Structure-based design, synthesis and structure-activity relationships of dibenzosuberyl- and benzoate-substituted tropines as ligands for acetylcholine-binding protein

Using structure-based optimization procedures on in silico hits, dibenzosuberyl- and benzoate substituted tropines were designed as ligands for acetylcholine-binding protein (AChBP). This protein is a homolog to the ligand binding domain of the nicotinic acetylcholine receptor (nAChR). Distinct SAR is observed between two AChBP species variants and between the α7 and α4β2 nAChR subtype. The AChBP species differences are indicative of a difference in accessibility of a ligand-inducible subpocket. Hereby, we have identified a region that can be scrutinized to achieve selectivity for nicotinic receptor subtypes.     

Proteasome-independent HLA-B27 ligands arise mainly from small basic proteins

Many of the constitutive peptide ligands of HLA-B27, a molecule strongly associated with spondyloarthritis, are proteasome-independent. Stable isotope tagging, mass spectrometry, and epoxomicin-mediated inhibition were used to determine their percentage, structural features, and parental proteins. Of 104 molecular species examined, 29.8% were proteasome-independent, paralleling the level of HLA-B27 re-expression in the presence of epoxomicin after acid stripping. Proteasome-dependent and -independent ligands differed little in peptide motifs, flanking sequences, and cellular localization of the parental proteins. In contrast, whereas the former set arose from proteins whose size and isoelectric point distribution largely reflected those in the human proteome, proteasome-independent ligands, other than a few matching signal sequences, were almost totally derived from small (about 6-16.5 kDa) and basic proteins, which account for only 6.6% of the human proteome. Thus, a non-proteasomal proteolytic pathway with strong preference for small proteins is responsible for a significant fraction of the HLA-B27-bound peptide repertoire.     

Structure-based protein design with deep learning

Since the first revelation of proteins functioning as macromolecular machines through their three dimensional structures, researchers have been intrigued by the marvelous ways the biochemical processes are carried out by proteins. The aspiration to understand protein structures has fueled extensive efforts across different scientific disciplines. In recent years, it has been demonstrated that proteins with new functionality or shapes can be designed via structure-based modeling methods, and the design strategies have combined all available information — but largely piece-by-piece — from sequence derived statistics to the detailed atomic-level modeling of chemical interactions. Despite the significant progress, incorporating data-derived approaches through the use of deep learning methods can be a game changer. In this review, we summarize current progress, compare the arc of developing the deep learning approaches with the conventional methods, and describe the motivation and concepts behind current strategies that may lead to potential future opportunities.     

Structure-based de novo design of ligands using a three-dimensional model of the insulin receptor

For the first time, a three-dimensional model of the insulin receptor is used in the de novo design of novel ligands that potentially mimic interactions of insulin at its receptor. Compound 4 competed with insulin as seen in autophosphorylation assays and inhibited up to 68% of IR autophosphorylation at 300 microM of 4 in 3T3IR cells induced by 1 nM insulin. This model provides a basis for the design of potent insulin receptor ligands.     

A Yersinia pseudotuberculosis protein which cross-reacts with HLA-B27

The most-debated question in the investigation of the spondyloarthropathies has been whether there is molecular mimicry between host HLA-B27 antigens and the arthritis-causing pathogens. We have generated a monoclonal anti-HLA-B27 antibody in our laboratory and have used a radioimmunoassay to screen a panel of bacterial species. Two strains of Yersinia pseudotuberculosis were found to be highly reactive. The cross-reactive Yersinia component was identified by Western blot to be a 19,000 component. A preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis chromatography apparatus was constructed to isolate milligram quantities of this component. To verify that the component carried the HLA-B27-specific epitope, rabbits were hyperimmunized with the purified materials. Affinity-purified antibodies from one of the immunized rabbits indeed carried anti-HLA-B27 activity. Last, antibodies generated against synthetic peptides derived from the HLA-B27.1 amino acid sequence were tested against the Yersinia component. Positive reactivity was found with antibodies generated against a peptide spanning residues 69-83 of the HLA-B27.1 protein. Since this resides in the segment responsible for the allotypic specificity of the antigen, these experiments establish the presence of molecular mimicry to a high degree of confidence.     

