The physical and chemical properties of eumelanin

In this article, we review the current state of knowledge concerning the physical and chemical properties of the eumelanin pigment. We examine properties related to its photoprotective functionality, and draw the crucial link between fundamental molecular structure and observable macroscopic behaviour. Where necessary, we also briefly review certain aspects of the pheomelanin literature to draw relevant comparison. A full understanding of melanin function, and indeed its role in retarding or promoting the disease state, can only be obtained through a full mapping of key structure-property relationships in the main pigment types. We are engaged in such an endeavor for the case of eumelanin.

Toxin insights into nicotinic acetylcholine receptors

Venomous species have evolved cocktails of bioactive peptides to facilitate prey capture. Given their often exquisite potency and target selectivity, venom peptides provide unique biochemical tools for probing the function of membrane proteins at the molecular level. in the field of the nicotinic acetylcholine receptors (nAChRs), the subtype specific snake alpha-neurotoxins and cone snail alpha-conotoxins have been widely used to probe receptor structure and function in native tissues and recombinant systems. However, only recently has it been possible to generate an accurate molecular view of these nAChR-toxin interactions. Crystal structures of AChBP, a homologue of the nAChR ligand binding domain, have now been solved in complex with alpha-cobratoxin, alpha-conotoxin PnIA and alpha-conotoxin Iml. The orientation of all three toxins in the ACh binding site confirms many of the predictions obtained from mutagenesis and docking simulations on homology models of mammalian nAChR. The precise understanding of the molecular determinants of these complexes is expected to contribute to the development of more selective nAChR modulators. In this commentary, we review the structural data on nAChR-toxin interactions and discuss their implications for the design of novel ligands acting at the nAChR. (c) 2006 Elsevier Inc. All rights reserved.

N-type calcium channel blockers: Novel therapeutics for the treatment of pain

Highly selective Cav2.2 voltage-gated calcium channel (VGCC) inhibitors have emerged as a new class of therapeutics for the treatment of chronic and neuropathic pain. Cone snail venoms provided the first drug in class with FDA approval granted in 2005 to Prialt (ω-conotoxin MVIIA, Elan) for the treatment of neuropathic pain. Since this pioneering work, major efforts underway to develop alternative small molecule inhibitors of Cav2.2 calcium channel have met with varied success. This review focuses on the properties of the Cav2.2 calcium channel in different pain states, the action of ω-conotoxins GVIA, MVIIA and CVID, describing their structure-activity relationships and potential as leads for the design of improved Cav2.2 calcium channel therapeutics, and finally the development of small molecules for the treatment of chronic pain.

Design, Synthesis, Potency and Cytoselectivity of Anticancer Agents Derived by Parallel Synthesis from alpha-Aminosuberic Acid

Chemotherapy in the last century was characterized by cytotoxic drugs that did not discriminate between cancerous and normal cell types and were consequently accompanied by toxic side effects that were often dose limiting. The ability of differentiating agents to selectively kill cancer cells or transform them to a nonproliferating or normal phenotype could lead to cell- and tissue-specific drugs without the side effects of current cancer chemotherapeutics. This may be possible for a new generation of histone deacetylase inhibitors derived from amino acids. Structure-activity relationships are now reported for 43 compounds derived from 2-aminosuberic acid that kill a range of cancer cells, 26 being potent cytotoxins against MM96L melanoma cells (IC50 20 nM-1 mu M), while 17 were between 5- and 60-fold more selective in killing MM96L melanoma cells versus normal (neonatal foreskin fibroblasts, NFF) cells. This represents a 10- to 100-fold increase in potency and up to a 10-fold higher selectivity over previously reported compounds derived from cysteine (J. Med. Chem. 2004, 47, 2984). Selectivity is also an underestimate, because the normal cells, NFF, are rarely all killed by the drugs that also induce selective blockade of the cell cycle for normal but not cancer cells. Selected compounds were tested against a panel of human cancer cell lines (melanomas, prostate, breast, ovarian, cervical, lung, and colon) and found to be both selective and potent cytotoxins (IC50 20 nM-1 mu M). Compounds in this class typically inhibit human histone deacetylases, as evidenced by hyperacetylation of histones in both normal and cancer cells, induce expression of p21, and differentiate surviving cancer cells to a nonproliferating phenotype. These compounds may be valuable leads for the development of new chemotherapeutic agents.

