Primary Drug Target Research Articles

The Global Problem of Antibiotic Resistance

Amid the recent attention justly focused on the potential problem of microbial sources for weapons of bioterrorism, it is also apparent that human pathogens frequently isolated from infections in patients from community and hospital sources have been growing more resistant to commonly used antibiotics. Much of the growth of multiple-drug-resistant (MDR) bacterial pathogens can be contributed to the overuse of broad-spectrum antimicrobial products. However, an equally troubling and often overlooked component of the problem involves the elegant ways in which pathogenic bacteria continually evolve complex genetic systems for acquiring and regulating an endless array of antibiotic-resistance mechanisms. Efforts to develop new antimicrobials have over the past two decades been woefully behind the rapid evolution of resistance genes developing among both gram-positive and gram-negative pathogens. Several new agents that are best suited for use in the hospital environment have been developed to combat staphylococci resistant to beta-lactam antimicrobials following acquisition of the mecA gene. However, the dramatic spread in the US of the now common community strain of Staphylococcus aureus USA300 has shifted the therapeutic need for new antibiotics useful against MRSA to the community. As the pharmaceutical industry focused on discovering new agents for use against MRSA, hospitals in many parts of the world have seen the emergence of gram-negative pathogens such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae that are clinically resistant to almost all available antimicrobials. Such MDR isolates usually contain multiple-resistance determinants, including loss of outer membrane porins via gene inactivation by chromosomally encoded insertion sequences, up-regulation of inate efflux pumps, as well as acquisition of drug-inactivating enzymes whose genes are encoded on self-transmissible plasmids, integrons, and complex transposable elements. These determinants confer a complex resistance phenotype that is often superimposed on mutations in the primary drug target in the cell. The continued evolution of such a complex array of antibiotic-resistance genes presents a formidable challenge at a time when large pharmaceutical companies have scaled down their presence in the anti-infectives arena.

Dephosphorylation of Threonine 38 Is Required for Nuclear Translocation and Activation of Human Xenobiotic Receptor CAR (NR1I3)

Upon activation by therapeutics, the nuclear xenobiotic/ constitutive active/androstane receptor (CAR) regulates various liver functions ranging from drug metabolism and excretion to energy metabolism. CAR can also be a risk factor for developing liver diseases such as hepatocellular carcinoma. Here we have characterized the conserved threonine 38 of human CAR as the primary residue that regulates nuclear translocation and activation of CAR. Protein kinase C phosphorylates threonine 38 located on the alpha-helix spanning from residues 29-42 that constitutes a part of the first zinc finger and continues into the region between the zinc fingers. Molecular dynamics study has revealed that this phosphorylation may destabilize this helix, thereby inactivating CAR binding to DNA as well as sequestering it in the cytoplasm. We have found, in fact, that helix-stabilizing mutations reversed the effects of phosphorylation. Immunohistochemical study using an anti-phospho-threonine 38 peptide antibody has, in fact, demonstrated that the classic CAR activator phenobarbital dephosphorylates the corresponding threonine 48 of mouse CAR in the cytoplasm of mouse liver and translocates CAR into the nucleus. These results define CAR as a cell signal-regulated constitutive active nuclear receptor. These results also provide phosphorylation/dephosphorylation of the threonine as the primary drug target for CAR activation.

Location of the Antidepressant Binding Site in the Serotonin Transporter

The serotonin transporter (SERT) regulates extracellular levels of serotonin (5-hydroxytryptamine, 5HT) in the brain by transporting 5HT into neurons and glial cells. The human SERT (hSERT) is the primary target for drugs used in the treatment of emotional disorders, including depression. hSERT belongs to the solute carrier 6 family that includes a bacterial leucine transporter (LeuT), for which a high resolution crystal structure has become available. LeuT has proved to be an excellent model for human transporters and has advanced the understanding of solute carrier 6 transporter structure-function relationships. However, the precise structural mechanism by which antidepressants inhibit hSERT and the location of their binding pockets are still elusive. We have identified a residue (Ser-438) located within the 5HT-binding pocket in hSERT to be a critical determinant for the potency of several antidepressants, including the selective serotonin reuptake inhibitor citalopram and the tricyclic antidepressants imipramine, clomipramine, and amitriptyline. A conservative mutation of Ser-438 to threonine (S438T) selectively increased the K(i) values for these antidepressants up to 175-fold. The effects of introducing a protein methyl group into the 5HT-binding pocket by S438T were absent or reduced for analogs of these antidepressants lacking a single methyl group. This suggests that these antidepressants interact directly with Ser-438 during binding to hSERT, implying an overlapping localization of substrate- and inhibitor-binding sites in hSERT suggesting that antidepressants function by a mechanism that involves direct occlusion of the 5HT-binding site.

