Structure-based Design Approach Research Articles

Application and Limitations of X‐Ray Crystallographic Data in Structure‐Based Ligand and Drug Design.

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Design of Potent Peptide Mimetics of Brain-derived Neurotrophic Factor

Brain-derived neurotrophic factor (BDNF) has potential for the treatment of human neurodegenerative diseases. However, the general lack of success of neurotrophic factors in clinical trials has led to the suggestion that low molecular weight neurotrophic drugs may be better agents for therapeutic use. Here we describe small, dimeric peptides designed to mimic a pair of solvent-exposed loops important for the binding and activation of the BDNF receptor, trkB. The monomer components that make up the dimers were based on a monocyclic monomeric peptide mimic of a single loop of BDNF (loop 2) that we had previously shown to be an inhibitor of BDNF-mediated neuronal survival (O'Leary, P. D., and Hughes, R. A. (1998) J. Neurochem. 70, 1712-1721). Bicyclic dimeric peptides behaved as partial agonists with respect to BDNF, promoting the survival of embryonic chick sensory neurons in culture. We reasoned that the potency and/or efficacy of these compounds might be improved by reducing the conformational flexibility about their dimerizing linker. Thus, we designed a highly conformationally constrained tricyclic dimeric peptide and synthesized it using an efficient, quasi-one-pot approach. Although still a partial BDNF-like agonist, the tricyclic dimer was particularly potent in promoting neuronal survival in vitro (EC50 11 pm). The peptides described here, which are greatly reduced in size compared with the parent protein, could serve as useful lead compounds for the development of true neurotrophic drugs and indicate that the structure-based design approach could be used to obtain potent mimetics of other growth factors that dimerize their receptors.

Application and limitations of X-ray crystallographic data in structure-based ligand and drug design.

Structure-based design usually focuses upon the optimization of ligand affinity. However, successful drug design also requires the optimization of many other properties. The primary source of structural information for protein-ligand complexes is X-ray crystallography. The uncertainties introduced during the derivation of an atomic model from the experimentally observed electron density data are not always appreciated. Uncertainties in the atomic model can have significant consequences when this model is subsequently used as the basis of manual design, docking, scoring, and virtual screening efforts. Docking and scoring algorithms are currently imperfect. A good correlation between observed and calculated binding affinities is usually only observed only when very large ranges of affinity are considered. Errors in the correlation often exceed the range of affinities commonly encountered during lead optimization. Some structure-based design approaches now involve screening libraries by using technologies based on NMR spectroscopy and X-ray crystallography to discover small polar templates, which are used for further optimization. Such compounds are defined as leadlike and are also sought by more traditional high-throughput screening technologies. Structure-based design and HTS technologies show important complementarity and a degree of convergence.

Non-Covalent Inhibitors of the 20S Proteasome

Peptidomimetics have been commonly used as lead compounds to design inhibitors with high affinity and specificity for a particular enzyme. The discovery that a 2-aminobenzylstatine derivative originally designed to target an aspartyl protease was able to inhibit specifically and non-covalently the chymotrypsin-like activity of the 20S proteasome represented a unique starting point for our medicinal chemistry endeavor for this target. Utilizing a structure-based design approach, we have been able to improve the potency of this new class of proteasome inhibitors without affecting its in vitro selectivity profile.

Structure-based design approaches to cell wall biosynthesis inhibitors.

This review summarizes some of the published attempts to incorporate protein and NMR structures in the design of new antibiotics that specifically target Cell Wall biosynthesis. Most of the steps involved in peptidglycan synthesis have been investigated as potential strategies against cell wall inhibition. Structural information has been most useful in the design of molecules in the Mur enzyme pathway, penicillin binding proteins and lactamases, as well as proteins that are part of the final steps of transglycosylation - in particular, d-Ala-d-Ala ligase. Several unique issues exist in the design of effective antibacterials, such as the significant differences in protein structure between organisms, such as the case of MurB in which a large amino acid loop that occupies the active site of the E. Coli is gone in the Staph aureus enzyme. Additionally, bacterial resistance is an important issue, and in some cases, structural information can be used to understand the source of this resistance. For example, mutations within the d-Ala-d-Ala ligases lead to the inability of Vancomycin antibiotics to bind.