Structural determinants for binding to CGRP receptors expressed by human SK-N-MC and Col 29 cells:Studies with chimeric and other peptides

1. Structure-activity relationships for the binding of human α-calcitonin gene-related peptide 8-37 (hαCGRP8-37) have been investigated at the CGRP receptors expressed by human SK-N-MC (neuroblastoma) and Col 29 (colonic epithelia) cells by radioligand binding assays and functional assays (hαCGRP stimulation of adenylate cyclase). 2. On SK-N-MC cells the potency order was hαCGRP8-37 > hαCGRP19-37 = AC187 > rat amylin8-37 > hα[Tyr0]-CGRP28-37 (apparent pKBS of 7.49 ± 0.25, 5.89 ± 0.20, 6.18 ± 0.19, 5.85 ± 0.19 and 5.25 ± 0.07). The SK-N-MC receptor appeared CGRP1-like. 3. On Col 29 cells, only hαCGRP8-37 of the above compounds was able to antagonize the actions of hαCGRP (apparent pKB = 6.48 ± 0.28). Its receptor appeared CGRP2-like. 4. hα[Ala11,18]-CGRP8-37, where the amphipathic nature of the N-terminal α-helix has been reduced, bound to SK-N-MC cells a 100 fold less strongly than hαCGRP8-37. 5. On SK-N-MC cells, hαCGRP(8-18, 28-37) (M433) and mastoparan-hαCGRP28-37 (M432) had apparent pKBS of 6.64 ± 0.16 and 6.42 ± 0.26, suggesting that residues 19-27 play a minor role in binding. The physico-chemical properties of residues 8-18 may be more important than any specific side-chain interactions. 6. M433 was almost as potent as hαCGRP8-37 on Col 29 cells (apparent pKB = 6.17 ± 0.20). Other antagonists were inactive.

Recent advances in the development of tissue transglutaminase (TG2) inhibitors

Tissue transglutaminase (TG2) is a Ca2+-dependent enzyme and probably the most ubiquitously expressed member of the mammalian transglutaminase family. TG2 plays a number of important roles in a variety of biological processes. Via its transamidating function, it is responsible for the cross-linking of proteins by forming isopeptide bonds between glutamine and lysine residues. Intracellularly, Ca2+ activation of the enzyme is normally tightly regulated by the binding of GTP. However, upregulated levels of TG2 are associated with many disease states like celiac sprue, certain types of cancer, fibrosis, cystic fibrosis, multiple sclerosis, Alzheimer's, Huntington's and Parkinson's disease. Selective inhibitors for TG2 both cell penetrating and non-cell penetrating would therefore serve as novel therapeutic tools for the treatment of these disease states. Moreover, they would provide useful tools to fully elucidate the cellular mechanisms TG2 is involved in and help comprehend how the enzyme is regulated at the cellular level. The current paper is intended to give an update on the recently discovered classes of TG2 inhibitors along with their structure-activity relationships. The biological properties of these derivatives, in terms of both activity and selectivity, will also be reported in order to translate their potential for future therapeutic developments. © 2011 Springer-Verlag.

Removing the redundancy from randomised gene libraries

Amino acid substitution plays a vital role in both the molecular engineering of proteins and analysis of structure-activity relationships. High-throughput substitution is achieved by codon randomisation, which generates a library of mutants (a randomised gene library) in a single experiment. For full randomisation, key codons are typically replaced with NNN (64 sequences) or NNG CorT (32 sequences). This obligates cloning of redundant codons alongside those required to encode the 20 amino acids. As the number of randomised codons increases, there is therefore a progressive loss of randomisation efficiency; the number of genes required per protein rises exponentially. The redundant codons cause amino acids to be represented unevenly; for example, methionine is encoded just once within NNN, whilst arginine is encoded six times. Finally, the organisation of the genetic code makes it impossible to encode functional subsets of amino acids (e.g. polar residues only) in a single experiment. Here, we present a novel solution to randomisation where genetic redundancy is eliminated; the number of different genes equals the number of encoded proteins, regardless of codon number. There is no inherent amino acid bias and any required subset of amino acids may be encoded in one experiment. This generic approach should be widely applicable in studies involving randomisation of proteins. © 2003 Elsevier Ltd. All rights reserved.

