Countering cooperative effects in protease inhibitors using constrained b-strand-mimicking templates in focused combinatorial libraries

A major problem in de novo design of enzyme inhibitors is the unpredictability of the induced fit, with the shape of both ligand and enzyme changing cooperatively and unpredictably in response to subtle structural changes within a ligand. We have investigated the possibility of dampening the induced fit by using a constrained template as a replacement for adjoining segments of a ligand. The template preorganizes the ligand structure, thereby organizing the local enzyme environment. To test this approach, we used templates consisting of constrained cyclic tripeptides, formed through side chain to main chain linkages, as structural mimics of the protease-bound extended beta-strand conformation of three adjoining amino acid residues at the N- or C-terminal sides of the scissile bond of substrates. The macrocyclic templates were derivatized to a range of 30 structurally diverse molecules via focused combinatorial variation of nonpeptidic appendages incorporating a hydroxyethylamine transition-state isostere. Most compounds in the library were potent inhibitors of the test protease (HIV-1 protease). Comparison of crystal structures for five protease-inhibitor complexes containing an N-terminal macrocycle and three protease-inhibitor complexes containing a C-terminal macrocycle establishes that the macrocycles fix their surrounding enzyme environment, thereby permitting independent variation of acyclic inhibitor components with only local disturbances to the protease. In this way, the location in the protease of various acyclic fragments on either side of the macrocyclic template can be accurately predicted. This type of templating strategy minimizes the problem of induced fit, reducing unpredictable cooperative effects in one inhibitor region caused by changes to adjacent enzyme-inhibitor interactions. This idea might be exploited in template-based approaches to inhibitors of other proteases, where a beta-strand mimetic is also required for recognition, and also other protein-binding ligands where different templates may be more appropriate.

Molecular characterization of the toxic cyanobacterium Cylindrospermopsis raciborskii and design of a species-specific PCR

Cylindrospermopsis raciborskii is a toxic-bloom-forming cyanobacterium that is commonly found in tropical to subtropical climatic regions worldwide, but it is also recognized as a common component of cyanobacterial communities in temperate climates. Genetic profiles of C. raciborskii were examined in 19 cultured isolates originating from geographically diverse regions of Australia and represented by two distinct morphotypes. A 609-bp region of rpoC1, a DNA-dependent RNA polymerase gene, was amplified by PCR from these isolates with cyanobacterium-specific primers. Sequence analysis revealed that all isolates belonged to the same species, including morphotypes with straight or coiled trichomes. Additional rpoC1 gene sequences obtained for a range of cyanobacteria highlighted clustering of C. raciborskii with other heterocyst-producing cyanobacteria (orders Nostocales and Stigonematales). In contrast, randomly amplified polymorphic DNA and short tandemly repeated repetitive sequence profiles revealed a greater level of genetic heterogeneity among C. raciborskii isolates than did rpoC1 gene analysis, and unique band profiles were also found among each of the cyanobacterial genera examined. A PCR test targeting a region of the rpoC1 gene unique to C. raciborskii was developed for the specific identification of C. raciborskii from both purified genomic DNA and environmental samples. The PCR was evaluated with a number of cyanobacterial isolates, but a PCR-positive result was only achieved with C, raciborskii. This method provides an accurate alternative to traditional morphological identification of C. raciborskii.

Bioprocess-centered molecular design (BMD) for the efficient production of an interfacially active peptide

The efficient expression and purification of an interfacially active peptide (mLac21) was achieved by using bioprocess-centered molecular design (BMD), wherein key bioprocess considerations are addressed during the initial molecular biology work. The 21 amino acid mLac21 peptide sequence is derived from the lac repressor protein and is shown to have high affinity for the oil-water interface, causing a substantial reduction in interfacial tension following adsorption. The DNA coding for the peptide sequence was cloned into a modified pET-31(b) vector to permit the expression of mLac21 as a fusion to ketosteroid isomerase (KSI). Rational iterative molecular design, taking into account the need for a scaleable bioprocess flowsheet, led to a simple and efficient bioprocess yielding mLac21 at 86% purity following ion exchange chromatography (and >98% following chromatographic polishing). This case study demonstrates that it is possible to produce acceptably pure peptide for potential commodity applications using common scaleable bioprocess unit operations. Moreover, it is shown that BMD is a powerful strategy that can be deployed to reduce bioseparation complexity. (C) 2004 Wiley Periodicals, Inc.