Hepatitis C virus NS3 serine protease as a drug discovery target

Hepatitis C virus NS3 serine protease (HCV-Pr) is an extensively studied enzyme for drug intervention. The target presented serious challenges in early screening efforts, however, wish the lack of prominent active site features rendering traditional nonpeptidic serine protease inhibitor motifs and high-throughput screening campaigns ineffectual. In contrast, the peptidomimetic structure-based design approach has proven uccessful in the discovery of potent inhibitors of HCV Pr. Subsequent rational design efforts have led to the identification of an inhibitor that demonstrates efficacy in man, validating the years of research. This review summarizes why HCV Pr provides a viable drug discovery target despite the many obstacies, and details the breakthroughs in protein production and essay development that have facilitated inhibitor advances. The latest inhibitors in preclinical and clinical research and development are also presented, along with a discussion of how the recent HCV Pr clinical candidate chalienges much of the dogma surrounding peptidomimetic design. In addition, future issues such as resistance, genotype coverage and HIV-HCV coinfected individuals are considered.

Structure-based inhibitor design targeting HIV-1 integrase.

HIV integrase (IN) is a viral-encoded protein that catalyzes the breaking and joining reactions that mediate integration of viral DNA into the host genome. Therefore, IN offers a unique target for the development of novel anti-HIV and anti-AIDS therapeutics. To take advantage of this potential, drug discovery efforts via structure-based design approaches have been undertaken. Presented is a review of computer-aided drug design efforts targeting HIV IN. Included is an overview of the life-cycle of HIV, with emphasis on the mechanism of action of IN, biological assays for measuring IN activity and identifying IN inhibitors, and the appropriate cell-based assays required for determining the antiviral activity of IN inhibitors. This is followed by a review of the available three-dimensional structures of HIV IN. Structure-based drug design efforts are then critiqued, including both ligand-based (e.g. pharmacophore) and target-based (e.g. docking) methods. Results from recent computational chemistry studies of IN are also discussed.

A structure-based design approach for the identification of novel inhibitors: application to an alanine racemase.

We report a new structure-based strategy for the identification of novel inhibitors. This approach has been applied to Bacillus stearothermophilus alanine racemase (AlaR), an enzyme implicated in the biosynthesis of the bacterial cell wall. The enzyme catalyzes the racemization of L- and D-alanine using pyridoxal 5'-phosphate (PLP) as a cofactor. The restriction of AlaR to bacteria and some fungi and the absolute requirement for D-alanine in peptidoglycan biosynthesis make alanine racemase a suitable target for drug design. Unfortunately, known inhibitors of alanine racemase are not specific and inhibit the activity of other PLP-dependent enzymes, leading to neurological and other side effects. This article describes the development of a receptor-based pharmacophore model for AllaR, taking into account receptor flexibility (i.e. a 'dynamic' pharmacophore model). In order to accomplish this, molecular dynamics (MD) simulations were performed on the full AlaR dimer from Bacillus stearothermophilus (PDB entry, 1 sft) with a D-alanine molecule in one active site and the non-covalent inhibitor, propionate, in the second active site of this homodimer. The basic strategy followed in this study was to utilize conformations of the protein obtained during MD simulations to generate a dynamic pharmacophore model using the property mapping capability of the LigBuilder program. Compounds from the Available Chemicals Directory that fit the pharmacophore model were identified and have been submitted for experimental testing. The approach described here can be used as a valuable tool for the design of novel inhibitors of other biomolecular targets.

An evolutionary approach for structure-based design of natural and non-natural peptidic ligands.