A combinatorial approach to the chemical synthesis and biological evaluation of 3,4,5-trisubstiituted furan-2(5H)-ones

Many important natural products contain the furan-2(5H)-one structure. The structure of this molecule lends itself to manipulation using combinatorial techniques due to the presence of more than one site for the attachment of different suhstituents. By developing different reaction schemes at the three sites available for attachment on the furan-2(5H)-one scaffold, combinatorial chemistry techniques can be employed to assemble libraries of novel furan 2(5H)-ones. These libraries can then be entered into various biological screening programmes. This approach will enable a vast diversity or compounds to be examined, in the hope or finding new biologically active Iead structures. The work in this thesis has investigated the potential that combinatorial chemistry has in the quest for new biologically active lead structures based on the furan-2(5H)-one structure. Different reactions were investigated with respect to their suitability for inclusion in a library. Once sets of reactions at the various sites had been established, the viability of these reactions in the assembly of combinatorial libraries was investigated. Purification methods were developed, and the purified products entered into suitable biological screening tests. Results from some of these tests were optimised using structure activity relationships, and the resulting products re-screened. The screening tests performed were for anticancer and antimicrobial activity, cholecystokinin (CCK-B) antagonism and anti-inflammatory activity (in the quest for novel cyclo-oxygenase (COX-2) selective non-steroidal anti-inflammatory drugs). It has been shown that many reactions undergone by the furan-2(5H)-one structure are suitable for the assembly of a combinatorial library. Investigation into the assembly of different libraries has been carried out with initial screening results included. From this work, further investigation into combinatorial library assembly and structure activity relationships of screened reaction products can be undertaken.

Investigating the modulation of oral drug absorption using in vitro models

Dipeptides can be absorbed into cells via the dipeptide transporter (which also transported tripeptides and dipeptide derivatives). The optimum conditions for measuring the inhibition of Gly-Pro uptake in Caco-2 cells were identified. A number of structure-activity relationships were identified. These included the effects of increasing the amino-acid chain-length, and the presence of a thiol or hydroxyl group in the side-chain increased IC50 while the presence of a hydroxyl group did not. The benzyl esters had lower or equal IC50 values compared to the parent dipeptides while the methyl esters had higher values. These results indicated that while molecular properties did affect IC50, the size, charge and composition of three particular groups caused the most significant effects, supporting the structure-activity relationship identified. An assay was developed using calcein-AM to show the inhibition of p-glycoprotein activity. There was no significant change due to the presence of mannitol but there was in the presence of clyclosporin A (p<0.01). Incubating the cells with the test solution for 30 minutes before the addition of the ester resulted in a significant (p<0.001) difference. The assay was specific for p-glycoprotein, as the presence MRP inhibitors had no effect (p>0.05). The modified protocol allowed the identification of p-glycoprotein inhibitors quickly and simply using a cell suspension of unmodified cells. The clinically relevant buffering of grapefruit juice to pH 7 led to a four-fold increase in intracellular calcein and hence significant inhibition of p-glycoprotein. Buffered orange and lemon juices had no effect on the assay. Flavone derivatives had previously been found to be inhibitors of CYP3A4 yet neither naringin nor naringenin had any significant effect at concentrations found in grapefruit juice. Of the other (non-grapefruit) flavone derivatives tested, hesperidin, found in orange juice, had no significant effect, kaempferol and rutin also had no effect while genistein significantly inhibited p-glycoprotein (results that support previous studies). Hydroxycinnamic acids had no effect on p-glycoprotein. Studies on other compounds found that the balance between inhibiting p-glycoprotein and disrupting cell membranes depends on the compound containing an oxygen atom and the size of the negative charge on it, as well as three-dimensional arrangement of the atoms.

High-performance liquid chromatography and pharmacokinetics of some antibiotics

The principles of High Performance Liquid Chromatography (HPLC) and pharmacokinetics were applied to the use of several clinically-important drugs at the East Birmingham Hospital. Amongst these was gentamicin, which was investigated over a two-year period by a multi-disciplinary team. It was found that there was considerable intra- and inter-patient variation that had not previously been reported and the causes and consequences of such variation were considered. A detailed evaluation of available pharmacokinetic techniques was undertaken and 1- and 2-compartment models were optimised with regard to sampling procedures, analytical error and model-error. The implications for control of therapy are discussed and an improved sampling regime is proposed for routine usage. Similar techniques were applied to trimethoprim, assayed by HPLC, in patients with normal renal function and investigations were also commenced into the penetration of drug into peritoneal dialysate. Novel assay techniques were also developed for a range of drugs including 4-aminopyridine, chloramphenicol, metronidazole and a series of penicillins and cephalosporins. Stability studies on cysteamine, reaction-rate studies on creatinine-picrate and structure-activity relationships in HPLC of aminopyridines are also reported.