Mutational ‘hot-spots’ in mammalian, bacterial and protozoal dihydrofolate reductases associated with antifolate resistance: Sequence and structural comparison

Human dihydrofolate reductase (DHFR) is a primary target for antifolate drugs in cancer treatment, while DHFRs from Plasmodium falciparum, Plasmodium vivax and various bacterial species are primary targets in the treatment of malaria and bacterial infections. Mutations in each of these DHFRs can result in resistance towards clinically relevant antifolates. We review the structural and functional impact of active-site mutations with respect to enzyme activity and antifolate resistance of DHFRs from mammals, protozoa and bacteria. The high structural homology between DHFRs results in a number of cross-species, active-site 'hot-spots' for broad-based antifolate resistance. In addition, we identify mutations that confer species-specific resistance, or antifolate-specific resistance. This comparative review of antifolate binding in diverse species provides new insights into the relationship between antifolate design and the development of mutational resistance. It also presents avenues for designing antifolate-resistant mammalian DHFRs as chemoprotective agents.

Role of the cholinergic system in the pathology and treatment of schizophrenia

Schizophrenia is a devastating psychiatric disorder; the diagnosis probably encompasses a number of illnesses with similar clinical presentations, complicating both treatment and studies into the pathology of the disorder. The development of antipsychotic medications revolutionized treatment for the disorder. However, there are still symptom domains that are relatively untouched by the drugs currently available; these are the cognitive deficits and the negative symptoms. Basic research strongly supports a role of acetylcholine in cognitive processes, making it a primary target for drugs designed to improve this most impairing symptom. In addition, the fact that acetylcholine can modulate dopamine release makes the cholinergic system a target for novel antipsychotic drugs, ideally without the side-effect profiles that contribute to patient noncompliance seen with current antipsychotic drugs. Thus far, the results of clinical trials with cholinomimetics have been equivocal; where beneficial effects are seen they are, by and large, modest rather than compelling. These trials and studies investigating the role of both nicotinic and muscarinic receptors in the pathology of the disorder, published during the last 5 years, are reviewed to ascertain whether there is a role for the cholinergic system in the treatment of schizophrenia.

Functional variation in the 3′ untranslated region of the serotonin transporter in human and rhesus macaque

The serotonin system underlies a wide variety of behavioral traits and its dysregulation is the cause of numerous neuropsychiatric disorders. Among genes involved in the system, the serotonin transporter (SERT) is integral and has been repeatedly shown to be associated with disease as well as being a primary drug target. In addition to promoter region variation, we identify here variation in a regulatory region in the 3' untranslated region (UTR) of the SERT gene in both humans and rhesus macaques. We comprehensively survey the 3' UTR of SLC6A4 in Indian-origin rhesus macaques to identify three single nucleotide polymorphisms (SNPs) creating two haplotypes, both derived from an ancestral sequence, that represent the vast majority of the alleles in the population. Through the use of a luciferase reporter gene assay, we are able to show that not only do these alleles have differential effects on gene expression, modulated through changes in messenger RNA stability, but that different commonly occurring SNPs in the human 3' UTR also have similar effects. This finding not only offers additional insight into the regulation, and thus dysregulation, of SERT expression, but also suggests the role of natural selection in maintaining both high and low SERT expression levels broadly across populations of multiple primate species.

Crystal engineering of HIV-1 reverse transcriptase for structure-based drug design

HIV-1 reverse transcriptase (RT) is a primary target for anti-AIDS drugs. Structures of HIV-1 RT, usually determined at ∼2.5–3.0 Å resolution, are important for understanding enzyme function and mechanisms of drug resistance in addition to being helpful in the design of RT inhibitors. Despite hundreds of attempts, it was not possible to obtain the structure of a complex of HIV-1 RT with TMC278, a nonnucleoside RT inhibitor (NNRTI) in advanced clinical trials. A systematic and iterative protein crystal engineering approach was developed to optimize RT for obtaining crystals in complexes with TMC278 and other NNRTIs that diffract X-rays to 1.8 Å resolution. Another form of engineered RT was optimized to produce a high-resolution apo-RT crystal form, reported here at 1.85 Å resolution, with a distinct RT conformation. Engineered RTs were mutagenized using a new, flexible and cost effective method called methylated overlap-extension ligation independent cloning. Our analysis suggests that reducing the solvent content, increasing lattice contacts, and stabilizing the internal low-energy conformations of RT are critical for the growth of crystals that diffract to high resolution. The new RTs enable rapid crystallization and yield high-resolution structures that are useful in designing/developing new anti-AIDS drugs.