A new computational approach (PEP) is presented for the structure-based design of linear polymeric ligands consisting of any type of amino acid. Ligands are grown from a seed by iteratively adding amino acids to the growing construct. The search in chemical space is performed by a build-up approach which employs all of the residues of a user-defined library. At every growing step, a genetic algorithm is used for conformational optimization of the last added monomer inside the binding site of a rigid target protein. The binding energy with electrostatic solvation is evaluated to select sequences for further growing. PEP is tested on the peptide substrate binding site of the insulin receptor tyrosine kinase and farnesyltransferase. In both test cases, the peptides designed by PEP correspond to the sequence motifs of known substrates. For tyrosine kinase, tyrosine residues are suggested at position P and P+2. While the tyrosine at P is in agreement with the experimental data, the one at P+2 is a prediction which awaits experimental validation. For farnesyltransferase, it is shown that electrostatic solvation is necessary for the correct design of peptidic inhibitors.

Antagonists of the Src homology 2 (SH2) domains of Grb2, Src, Lck and ZAP-70.

Src homology 2 (SH2) domains are protein modules that mediate intracellular protein-protein interactions in signal transduction pathways. The specific association of an SH2 domain with a phosphotyrosine-containing sequence of another protein induces a cascade of molecular interactions that effect a wide range of cellular processes. Alterations in these signaling pathways have been associated with the development and progression of a broad range of pathologies. Because of the regulatory role of SH2 domains in these signal transduction pathways, specific SH2 domains can be ideal targets for intervention with therapeutic agents in many different disease indications (e.g. cancer, osteoporosis, disorders of the immune and cardiovascular systems). Among the SH2 domains pursued as drug discovery targets in the last few years are those of Grb2, Src, Lck and ZAP-70. This review focuses on contributions in the design and synthesis of antagonists of these particular SH2 domains. Specific examples have been selected to illustrate how structure-based design approaches have been used to progress in this area of research.

Recognition of small molecules by a ribonucleopeptide.

In this research, we have design an ATP binding domain that consists of an RNA subunit and a peptide subunit by means of structure-based design approach and in vitro selection method. The RNA subunit is designed to consist of two functional domains; an ATP binding domain with 20 randomized nucleotides and an adjacent stem region that serves as a binding site for the RNA-binding peptide. RNA-peptide receptors to ATP were isolated from a pool of different RNAs by selection with affinity column and enzymatic amplification. Effects of the RNA-binding peptide for the specific ATP binding to the selected RNA-peptide receptors are discussed.

A structure-based design approach to the development of novel, reversible AChE inhibitors.

Chimeras of tacrine and m-(N,N,N-Trimethylammonio)trifluoroacetophenone (1) were designed as novel, reversible inhibitors of acetylcholinesterase. On the basis of the X-ray structure of the apoenzyme, a molecular modeling study determined the favored attachment positions on the 4-aminoquinoline ring (position 3 and the 4-amino nitrogen) and the favored lengths of a polymethylene link between the two moieties (respectively 5-6 and 4-5 sp(3) atoms). Seven compounds matching these criteria were synthesized, and their inhibitory potencies were determined to be in the low nanomolar range. Activity data for close analogues lacking some of the postulated key features showed that our predictions were correct. In addition, a subsequent crystal structure of acetylcholinesterase complexed with the most active compound 27 was in good agreement with our model. The design strategy is therefore validated and can now be developed further.

Rational design of potent and selective EGFR tyrosine kinase inhibitors as anticancer agents.