基于结构的CRISPR蛋白xCas9的优化设计

酿脓链球菌(Streptococcus pyogenes Cas9,SpCas9)已成为强大的基因组编辑工具,但其可识别的前间隔序列临近基序(Protospacer adjacent motifs,PAMs)范围有限,且存在脱靶效应.为解决这些问题,文中提出一种对SpCas9的定向进化突变体xCas9进行优化的理性方法...     

Structure-based design of protein tyrosine phosphatase-1B inhibitors

Using structure-based design, a new class of inhibitors of protein tyrosine phosphatase-1B (PTP1B) has been identified, which incorporate the 1,2,5-thiadiazolidin-3-one-1,1-dioxide template.     

Structure-based design of a thiazolidinedione which targets the mitochondrial protein mitoNEET

Several PPAR-gamma agonists containing a thiazolidinedione moiety (referred to as glitazones) have been proposed to be neuroprotective and appear to alter mitochondrial function. Recently, a search for mitochondrial proteins that bind pioglitazone identified a novel protein, mitoNEET, which was later shown to regulate the oxidative capacity of the mitochondria. This identified an alternative target for the glitazones suggesting a possible new drug target for the treatment of neurodegenerative diseases. Molecular docking studies employing the reported crystal structure revealed five possible binding pockets on mitoNEET. We focused on two sites based on their physical characteristics. Using binding information gained from the analysis of two glitazones docked in these pockets, we designed and synthesized a ligand (NL-1) that would preferentially bind to site 1. Based on [³H]-binding data of the glitazones and comparisons to computer generated K_is, we were able to predict that site 1 was likely the target of the glitazones. NL-1 uncoupled isolated mitochondrial complex I respiration with an IC₅₀ of 2.4 μM and inhibited state III respiration up to 45%. To investigate the ability of NL-1 to block rotenone initiated free radicals from complex I, we found it was able to protect the human neuronal cell line SH-SY5Y against rotenone induced cell death. These data demonstrate that mitoNEET is a viable target for the design and synthesis of novel therapeutic agents aimed at altering mitochondrial function.     

HLA-B27-Homodimer-Specific Antibody Modulates the Expansion of Pro-Inflammatory T-Cells in HLA-B27 Transgenic Rats

Objectives

HLA-B27 is a common genetic risk factor for the development of Spondyloarthritides (SpA). HLA-B27 can misfold to form cell-surface heavy chain homodimers (B27₂) and induce pro-inflammatory responses that may lead to SpA pathogenesis. The presence of B27₂ can be detected on leukocytes of HLA-B27+ Ankylosing spondylitis (AS) patients and HLA-B27 transgenic rats. We characterized a novel B27₂–specific monoclonal antibody to study its therapeutic use in HLA-B27 associated disorders.

Methods

The monoclonal HD5 antibody was selected from a phage library to target cell-surface B27₂ homodimers and characterized for affinity, specificity and ligand binding. The immune modulating effect of HD5 was tested in HLA-B27 transgenic rats. Onset and progression of disease profiles were monitored during therapy. Cell-surface B27₂ and expansion of pro-inflammatory cells from blood, spleen and draining lymph nodes were assessed by flow cytometry.

Results

HD5 bound B27₂ with high specificity and affinity (K^d = 0.32 nM). HD5 blocked cell-surface interaction of B27₂ with immune regulatory receptors KIR3DL2, LILRB2 and Pirb. In addition, HD5 modulated the production of TNF from CD4+ T-cells by limiting B27₂ interactions in vitro. In an HLA-B27 transgenic rat model repetitive dosing of HD5 reduced the expansion of pro-inflammatory CD4+ T-cells, and decreased the levels of soluble TNF and number of cell-surface B27₂ molecules.