Dynamic binding orientations direct activity of HIV reverse transcriptase

The reverse transcriptase of human immunodeficiency virus (HIV) catalyses a series of reactions to convert the single-stranded RNA genome of HIV into double-stranded DNA for host-cell integration. This task requires the reverse transcriptase to discriminate a variety of nucleic-acid substrates such that active sites of the enzyme are correctly positioned to support one of three catalytic functions: RNA-directed DNA synthesis, DNA-directed DNA synthesis and DNA-directed RNA hydrolysis. However, the mechanism by which substrates regulate reverse transcriptase activities remains unclear. Here we report distinct orientational dynamics of reverse transcriptase observed on different substrates with a single-molecule assay. The enzyme adopted opposite binding orientations on duplexes containing DNA or RNA primers, directing its DNA synthesis or RNA hydrolysis activity, respectively. On duplexes containing the unique polypurine RNA primers for plus-strand DNA synthesis, the enzyme can rapidly switch between the two orientations. The switching kinetics were regulated by cognate nucleotides and non-nucleoside reverse transcriptase inhibitors, a major class of anti-HIV drugs. These results indicate that the activities of reverse transcriptase are determined by its binding orientation on substrates.

TNOX (ENOX2) Target for Chemosensitization-Low-Dose Responses in the Hormetic Concentration Range

An emerging concept of cancer chemotherapy is that of chemosensitization. Most often applied to the treatment of drug-resistance cancers, chemosensitization has utility when such cancers are rendered drug sensitive through treatment with the sensitizing agent. A particularly striking example of chemosensitization is that encountered with the synthetic isoflavene phenoxodiol where patients with taxane- and/or platinum-resistant ovarian carcinoma once again become sensitive to these drugs following treatment with phenoxodiol. The latter appears to be a true chemosensitization in that the phenoxodiol need not be co-administered with the taxane or platinum drugs. Sensitivity is retained many weeks after the phenoxodiol has been cleared from the system. The response appears to be mediated through the primary drug target of phenoxodiol, a cancer-specific cell surface ECTO-NOX or ENOX protein designated tNOX (ENOX2). The ENOX protein family has been previously recognized as a hormetic target. The hypothesis under investigation is that chemosensitization and low dose synergies are most obvious in the hormetic range of concentrations and that the two phenomena, hormesis and chemosensitization, may be related mechanistically.

Comparative characterization of cellular and molecular anti-restenotic profiles of paclitaxel and sirolimus

Pleiotropic anti-restenotic properties of drugs that are eluted from coated stents are critical for efficacy and safety. Little is known about comparative drug properties in appropriate human coronary target cell lines for the two compounds that are utilized on FDA-approved drug-eluting stent (DES) platforms, paclitaxel (PTX) and sirolimus (SRL). Target cell lines that play a pivotal role for the pathogenesis of restenosis and vascular healing include human coronary artery smooth muscle (CASMC) and endothelial cells (CAEC). PTX and SRL inhibited CASMC and CAEC proliferation and migration efficiently. However, there was a differential effect on proliferation and migration in CAEC with a more profound inhibition of both parameters by PTX, even at low dosages. Induction of cytotoxicity and apoptosis was pronounced in PTX- and very modest in SRL-treated CASMC and CAEC. PTX increased eNOS activity and nitric oxide (NO) release from CAEC. Neutrophilic leukocyte activation and transmigration, which should be avoided since it may precipitate adverse coronary events such as restenosis and stent thrombosis, was suppressed by SRL, whereas PTX tended to increase neutrophilic leucocyte activity. Therefore, although the primary drug target, inhibition of mitogen-mediated CASMC proliferation, is effectively accomplished by both drugs, auxiliary pharmacological properties that are crucial for the anti-restenotic drug effect and vascular healing are considerably different between PTX and SRL. In comparison with PTX, SRL shows minor interference with endothelial cell proliferation and migration, lower levels of cytotoxicity and apoptosis, a broader therapeutic range and distinctive immunosuppressive properties.

