-1 is amenable to chemical synthesis, allowing for the creation of synthetic variants. Alterations to the structure such as linearising the backbone or removing the disulfide bridge do not reduce the potency of SFTI-1 significantly, and minimising the peptide to as few as nine residues results in only a small decrease in reactivity. The creation of linear variants of SFTI-1 also provides a tool for investigating putative linear precursor peptides. The mechanism of biosynthesis of SFTI-1 is not yet known but it seems likely that it is a gene-coded product that has arisen from a precursor protein that may be evolutionarily related to classic Bowman-Birk inhibitors.

Enzymatic cyclization of a potent bowman-birk protease inhibitor, sunflower trypsin inhibitor-1, and solution structure of an acyclic precursor peptide.

The most potent known naturally occurring Bowman-Birk inhibitor, sunflower trypsin inhibitor-1 (SFTI-1), is a bicyclic 14-amino acid peptide from sunflower seeds comprising one disulfide bond and a cyclic backbone. At present, little is known about the cyclization mechanism of SFTI-1. We show here that an acyclic permutant of SFTI-1 open at its scissile bond, SFTI-1[6,5], also functions as an inhibitor of trypsin and that it can be enzymatically backbone-cyclized by incubation with bovine beta-trypsin. The resulting ratio of cyclic SFTI-1 to SFTI-1[6,5] is approximately 9:1 regardless of whether trypsin is incubated with SFTI-1[6,5] or SFTI-1. Enzymatic resynthesis of the scissile bond to form cyclic SFTI-1 is a novel mechanism of cyclization of SFTI-1[6,5]. Such a reaction could potentially occur on a trypsin affinity column as used in the original isolation procedure of SFTI-1. We therefore extracted SFTI-1 from sunflower seeds without a trypsin purification step and confirmed that the backbone of SFTI-1 is indeed naturally cyclic. Structural studies on SFTI-1[6,5] revealed high heterogeneity, and multiple species of SFTI-1[6,5] were identified. The main species closely resembles the structure of cyclic SFTI-1 with the broken binding loop able to rotate between a cis/trans geometry of the I7-P8 bond with the cis conformer being similar to the canonical binding loop conformation. The non-reactive loop adopts a beta-hairpin structure as in cyclic wild-type SFTI-1. Another species exhibits an iso-aspartate residue at position 14 and provides implications for possible in vivo cyclization mechanisms.