Increasing knowledge of the structure and function of the Epidermal Growth Factor Receptor (EGFR) subfamily of tyrosine kinases, and of their role in the initiation and progression of various cancers has led to the search for inhibitors of signaling molecules that may prove to be important in cancer therapy. The complex nature of EGFR biology allows for potential opportunities for EGFR inhibitors in a number of areas of cancer therapy, including proliferative, angiogenic, invasive, and metastatic aspects. Different approaches have been used to target either the extracellular ligand-binding domain of the EGFR or the intracellular tyrosine kinase region that results in interference with its signaling pathways that modulate cancer-promoting responses. Examples of these include a number of monoclonal antibodies, immunotoxins and ligand-binding cytotoxic agents that target the extracellular ligand binding region of EGFR, and small molecule inhibitors that target the intracellular kinase domain and act by interfering with ATP binding to the receptor. During the past 3 years, significant progress has been made towards the identification of new structural classes of small molecule inhibitors that show high potency and specificity towards EGFR. The search for new small molecules that inhibit kinases has included traditional approaches like the testing of natural products, random screening of chemical libraries, the use of classical structure-activity-relationship studies, and the incorporation of structure-based drug design and combinatorial chemistry techniques. There has been a significant improvement in the development of selective EGFR inhibitors with the use of a structure-based design approach employing a homology model of the EGFR kinase domain. Molecular modeling procedures have been used to generate novel molecules that are complementary in shape and electrostatics to the EGFR kinase domain topography. This review focuses on some examples of the successful use of this method.

Peptide and peptide mimetic inhibitors of antigen presentation by HLA-DR class II MHC molecules. Design, structure-activity relationships, and X-ray crystal structures.

Molecular features of ligand binding to MHC class II HLA-DR molecules have been elucidated through a combination of peptide structure-activity studies and structure-based drug design, resulting in analogues with nanomolar affinity in binding assays. Stabilization of lead compounds against cathepsin B cleavage by N-methylation of noncritical backbone NH groups or by dipeptide mimetic substitutions has generated analogues that compete effectively against protein antigens in cellular assays, resulting in inhibition of T-cell proliferation. Crystal structures of four ternary complexes of different peptide mimetics with the rheumatoid arthritis-linked MHC DRB10401 and the bacterial superantigen SEB have been obtained. Peptide-sugar hybrids have also been identified using a structure-based design approach in which the sugar residue replaces a dipeptide. These studies illustrate the complementary roles played by phage display library methods, peptide analogue SAR, peptide mimetics substitutions, and structure-based drug design in the discovery of inhibitors of antigen presentation by MHC class II HLA-DR molecules.

ATP site-directed competitive and irreversible inhibitors of protein kinases

Several tyrosine and serine/threonine protein kinases have emerged in the last few years as attractive targets in the search for new therapeutic agents being applicable in many different disease indications. Initially, inhibition of these protein kinases by ATP site-directed inhibitors was considered less prone to success, but medicinal chemists from both academia and industry have been able to impart potency and selectivity to a limited number of scaffolds by modulating and fine-tuning the interactions of the modified template with the ATP binding site of the selected kinase. The chemical templates that have been used in the synthesis of ATP site-directed protein kinase inhibitors are reviewed with emphasis on the kinase inhibitors that have entered or are about to enter clinical trials. Examples have been selected to illustrate how structure-based design approaches and new methods to increase compound diversity have had an impact on this area of research. © 2000 John Wiley & Sons, Inc. Med Res Rev, 20, No. 1, 28–57, 2000

ATP site-directed competitive and irreversible inhibitors of protein kinases.

Several tyrosine and serine/threonine protein kinases have emerged in the last few years as attractive targets in the search for new therapeutic agents being applicable in many different disease indications. Initially, inhibition of these protein kinases by ATP site-directed inhibitors was considered less prone to success, but medicinal chemists from both academia and industry have been able to impart potency and selectivity to a limited number of scaffolds by modulating and fine-tuning the interactions of the modified template with the ATP binding site of the selected kinase. The chemical templates that have been used in the synthesis of ATP site-directed protein kinase inhibitors are reviewed with emphasis on the kinase inhibitors that have entered or are about to enter clinical trials. Examples have been selected to illustrate how structure-based design approaches and new methods to increase compound diversity have had an impact on this area of research.