Drug therapy of the metabolic syndrome: minimizing the emerging crisis in polypharmacy

The metabolic syndrome--a collection of factors associated with increased risk for cardiovascular disease and diabetes--is becoming increasingly common, largely as a result of the increase in the prevalence of obesity. Although it is generally agreed that first-line clinical intervention for the metabolic syndrome is lifestyle change, this is insufficient to normalize the risk factors in many patients, and so residual risk could be high enough to justify drug therapy. However, at present there are no approved drugs that can reliably reduce all of the metabolic risk factors over the long term, and so there is growing interest in therapeutic strategies that might target multiple risk factors more effectively, thereby minimizing problems with polypharmacy. This review summarizes current understanding of the nature of the metabolic syndrome, and discusses each of the risk factors of the metabolic syndrome as possible primary drug targets; potential secondary or tertiary targets are also considered.

Recent Developments of Structure Based β-Secretase Inhibitors for Alzheimers Disease

The amyloid-beta (Abeta) peptide is the principal components of the senile plaques found in the brains of patients with Alzheimer's disease (AD). The poorly soluble 40-42 amino acid peptide, formed from the cleavage of the Abeta precursor protein (APP) by two proteases, is believed to play a central role in the pathogenesis of AD. Beta-secretase (memapsin 2, BACE1), a membrane-anchored aspartic protease, is responsible for the initial step of APP cleavage leading to the generation of Abeta. Identification and structural determination of beta-secretase have established it to be a primary drug target for AD therapy and stimulated active studies on the inhibitors of this protease. Here we review more recent developments in the design and testing of structure-based beta-secretase inhibitors.

Proteomics of Acute Myeloid Leukemia: Cytogenetic Risk Groups Differ Specifically in Their Proteome, Interactome and Posttranslational Protein Modifications.

Acute Myeloid Leukemia (AML) is characterized by specific cytogenetic aberrations which are strong determinants of prognostic outcome and therapeutic response. Because the clinical outcome in AML cytogenetic groups differs considerably, we hypothesized that cytogenetic risk groups of AML might differ specifically in their proteome, protein interaction pathways and posttranslational modifications (PTMs). Thus, we determined the proteome of 30 AML patients belonging to various cytogenetic groups based on two-dimensional gel electrophoresis and Nano LC coupled MALDI-TOF-TOF tandem mass spectrometry. We could identify substantial differences in the proteome, protein expression and peak pattern between cytogenetic risk groups of AML. The interactome analysis based on computational bioinformatics using Ingenuity analysis revealed major regulating networks: MAPK8 and MYC for complex aberrant karyotype AML, TP53 for t(8;21)-AML, TP53- MYC- PRKAC for 11q23-AML, JUN and MYC for inv(16)-AML. Most interestingly, peak explorer analysis revealed a modification of O-linked acetyl glucosamine of hnRNPH1 in AML patients with a 11q23 translocation, an acetylation of calreticulin in t(8;21) translocation AML, an increased intensity of dimethylated peptide of hnRNPA2/B1 in AML patients with translocations of t(8;21) and inv(16) in comparison to healthy bone marrow. We show for the first time that cytogenetic risk groups of AML differ specifically both in their proteome, interactome and PTMs. These findings lead to a new thinking about the pathogenesis of AML and has major therapeutic implications because PTMs are the primary drug targets.

Allelic Expression Imbalance of Human mu Opioid Receptor (OPRM1) Caused by Variant A118G

As a primary target for opioid drugs and peptides, the mu opioid receptor (OPRM1) plays a key role in pain perception and addiction. Genetic variants of OPRM1 have been implicated in predisposition to drug addiction, in particular the single nucleotide polymorphism A118G, leading to an N40D substitution, with an allele frequency of 10-32%, and uncertain functions. We have measured allele-specific mRNA expression of OPRM1 in human autopsy brain tissues, using A118G as a marker. In 8 heterozygous samples measured, the A118 mRNA allele was 1.5-2.5-fold more abundant than the G118 allele. Transfection into Chinese hamster ovary cells of a cDNA representing only the coding region of OPRM1, carrying adenosine, guanosine, cytidine, and thymidine in position 118, resulted in 1.5-fold lower mRNA levels only for OPRM1-G118, and more than 10-fold lower OPRM1 protein levels, measured by Western blotting and receptor binding assay. After transfection and inhibition of transcription with actinomycin D, analysis of mRNA turnover failed to reveal differences in mRNA stability between A118 and G118 alleles, indicating a defect in transcription or mRNA maturation. These results indicate that OPRM1-G118 is a functional variant with deleterious effects on both mRNA and protein yield. Clarifying the functional relevance of polymorphisms associated with susceptibility to a complex disorder such as drug addiction provides a foundation for clinical association studies.