Design, development, and use of molecular primers and probes for the detection of Gluconacetobacter species in the pink sugarcane mealybug

Molecular tools for the species-specific detection of Gluconacetobacter sacchari, Gluconacetobacter diazotrophicus, and Gluconacetobacter liquefaciens from the pink sugarcane mealybug (PSMB) Saccharicoccus sacchari Cockerell (Homiptera: Pseudococcidae) were developed and used in polymerase chain reactions (PCR) and in fluorescence in situ hybridizations (FISH) to better understand the microbial diversity and the numerical significance of the acetic acid bacteria in the PSMB microenvironment. The presence of these species in the PSMB occurred over a wide range of sites, but not in all sites in sugarcane-growing areas of Queensland, Australia, and was variable over time. Molecular probes for use in FISH were also designed for the three acetic acid bacterial species, and shown to be specific only for the target species. Use of these probes in FISH of squashed whole mealybugs indicated that these acetic acid bacteria species represent only a small proportion of the microbial population of the PSMB. Despite the detection of Glac. sacchari, Glac. diazotrophicus, and Glac. liquefaciens by PCR from different mealybugs isolated at various times and from various sugarcane-growing areas in Queensland, Australia, these bacteria do not appear to be significant commensals in the PSMB environment.

Design strategies for controlling the molecular weight and rate using reversible addition-fragmentation chain transfer mediated living radical polymerization

Living radical polymerization has allowed complex polymer architectures to be synthesized in bulk, solution, and water. The most versatile of these techniques is reversible addition-fragmentation chain transfer (RAFT), which allows a wide range of functional and nonfunctional polymers to be made with predictable molecular weight distributions (MWDs), ranging from very narrow to quite broad. The great complexity of the RAFT mechanism and how the kinetic parameters affect the rate of polymerization and MWD are not obvious. Therefore, the aim of this article is to provide useful insights into the important kinetic parameters that control the rate of polymerization and the evolution of the MWD with conversion. We discuss how a change in the chain-transfer constant can affect the evolution of the MWD. It is shown how we can, in principle, use only one RAFT agent to obtain a poly-mer with any MWD. Retardation and inhibition are discussed in terms of (1) the leaving R group reactivity and (2) the intermediate radical termination model versus the slow fragmentation model. (c) 2005 Wiley Periodicals, Inc.

Molecular recognition of macrocyclic peptidomimetic inhibitors by HIV-1 protease

High-resolution crystal structures are described for seven macrocycles complexed with HIV-1 protease (HIVPR). The macrocycles possess two amides and an aromatic group within 15-17 membered rings designed to replace N- or C-terminal tripeptides from peptidic inhibitors of HIVPR. Appended to each macrocycle is a transition state isostere and either an acyclic peptide, nonpeptide, or another macrocycle. These cyclic analogues are potent inhibitors of HIVPR, and the crystal structures show them to be structural mimics of acyclic peptides, binding in the active site of HIVPR via the same interactions. Each macrocycle is restrained to adopt a P-strand conformation which is preorganized for protease binding. An unusual feature of the binding of C-terminal macrocyclic inhibitors is the interaction between a positively charged secondary amine and a catalytic aspartate of HIVPR. A bicyclic inhibitor binds similarly through its secondary amine that lies between its component N-terminal and C-terminal macrocycles. In contrast, the corresponding tertiary amine of the N-terminal macrocycles does not interact with the catalytic aspartates. The amine-aspartate interaction induces a 1.5 Angstrom N-terminal translation of the inhibitors in the active site and is accompanied by weakened interactions with a water molecule that bridges the ligand to the enzyme, as well as static disorder in enzyme flap residues. This flexibility may facilitate peptide cleavage and product dissociation during catalysis. Proteases [Aba(67,95)]HIVPR and [Lys(7),Ile(33),Aba(67,95)]- HIVPR used in this work were shown to have very similar crystal structures.

Applications of NMR in drug design: Sructure-activity relationships in disulfide-rich peptides

NMR is a powerful technique for determining structures of biologically active molecules in solution. In recent years. our laboratory has focussed on the structure determination of small disulfide-rich proteins from both plants and animals which are valuable targets in drug design applications. This article will review these structural studies and their implications in drug design.

