Rohanah Hussain

Identification and Characterization of Novel Lipophilic Antimicrobial Peptides Derived from Naturally Occurring Proteins

Microbes are increasingly developing defensive mechanisms against known drugs via mutations. There are signs of emergence of superbugs immune to most known antibiotics available. The need for a new class of drugs to counteract this problem is of paramount importance for continued general well being of mankind. A new class of drugs, antimicrobial peptides, has not been fully exploited primarily due to high cytotoxicity, poor lipophilicity preventing systemic distribution and stability. We have synthesised 9-amino acid residue cationic peptides RH01 and RH02 lipidated with myristoyl and octyl groups respectively. These peptides exhibited potent antimicrobial activity and low cytotoxicity. The lipopeptide RH01 has antimicrobial activity against a broad range of microorganisms including bacteria, yeast and filamentous fungi with greatest activity toward Gram-positive bacteria, including S. aureus MRSA stain, MIC’s ranging between 2–8xa0μM. The MIC for Gram-negative bacteria was higher ranging from between 30–250xa0μM. RH01 also had antimicrobial activity towards fungi showing good activity against the pathogenic yeast Candida albicans but was less active towards the filamentous fungi Aspergillus niger. The antimicrobial activity of RH01 as a measure of Ki(50) for E. coli and S. aureus was 35–60xa0μM and 3–7xa0μM, respectively. In-house data showed the compound is bactericidal even at higher bacteria concentration. The octylated lipopeptide RH02 has similar activities towards S. aureus (3.3xa0μM) and E coli (53.3xa0μM) as the myristolated RH01. There was no haemolytic activity of the lipopeptide RH01 towards human blood. Acute intravenous toxicity study in mice showed that both RH01 and RH02 induced no macroscopic abnormalities at their highest non-lethal dose of 75xa0mg/kg and 150xa0mg/kg bodyweight, respectively.

TOAC: a useful Cα-tetrasubstituted α-amino acid for peptide conformational analysis by CD spectroscopy in the visible region. Part I

Doubly labelled 4-amino-4-carboxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TOAC)-containing trichogin analogues showed a correlation between the CD intensity of the TOAC transition and their conformation. The helical-inducing property of the TOAC residue is position dependent and, apart from the N-terminal position, better than that of Aib.

Applications of Circular Dichroism

Circular dichroism (CD) spectroscopy is the technique of choice to study chiral molecules in solution, in particular biologically important molecules such as proteins, nucleic acids, carbohydrates, and therapeutic drugs. An important application of CD spectroscopy is the determination of the equilibrium dissociation constant, Kd, of binding interactions between a macromolecular host and a ligand. The Kd can be calculated by analyzing CD data at fixed wavelength versus ligand concentration using a nonlinear regression method. Several examples of CD titrations are illustrated here to describe the ‘golden’ rules on how to determine the molecular binding interactions of nonimmobilized and nonlabeled systems of biologically important molecules by CD spectroscopy.

Circular Dichroism, Applications

Circular dichroism (CD) spectroscopy is the technique of choice to study chiral molecules in solution, in particular biologically important molecules such as proteins, nucleic acids, carbohydrates, and therapeutic drugs. An important application of CD spectroscopy is the determination of the equilibrium dissociation constant, K d , of binding interactions between a macromolecular host and a ligand. The K d can be calculated by analyzing CD data at fixed wavelength versus ligand concentration using a nonlinear regression method. Several examples of CD titrations are illustrated here to describe the ‘golden’ rules on how to determine the molecular binding interactions of nonimmobilized and nonlabeled systems of biologically important molecules by CD spectroscopy.

B23 circular dichroism beamline at the Diamond Light Source

Circular dichroism spectroscopy is a useful and versatile tool to obtain low-resolution structural information about proteins, biopolymers and other chiral materials in solution. The first UV–VIS beamline dedicated to circular dichroism, B23, at Diamond Light Source Ltd., a third-generation synchrotron facility in the UK, has recently become operational and is now available for the user community. Herein we summarize the main characteristics of the beamline and some possible applications.

The use of CD spectroscopy for preliminary detection of drug bioavailability and interaction

Pharmacokinetics, bioavailability and interactions of pharmaceuticals are important features for the formulation and drug dosage determination. Bioavailability of a free drug in vivo is vital for its therapeutic and/or prophylactic actions. Once administered into the body the drug is partitioned to various biological system compartments and is also bound to circulating carrier proteins. One of the major carrier proteins in the body is human serum albumin, HSA. Drugs bound to HSA are thus not available to reach the target site hence affecting the efficacy of the drugs in their pharmacological action. This is especially critical for a drug with a very small therapeutic margin such as cytotoxic drugs for chemotherapy or hypoalbuminemic patients. The amount of free drugs available in w o is also affected by other ligands competing on the same binding sites influencing the drugs efficacy. A model drug, diazepam, an antidepressant, and a synthetic lipopeptide PKC inhibitor, (2B) (K(Pm)FARKGALRQKNK(Pm); Pm, p h t o y l ) binding to HSA are explored and discussed below. Interaction of non-chiral drugs to proteins is readily achieved in solution by circular dichroism (CD). The screening of binding properties of nonchiral ligands in solution is facilitated by the fact that only the ligand bound species show induced CD. The ordinary UV spectrum of the ligand-host complex will have contributions from both free and bound ligand species. The CD, on the other hand, will be induced only for the ligand species bound to the host chiral binding sites. Induced CD is thus a powerful means of identifying, in solution, nonchiral ligands bound to chiral hosts. Here we present the induced CD of diazepam (DZ) bound to fatty acid free human serum albumin (HSAff) (Fig. I). Above 3201x11, HSA has no CD contribution therefore the CD profile of HSA+DZ (1: 1) mixture is due only to the bound DZ (Fig. 1). NaCaprylate (CA) is normally used to stabilise HSA. CA is transparent in the near UV region (250-3501x11). On adding CA to the HSA-DZ [1:1] complex, an overall decreased HSA is a lipid carrier. intensity of the induced CD of DZ was observed indicative of DZ displacement by CA (Fig. 1). The competition study also provided the means of assessing the number of DZ bound species to HSA and other model drugs such as diclofenac. The fact that the profile of the induced CD of DZ, a negative band at about 320nm and two positive bands at about 290nm and 2601x11, decreased cooperatively with the same rate upon additions of CA was interpreted to be the evidence for only one single bound species of diazepam. Similar observation was obtained when palmitic acid (PA) was added to HSAffDZ mixtures at various molar ratios of PA (Fig. 1). A reduction of bound DZ was also detected when 2B was added to the HSAff-DZ mixture (Fig. 1 ) indicating the Pm moiety of the 2B is competing with DZ on the same HSA:DZ binding site thus affecting the amount of free DZ and 2B.