Ampicillin/penicillin-binding protein interactions as a model drug-target system to optimize affinity pull-down and mass spectrometric strategies for target and pathway identification

The identification and validation of the targets of active compounds identified in cell-based assays is an important step in preclinical drug development. New analytical approaches that combine drug affinity pull-down assays with mass spectrometry (MS) could lead to the identification of new targets and druggable pathways. In this work, we investigate a drug-target system consisting of ampicillin- and penicillin-binding proteins (PBPs) to evaluate and compare different amino-reactive resins for the immobilization of the affinity compound and mass spectrometric methods to identify proteins from drug affinity pull-down assays. First, ampicillin was immobilized onto various amino-reactive resins, which were compared in the ampicillin-PBP model with respect to their nonspecific binding of proteins from an Escherichia coli membrane extract. Dynal M-270 magnetic beads were chosen to further study the system as a model for capturing and identifying the targets of ampicillin, PBPs that were specifically and covalently bound to the immobilized ampicillin. The PBPs were identified, after in situ digestion of proteins bound to ampicillin directly on the beads, by using either one-dimensional (1-D) or two-dimensional (2-D) liquid chromatography (LC) separation techniques followed by tandem mass spectrometry (MS/MS) analysis. Alternatively, an elution with N-lauroylsarcosine (sarcosyl) from the ampicillin beads followed by in situ digestion and 2-D LC-MS/MS analysis identified proteins potentially interacting noncovalently with the PBPs or the ampicillin. The in situ approach required only little time, resources, and sample for the analysis. The combination of drug affinity pull-down assays with in situ digestion and 2-D LC-MS/MS analysis is a useful tool in obtaining complex information about a primary drug target as well as its protein interactors.

Serotonin transporter: Evolution and impact of polymorphic transcriptional regulation

The serotonin transporter (SERT) is the primary drug target in the current antidepressant therapy. A functional polymorphism in the 2nd intron of the 5HTT gene encoding the SERT has been identified and associated with susceptibility to affective disorders and treatment response to antidepressants. This study addresses the possible impact of the variable number of tandem repeats (VNTR) to behavior and disease by examining the evolutionary origin and mechanisms of differential transcriptional regulation of SERT. We trace the evolutionary origin of the VNTR and show that it is present and varies extensively across the great apes and monkeys as well as in rodents while it is absent in non-mammals. As in humans, the VNTR sequence may be polymorphic within species and thus it may underlie both inter- and intraspecies differences. Also, we find new putative binding sites for several transcription factors in the VNTRs of all mammalian SERT genes. The number of these putative binding sites varies proportionally to the length of the VNTR. We propose that the intronic VNTR have been selectively targeted through mammalian evolution to finetune transcriptional regulation of the serotonin expression.

412 A phase I and pharmacokinetic study of AEE788, a novel multi-targeted inhibitor of ErbB and VEGF receptor family tyrosine kinases

The quest to develop computational drug target identification methods in complex diseases like cancer is growing in recent years. Feedback, feed-forward loops and cross-talks observed among the MAPK pathways led to the definition of a network of MAPK pathways and considered for single or multiple therapeutic interventions. We developed a computational method to identify clusters of drug targets by analysing the directed network's topological properties and the individual node's functional roles. We aim to identify the primary drug target nodes possessing more cancerous properties and less number of cellular functional roles. For every primary drug targets, we collect the alternate substrate activating nodes for local resistance analysis. Alternate substrate activation free nodes identified as single drug target are SOS, ATF1, BAD, GAB1, LAD, NFAT4, ATF2, MEF2, eEF2K, 4EBP1 and HSP27. Among the remaining identified nodes and their corresponding alternate substrate activating nodes with their cancer retaining and side effects causing properties studied as three different classes-single, multiple and dangerous targets. C-Raf1 and MAPKAP-K observed as a single efficient target due to the absence of resistance mechanism. Due to the resistance mechanism observed among the targeted M3/6, GADD45, and MKK6 multiple target intervention of their corresponding alternate nodes might prove to be the efficient targets. Targeted effect on MLK3, ZAK, DLK and MLTKa/b will impair the network due to intertwined and proximity nature among themselves.