Asymmetric MRI magnet design using a hybrid numerical method

This paper describes a hybrid numerical method for the design of asymmetric magnetic resonance imaging magnet systems. The problem is formulated as a field synthesis and the desired current density on the surface of a cylinder is first calculated by solving a Fredholm equation of the first kind. Nonlinear optimization methods are then invoked to fit practical magnet coils to the desired current density. The field calculations are performed using a semi-analytical method. A new type of asymmetric magnet is proposed in this work. The asymmetric MRI magnet allows the diameter spherical imaging volume to be positioned close to one end of the magnet. The main advantages of making the magnet asymmetric include the potential to reduce the perception of claustrophobia for the patient, better access to the patient by attending physicians, and the potential for reduced peripheral nerve stimulation due to the gradient coil configuration. The results highlight that the method can be used to obtain an asymmetric MRI magnet structure and a very homogeneous magnetic field over the central imaging volume in clinical systems of approximately 1.2 m in length. Unshielded designs are the focus of this work. This method is flexible and may be applied to magnets of other geometries. (C) 1999 Academic Press.

Molecular immunology databases and data repositories

Over recent years databases have become an extremely important resource for biomedical research. Immunology research is increasingly dependent on access to extensive biological databases to extract existing information, plan experiments, and analyse experimental results. This review describes 15 immunological databases that have appeared over the last 30 years. In addition, important issues regarding database design and the potential for misuse of information contained within these databases are discussed. Access pointers are provided for the major immunological databases and also for a number of other immunological resources accessible over the World Wide Web (WWW). (C) 2000 Elsevier Science B.V. All rights reserved.

Molecular diversity through sugar scaffolds

Monosaccharides provide an excellent platform to tailor molecular diversity by appending desired substituents at selected positions around the sugar scaffold. The presence of five functionalized and stereo-controlled centres on the sugar scaffolds gives the chemist plenty of scope to custom design molecules to a pharmacophore model. This review focuses on the peptidomimetic developments in this area, as well as the concept of tailoring structural and functional diversity in a library using carbohydrate scaffolds and how this can lead to increased hit rates and rapid identification of leads, which has promising prospects for drug development.

Molecular epidemiology: A multidisciplinary approach to understanding parasitic zoonoses

Sound application of molecular epidemiological principles requires working knowledge of both molecular biological and epidemiological methods. Molecular tools have become an increasingly important part of studying the epidemiology of infectious agents. Molecular tools have allowed the aetiological agent within a population to be diagnosed with a greater degree of efficiency and accuracy than conventional diagnostic tools. They have increased the understanding of the pathogenicity, virulence, and host-parasite relationships of the aetiological agent, provided information on the genetic structure and taxonomy of the parasite and allowed the zoonotic potential of previously unidentified agents to be determined. This review describes the concept of epidemiology and proper study design, describes the array of currently available molecular biological tools and provides examples of studies that have integrated both disciplines to successfully unravel zoonotic relationships that would otherwise be impossible utilising conventional diagnostic tools. The current limitations of applying these tools, including cautions that need to be addressed during their application are also discussed.(c) 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

The design of planar gradient coils. Part I: A winding path correction method

In this work a new approach for designing planar gradient coils is outlined for the use in an existing MRI apparatus. A technique that allows for gradient field corrections inside the diameter-sensitive volume is deliberated. These corrections are brought about by making changes to the wire paths that constitute the coil windings, and hence, is called the path correction method. The existing well-known target held method is used to gauge the performance of a typical gradient coil. The gradient coil design methodology is demonstrated for planar openable gradient coils that can be inserted into an existing MRI apparatus. The path corrected gradient coil is compared to the coil obtained using the target field method. It is shown that using a wire path correction with optimized variables, winding patterns that can deliver high magnetic gradient field strengths and large imaging regions can be obtained.

Zipping up transcription factors: Rational design of anti-Jun and anti-Fos peptides

Various members of the bZip and bHLH-Zip families of eukaryotic transcription factors, including Jun, Fos, and Myc, have been identified as oncoproteins; mutation or deregulated expression of these proteins leads to certain types of cancer. These proteins can only bind to their cognate DNA enhancer sites following homodimerization, or heterodimerization with another family member, via their leucine zipper domain. Thus, a novel anticancer strategy would be to inhibit dimerization of these proteins, thereby blocking their DNA binding and transactivation functions. In this paper we show that it is possible to rationally design leucine zipper peptides that bind with high affinity to the leucine zipper dimerization domains of c-Jun and c-Fos, thus preventing the formation of functional c-Jun homodimers and c-Jun:c-Fos heterodimers; we refer to such peptides as superzippers (SZs). In vivo, c-Jun:SZ and c-Fos:SZ heterodimers should be nonfunctional as they lack one of the two basic domains that are essential for DNA binding. While the transport of a peptidic agent into cells often poses a severe obstacle to its therapeutic use, we show that a 46-residue leucine zipper peptide can be transported into HeLa cells by coupling it to a 17-residue carrier peptide from the Antennapedia homeodomain, thus paving the way for detailed studies of the therapeutic potential of superzipper peptides.