Ana Maria Soto

Antimicrobial peptides with atypical structural features from the skin of the Japanese brown frog Rana japonica

Japonicin-1 (FFPIGVFCKIFKTC) and japonicin-2 (FGLPMLSILPKALCILLKRKC), two peptides with differential growth-inhibitory activity against the Gram-negative bacterium, Escherichia coli and the Gram-positive bacterium Staphylococcus aureus, were isolated from an extract of the skin of the Japanese brown frog Rana japonica. Both peptides show little amino acid sequence similarity to previously characterized antimicrobial peptides isolated from the skins of Ranid frogs. Circular dichroism studies, however, demonstrate that japonicin-2 adopts an alpha-helical conformation in 50% trifluoroethanol in common with many other cationic antimicrobial peptides synthesized in amphibian skin. Peptides belonging to the brevinin-1, brevinin-2, and tigerinin families, previously identified in the skins of Asian Ranid frogs, were not detected but a temporin-related peptide (ILPLVGNLLNDLL.NH(2); temporin-1Ja), that atypically bears no net positive charge, was isolated from the extract. The minimum inhibitory concentrations (MICs) of the peptides against E. coli were japonicin-1, 30 microM; japonicin-2, 12 microM; and temporin-1Ja > 100 microM. The MICs against S. aureus were japonicin-1, > 100 microM; japonicin-2, 20 microM; and temporin-1Ja, > 100 microM.

Antimicrobial properties of the frog skin peptide, ranatuerin-1 and its [Lys-8]-substituted analog

The predicted conformation of ranatuerin-1 (SMLSVLKNLG(10)KVGLGFVACK(20)INK QC), an antimicrobial peptide first isolated from the skin of the bullfrog Rana catesbeiana, comprises three structural domains: alpha-helix (residues 1-8), beta-sheet (residues 11-16) and beta-turn (residues 20-25). Circular dichroism studies confirm significant alpha-helical character in 50% trifluoroethanol. Replacement of Cys-19 and Cys-25 by serine resulted only in decreased antimicrobial potency but deletion of either the cyclic heptapeptide region [residues (19-25)] or the N-terminal domain [residues (1-8)] produced inactive analogs. Substitution of the glycine residues in the central domain of the [Ser-19, Ser-25] analog by lysine produced inactive peptides despite increased alpha-helical content and cationicity. The substitution Asn-8-->Lys gave a ranatuerin-1 analog with increased alpha-helicity and cationicity and increased potency against a range of Gram-positive and Gram-negative bacteria and against C. albicans but only a small increase (21%) in hemolytic activity. In contrast, increasing alpha-helicity and hydrophobicity by the substitution Asn-22-->Ala resulted in a 3.5-fold increase in hemolytic activity. Effects on antimicrobial potencies of substitutions of neutral amino acids at positions 4, 18, 22, and 24 by lysine were less marked. Strains of pathogenic E. coli from different groups showed varying degrees of sensitivity to ranatuerin-1 (MIC between 5 and 40 microM) but [Lys-8] ranatuerin-1 showed increased potency (between 2- and 8-fold; P < 0.01) against all strains. The data demonstrate that [Lys-8] ranatuerin-1 shows potential as a candidate for drug development.

Binding of ethidium to DNA measured using a 2D diffusion-modulated gradient COSY NMR experiment.

The binding of ethidium bromide to a DNA hairpin (dU5‐hairpin) was investigated using a novel 2D diffusion‐modulated gradient correlation spectroscopy (DMG‐COSY) experiment to evaluate the applicability of this technique for studying the binding of drugs to DNA. The DMG‐COSY experiment includes a preparation period during which coherent magnetization is attenuated due to molecular self‐diffusion. Magnetization then evolves due to scalar coupling during an evolution delay, and is detected using gradient pulses for coherence selection. The time‐domain data are processed in an analogous manner as for gradient‐selected COSY experiments. The diffusion coefficient for uridine in DMSO solution was determined from the H5–H6 crosspeak intensities for a series of 2D DMG‐COSY experiments that differed in the magnitude of the gradient pulses applied during the preparation period of the DMG‐COSY experiment. The diffusion coefficient for uridine calculated from the DMG‐COSY experiments was identical (within experimental error) to that determined from 1D diffusion experiments (5.24×10−6 cm2/s at 26°C). The diffusion coefficients for ethidium bromide and for the dU5‐hairpin were first measured separately using the DMG‐COSY experiment, and then measured in the putative complex. The diffusion coefficient for free ethidium bromide (4.15×10−6 cm2/s at 26°C) was considerably larger than for the dU5‐hairpin (1.60×10−6 cm2/s at 26°C), as expected for the smaller molecule. The diffusion coefficient for ethidium was markedly decreased upon addition of the dU5‐hairpin, consistent with complex formation (1.22×10−6 cm2/s at 26°C). Complex formation of 1:1 stoichiometry between ethidium and the stem of the dU5‐hairpin was verified independently by fluorescence spectroscopy. These results demonstrate the utility of the DMG‐COSY experiment for investigating the binding of drugs to DNA in aqueous solution.