Engineering Biosensors by Introducing Fluorescent Allosteric Signal Transducers: Construction of a Novel Glucose Sensor

The development of biosensors based on genetically engineered proteins offers many potential advantages to sensors that rely on natural proteins only. Here we present how protein engineering techniques can be used to integrate optical signal transduction functions directly into proteins by incorporating environmentally sensitive fluorescent groups that are allosterically linked to a ligand binding site, using a structure-based rational design approach. The location of potential fluorescent allosteric signal transduction (FAST) sites were predicted in the periplasmic Glucose Binding Protein of Escherichia coli. Single cysteine mutations were constructed at these locations, which allowed site-specific, covalent coupling of environmentally sensitive fluorophores. The fluorescence of these conjugates was shown to be hyperbolically dependent upon glucose concentration, indicating that the behavior of the fluorophore is allosterically coupled to the degree of occupancy at the glucose binding site, as predicted....

Solution Structures of FcεRI α-Chain Mimics: A β-Hairpin Peptide and Its Retroenantiomer

A central event in the development of the allergic response is the interaction between immunoglobulin E (IgE) and its cellular high-affinity receptor FcεRI. Allergen-bound IgE mediates the allergic response by binding through its Fc region to its cellular receptor on mast cells and basophils, causing the release of chemical mediators. One strategy for the treatment of allergic disorders is the use of therapeutic compounds which would inhibit the interaction between IgE and FcεRI. Using a structure-based design approach, conformationally constrained synthetic peptides were designed to mimic a biologically active β-hairpin region of the α-chain of FcεRI. Two peptide mimics of the FcεRI α-chain were previously shown to inhibit IgE−FcεRI interactions, one a peptide comprised of l-amino acids, covalently cyclized by N- and C-terminal cysteine residues, and the other its retroenantiomer. In this paper the solution structures of these compounds are derived using NMR spectroscopy. The topochemical relationship between the retroenantiomeric compounds and the structural basis of their biological activity is described.

A CD4-CDR3 peptide analog inhibits both primary and secondary autoreactive CD4+ T cell responses in experimental allergic encephalomyelitis.

A structure-based design approach was used to develop a cyclized peptide analog of the murine CD4-CDR3-like region as a potential inhibitor of autoimmune CD4+ T cells responsible for the pathogenesis of experimental allergic encephalomyelitis (EAE). Our results indicate that this peptide, referred to as rD-mPGPtide, is able to significantly inhibit the clinical and pathologic symptoms of EAE in the SJL mouse model when administered on day 12 of induction. The optimum effective dosage range for the peptide, injected i.v., was between 0.125 and 0.5 mg and dosages of as high as 5 mg had no observable toxic effects. Treated mice had normal levels of lymphocytes less than 2 wk later and exhibited normal in vitro primary responses to alloantigen and secondary responses to keyhole limpet hemocyanin Ag. The specificity of the rD-mPGPtide treatment for autoreactive T cells was demonstrated by inhibiting proteolipid protein (p139-151)-induced EAE and finding that the lymph node T cells from these mice had suppressed responses to this Ag, but normal responses to alloantigen or other nominal Ag. Importantly, rD-mPGPtide was found to be effective on secondary T cell responses in an EAE rechallenge situation and was able to establish conditions for long-term resistance to further Ag exposure. Analysis of the cytokine profile of responding T cells during late effector stages of disease revealed that the levels of IFN-gamma and IL-4 are significantly reduced in rD-mPGPtide-treated mice. These results strongly suggest that the administration of a CD4-CDR3 peptide analog is an effective therapeutic approach for the inhibition of the CD4+ T cell-mediated autoimmune response in EAE.

Making New Drugs via Combinatorial Chemistry

ABSTRACT DRUG DEVELOPMENT, as a problemsolving enterprise, uses a combination of two different approaches—one deductive, the other inductive. The deductive approach is characterized by a shotgun approach of testing a host of compounds thought to have specific pharmacologic properties to see which have the greatest effect on the test subject. The process moves from generalized knowledge to specific application and has always been time-consuming and labor-intensive.The inductive approach, which is considered more intellectually challenging, starts with a structural understanding of a drug's binding site to more narrowly search for, or design and synthesize, its specific complement. This structure-based design approach moves from specific information to more generalized application.Structure-based design has made substantial strides in the past decade. Six months ago, for example, Jane E. Brody in the New York Times (November 7, 1995) described the work of a Cornell University research team that received a $3.3 million grant from