Characterisation of calcitonin generelated peptide (CGRP) and amylin receptors

The aims of this study were to examine the binding characteristics of the rat CGRP receptor and to further the classification of CGRP and amylin receptors in guinea-pig tissue preparations. Binding characteristics of CGRP were investigated on rat splenic, cerebellar and liver membrane preparations. Human-α-CGRP, rat-α-CGRP and the CGRP receptor analogues Tyrº -CGRPC28-37) and [Cys (ACM)2,7 ]-human CGRP and the CGRP receptor antagonist CGRPC8-37) were utilised in competitive radioligand binding experiments to identify possible CGRP receptor subtypes in these tissues. There appeared to be no significant differences between the rat CGRP receptors examined. A panel of monoclonal antibodies (Mabs) raised against CGRP were employed to investigate the structure-activity relationships of CGRP and its receptor. No differences between the tissue receptors were observed using this panel of Mabs. The effects of human-α, human-β, rat-α-CGRP, human and rat amylin and adrenomedullin(13-52) were examined on the spontaneously beating right atria and on electrically evoked twitch contractions of isolated guinea-pig ileum, vas deferens and left atria. All of the peptides caused concentration-dependent inhibition of twitch amplitude in the ileum and vas deferens. CGRP produced positive inotropic effects in the right and left atria and positive chronotropic effects in the right atria. A variety of CGRP receptor antagonists and putative amylin receptor antagonists were used to antagonise these effects. CGRP(8-37) is currently used as a basis for CGRP receptor classification (Dennis, et al., 1989). Based upon results obtained using CGRP(8-37) it has been shown that the guinea-pig ileum contains mainly CGRP 1 receptors and the vas deferens contain CGRP2 receptors. Amylin was shown to act at receptors distinct from those for CGRP and it is postulated that amylin has its own receptors in these preparations. Experiments using CGRP (19-37) and Tyrº -CGRP(28-37) indicate that human and rat CGRP act at distinct receptors in guinea-pig ileum and vas deferens. The amylin receptor antagonist amylin(8-37) and the putative antagonist AC187 provide evidence to suggest human and rat amylin also act at receptors able to distinguish between the two types of amylin.

The antimicrobial action of pseudin-2

The amphibian antimicrobial peptide pseudin-2 is a peptide derived from the skin of the South-American frog Pseudis paradoxa (Olson et al., 2001). This peptide possesses tremendous potential as a therapeutic lead since it has been shown to possess both antimicrobial as well insulin-releasing properties (Olson et al., 2001; Abdel-Wahab et al., 2008). This study aimed to develop pseudin-2’s potential by understanding and improving its properties as an antimicrobial agent. The structure-function relationships of pseudin-2 were explored using a combination of in-vitro and in-silico techniques, with an aim to predict how the structure of the peptide may be altered in order to improve its efficacy. A library of pseudin-2 mutants was generated by randomizing codons at positions 10, 14 and 18 of a synthetic gene, using NNK saturation mutagenesis. Analysis of these novel peptides broadly confirmed, in line with literature precedent, that anti-microbial activity increases with increased positive charge. Specifically, 2 positively-charged residues at positions 10 and 14 and a hydrophobic at position 18 are preferred. However, substitution at position 14 with some polar, non-charged residues also created peptides with antimicrobial activity. Interestingly, the pseudin-2 analogue [10-E, 14-Q, 18-L] which is identical to pseudin-2, except that the residues at positions 10 and 14 are switched, showed no anti-microbial activity at all. Molecular dynamics simulations of pseudin-2 showed that the peptide possesses two equilibrium structures in a membrane environment: a linear and a kinked a-helix which both embed into the membrane at an angle. Biophysical characterization using circular dichroism spectroscopy confirmed that the peptide is helical within the membrane environment whilst linear dichroism established that the peptide has no defined orientation within the membrane. Collectively, these data indicate that Pseudin-2 exerts its antimicrobial activity via the carpet model.

Empirical prediction of peptide octanol-water partition coefficients

Peptides are of great therapeutic potential as vaccines and drugs. Knowledge of physicochemical descriptors, including the partition coefficient P (commonly expressed in logarithm form: logP), is useful for screening out unsuitable molecules and also for the development of predictive Quantitative Structure-Activity Relationships (QSARs). In this paper we develop a new approach to the prediction of LogP values for peptides based on an empirical relationship between global molecular properties and measured physical properties. Our method was successful in terms of peptide prediction (total r2 = 0.641). The final model consisted of 5 physicochemical descriptors (molecular weight, number of single bonds, 2D-VDW volume, 2D-VSA hydrophobic and 2D-VSA polar). The approach is peptide specific and its predictive accuracy was high. Overall, 67% of the peptides were able to be predicted within +/-0.5 log units from the experimental values. Our method thus represents a novel prediction method with proven predictive ability.