Beta-lactams as versatile synthetic intermediates for the preparation of heterocycles of biological interest.

Since the advent of penicillin, the beta-lactam antibiotics have been the subject of much discussion and investigation, within both the scientific and public sectors. The primary biological targets of the beta-lactam antibacterial drugs are the penicillin binding proteins, a group of transpeptidases anchored within the bacterial cellular membrane, which mediate the final step of cell wall biosynthesis. The extensive use of common beta-lactam antibiotics such as penicillins and cephalosporins in medicine has resulted in an increasing number of resistant strains of bacteria through mutation and beta-lactamase gene transfer. Thus, a handful of nonconventional fused polycyclic beta-lactams have been described in the literature in order to overcome the defence mechanisms of the bacteria. In fact, tricyclic beta-lactam antibiotics, generally referred to as trinems, are a new class of synthetic antibacterial agents featuring good resistance to beta-lactamases and dehydropeptidases. In addition, recent discoveries have shown other biological properties of these compounds apart from their antibacterial action. In this sense, beta-lactams can serve as inhibitors of serine proteases, such as human leukocyte elastase (HLE) or thrombin, acyl-CoA cholesterol acyltransferase inhibitors and inhibitors of human cytomegalovirus. Additional impetus for research efforts on beta-lactam chemistry has been provided by the introduction of the beta-lactam synthon method, a term coined by Ojima 20 years ago, according to which 2-azetidinones can be employed as useful intermediates in organic synthesis. The usefulness of beta-lactams in the stereocontrolled synthesis of heterocycles of biological significance is based on the impressive variety of transformations, which can be derived from this system, due inter alia to a high chirality content that can be transferred into a variety of products. The cyclic 2-azetidinone skeleton has been extensively used as a template on which to build the heterocyclic structure fused to the four-membered ring, using the chirality and functionalisation of the beta-lactam nucleus as a stereocontrolling element. Alternatively, the direct one-pot generation of fused nitrogen heterocyclic systems from the nitrogen framework of 2-azetidinone derivatives has been achieved by selective bond breakage and rearrangement. It is our aim in this Review to highlight the state of the art in this endeavour, consisting either of the stereocontrolled synthesis of fused polycyclic beta-lactams of antibacterial interest, or stereoselective synthesis of different sized heterocycles of biological significance. Representative examples of the latter include indolizidines, pyrrolizidines, pirrolidines, pyrroles, taxoids and macrolide natural products.

Atypical Antipsychotics: Mechanism of Action

Atypical Antipsychotics: Mechanism of Action

Investigation of hydrophobic moment and hydrophobicity properties for transmembrane alpha-helices.

Integral membrane proteins are the primary targets of novel drugs but are largely without solved structures. As a consequence, hydrophobic moment plot methodology is often used to identify putative transmembrane α-helices of integral membrane proteins, based on their local maximum mean hydrophobic moment (<μH>) and the corresponding mean hydrophobicity (<H>). To calculate these properties, the methodology identifies an optimal eleven residue window (L = 11), assuming an amino acid angular frequency, θ, fixed at 100°.Using a data set of 403 transmembrane α-helix forming sequences, the relationship between <μH> and <H>, and the effect of varying of L and / or θ on this relationship, was investigated. Confidence intervals for correlations between <μH> and <H> are established. It is shown, using bootstrapping procedures that the strongest statistically significant correlations exist for small windows where 7 ≤ L ≤ 16. Monte Carlo analysis suggests that this correlation is dependent upon amino acid residue primary structure, implying biological function and indicating that smaller values of L give better characterisation of transmembrane sequences using <μH>. However, varying window size can also lead to different regions within a given sequence being identified as the optimal window for structure / function predictions. Furthermore, it is shown that optimal periodicity varies with window size; the optimum, based on <μH> over the range of window sizes, (7 ≤ L ≤ 16), was at θ = 102° for the transmembrane α-helix data set.