Shape-selective recognition of a model Okazaki fragment by geometrically-constrained bis-distamycins.

Okazaki fragments represent interesting targets for the design of anticancer drugs because of their selective occurrence during DNA replication, a process often elevated in aggressive malignancies. Structural studies have indicated a bend occurs in the helical axis at the junction region (JR) that joins the DNA duplex region (DDR) and the RNA-DNA hybrid duplex region (HDR) of model Okazaki fragments. To identify a structural motif that provides a shape complementary to the Okazaki fragment minor groove, we have investigated the binding of geometrically-constrained bis-distamycins to a model Okazaki fragment, [OKA], with a sequence derived from the genome of simian virus 40 (SV40). Both the JR and the DDR of [OKA] contain consecutive A/T base pairs that could accommodate distamycin binding. Of the six bis-distamycins selected for analysis, the two with a para configuration of the distamycins on the benzene or pyridine scaffold bound [OKA] tightly (Kd approximately 10(-6) M from gel-shift assays; Kd approximately 10(-8) M from deltaT(M)) while the four with a meta orientation did not bind. The two mono-distamycins studied also did not bind [OKA]. Molecular modeling of the complex between the para bis-distamycin MT-9 and [OKA] revealed MT-9 adopted an S- shape complementary to the minor groove of the model Okazaki fragment.

Structure-activity relationships of trout bradykinin ([Arg0,Trp5,Leu8]-bradykinin])

Abstract Trout bradykinin ([Arg0,Trp5,Leu8]-BK) produces sustained and concentration–dependent contractions of isolated longitudinal smooth muscle from trout stomach, although mammalian BK is without effect. Circular dichroism studies have demonstrated that trout BK, unlike mammalian BK, does not adopt a stable β-turn conformation, even in the presence of sodium dodecyl sulfate (SDS) or trifluoroethanol. The myotropic actions of a series of analogs in which each amino acid in trout BK was replaced by either alanine or the corresponding D-isomer were investigated. The peptides with Ala4, D-Pro3, D-Trp5, D-Ser6, and D-Pro7 substitutions were inactive and did not act as antagonists of trout BK. The analog with [Ala5] was a weak partial agonist. The substitution (Arg0 → Ala) led to >50-fold decrease in potency but, in contrast to the importance of Phe8 in both BK and desArg9-BK in activating the mammalian B2 and B1 receptors respectively, substitutions at Leu8 in trout BK had only a minor effect on potency. Antagonists to the mammalian B2 receptor generally contain a D-aromatic amino acid at position 7 of BK but the analog [Arg0,Trp5,D-Phe7,Leu8]-BK was a weak agonist at the trout receptor. Similarly, the potent nonpeptide mammalian B2 receptor antagonist FR173657 was without effect on the action of trout BK. These data suggest the hypothesis that the receptor binding conformation of trout BK is defined by the central region (residues 3–7) of the peptide but is adopted only upon interaction with the receptor. The bioactive conformation is probably stabilized by an ionic interaction between Arg0 in the peptide and an acidic residue in the receptor.

Shape-selective binding of geometrically-constrained bis-distamycins to a DNA duplex and a model Okazaki fragment of identical sequence

Abstract The binding of ligands to nucleic acids is of great interest for the control of gene expression and other nucleic acid mediated processes. We have evaluated the binding of several geometrically-constrained bis-distamycins to a model Okazaki fragment [OKA], or a DNA duplex having identical base sequence [DD], using gel-shift assays, optical spectroscopy and differential scanning calorimetry. In the case of covalent attachment of two distamycins to a central benzene ring, a similar binding profile was observed for [DD] as was observed for [OKA] (para binds [Kapp > 106 M−1], meta binds only weakly). For a central pyridyl ring, however, clear distinction between the binding to [DD] and binding to [OKA] was observed. While none of the three meta isomers having a central pyridyl ring bound [OKA], two of them (MT-17 and MT-12) bound [DD] [Kapp > 106 M−1]. These results demonstrate subtle differences in lexitropsin shape and placement of electronegative atoms may result in selective binding to a nucleic acid duplex based both on base sequence and chemical composition. Selective binding to DNA duplexes may be useful for designing ligands that regulate transcription, but do not interfere in other nucleic acid mediated processes.