On the hydrophobicity of peptides:comparing empirical predictions of peptide log P values

Peptides are of great therapeutic potential as vaccines and drugs. Knowledge of physicochemical descriptors, including the partition coefficient logP, is useful for the development of predictive Quantitative Structure-Activity Relationships (QSARs). We have investigated the accuracy of available programs for the prediction of logP values for peptides with known experimental values obtained from the literature. Eight prediction programs were tested, of which seven programs were fragment-based methods: XLogP, LogKow, PLogP, ACDLogP, AlogP, Interactive Analysis's LogP and MlogP; and one program used a whole molecule approach: QikProp. The predictive accuracy of the programs was assessed using r(2) values, with ALogP being the most effective (r( 2) = 0.822) and MLogP the least (r(2) = 0.090). We also examined three distinct types of peptide structure: blocked, unblocked, and cyclic. For each study (all peptides, blocked, unblocked and cyclic peptides) the performance of programs rated from best to worse is as follows: all peptides - ALogP, QikProp, PLogP, XLogP, IALogP, LogKow, ACDLogP, and MlogP; blocked peptides - PLogP, XLogP, ACDLogP, IALogP, LogKow, QikProp, ALogP, and MLogP; unblocked peptides - QikProp, IALogP, ALogP, ACDLogP, MLogP, XLogP, LogKow and PLogP; cyclic peptides - LogKow, ALogP, XLogP, MLogP, QikProp, ACDLogP, IALogP. In summary, all programs gave better predictions for blocked peptides, while, in general, logP values for cyclic peptides were under-predicted and those of unblocked peptides were over-predicted.

Biomimetic synthetic lubricants (Biosurfactants)

Poly(styrene-co-maleic anhydride) (PSMA) based copolymers are known to undergo conformational transition in response to environmental stimuli. This smart behaviour makes it possible to mimic the behaviour of native apoproteins. The primary aim of this study was to develop a better understanding of the structure-property relationships of various PSMA-based copolymers sought. The work undertaken in this thesis has revealed that the responsive behaviour of PSMA-based copolymers can be tailored by varying the molecular weight, hydrophobic (styrene) and hydrophilic (maleic acid) balance, and more so in the presence of additional hydrophobic, mono-partial ester moieties. Novel hydrophilic and hydrophobic synthetic surfactant protein analogues have successfully been prepared. These novel lipid solubilising agents possess a broad range of HLB (hydrophilic-lipophilic balance) values that have been estimated. NMR spectroscopy was utilised to confirm the structures for PSMA-based copolymers sought and proved useful in furthering understanding of the structure-property relationships of PSMA-based copolymers. The association of PSMA with the polar phospholipid, 2-dilauryl-sn-glycero-3- phosphocholine (DLPC) produces polymer-lipid complexes analogous to lipoprotein assemblies present in the blood plasma. NMR analysis reveals that the PSMA-based copolymers are not perfectly alternating. Regio-irregular structures, atactic and random monomer sequence distribution have been identified for all materials studied. Novel lipid solubilising agents (polyanionic surfactants) have successfully been synthesised from a broad range of PSMA-based copolymers with desired estimated HLB values that interact with polar phospholipids (DLPC/DPPC) uniquely. Very low static and dynamic surface tensions have been observed via the du Noϋy ring method and Langmuir techniques and correlate well with the estimated HLB values. Synthetic protein-lipid analogues have been successfully synthesised, that mimic the unique surface properties of native biological lubricants without the use of solvents. The novel PSMA-DLPC complexes have successfully been combined with hyaluronan (hyaluronic acid, HA). Today, the employment of HA is economically feasible, because it is readily available from bacterial fermentation processes in a thermally stable form - HyaCare®. The work undertaken in this thesis highlights the usage of HA in biolubrication applications and how this can be optimised and thus justified by carefully selecting the biological source, concentration, molecular weight, purity and most importantly by combining it with compatible boundary lubricating agents (polar phospholipids). Experimental evidence supports the belief that the combined HA and PSMA-DLPC complexes provide a balance of rheological, biotribological and surface properties that are composition dependent, and show competitive advantage as novel synthetic biological lubricants (biosurfactants).