Conclusion

HD5 predominantly inhibits early TNF production and expansion of pro-inflammatory CD4+ T-cells in HLA-B27 transgenic rats. Monoclonal antibodies targeting cell-surface B27₂ propose a new concept for the modulation of inflammatory responses in HLA-B27 related disorders.     

Structure-based design and protein X-ray analysis of a protein kinase inhibitor.

A 5-aryl-1H-pyrazole molecular scaffold was designed to ligate the ATP binding site of cyclin dependent kinase 2 (CDK2) on the basis of crystallographic information. A search of the compound collection of Novartis using this scaffold as substructure query led to the identification of PKF049-365 as a representative of a new class of inhibitors of the cell cycle kinases CDK1/2. The three-dimensional structure of CDK2 in complex with PKF049-365 was subsequently determined by protein crystallography and refined to 1.53 A resolution. The X-ray analysis confirmed the binding mode expected from the design hypothesis. In addition, it revealed an alternative binding orientation involving a second tautomeric form of the inhibitor that was not envisaged during the design stage.     

Structure-based thermodynamic design of peptide ligands: Application to peptide inhibitors of the aspartic protease endothiapepsin

The prediction of binding affinities from structure is a necessary requirement in the development of structure-based molecular design strategies. In this paper, a structural parameterization of the energetics previously developed in this laboratory has been incorporated into a molecular design algorithm aimed at identifying peptide conformations that minimize the Gibbs energy. This approach has been employed in the design of mutants of the aspartic protease inhibitor pepstatin A. The simplest design strategy involves mutation and/or chain length modification of the wild-type peptide inhibitor. The structural parameterization allows evaluation of the contribution of different amino acids to the Gibbs energy in the wild-type structure, and therefore the identification of potential targets for mutation in the original peptide. The structure of the wild-type complex is used as a template to generate families of conformational structures in which specific residues have been mutated. The most probable conformations of the mutated peptides are identified by systematically rotating around the side-chain and backbone torsional angles and calculating the Gibbs potential function of each conformation according to the structural parametrization. The accuracy of this approach has been tested by chemically synthesizing two different mutants of pepstatin A. In one mutant, the alanine at position five has been replaced by a phenylalanine, and in the second one a glutamate has been added at the carboxy terminus of pepstatin A. The thermodynamics of association of pepstatin A and the two mutants have been measured experimentally and the results compared with the predictions. The difference between experimental and predicted Gibbs energies for pepstatin A and the two mutants is 0.23 ± 0.06 kcal/mol. The excellent agreement between experimental and predicted values demonstrates that this approach can be used in the optimization of peptide ligands. Proteins 30:74–85, 1998. © 1998 Wiley-Liss, Inc.     

Structure-based identification of novel PPAR gamma ligands

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor with an important role in the glucose metabolism and a target for type 2 diabetes mellitus therapy. The recent findings relating the use of the receptor full agonist rosiglitazone and the incidence of myocardial infarction raised concerns regarding whether receptor activation can actually be useful for diabetes management. The discovery of MRL-24 and GQ-16, ligands that can partially activate PPARγ and prevent weight gain and fluid retention, showed that a submaximal receptor activation can be a goal in the development of new ligands for PPARγ. Additionally, two previously described receptor antagonists, SR-202 and BADGE, were also shown to improve insulin sensitivity and decrease TNF-α level, revealing that receptor antagonism may also be an approach to pursue. Here, we used a structure-based approach to screen the subset ‘Drugs-Now’ of ZINC database. Fifteen ligands were selected after visual inspection and tested for their ability to bind to PPARγ. A benzoimidazol acetate, a bromobenzyl-thio-tetrazol benzoate and a [[2-[(1,3-dioxoinden-2-ylidene)methyl]phenoxy]methyl]benzoate were identified as PPARγ ligands, with IC₅₀ values smaller than 10 μM. Molecular dynamic simulations showed that the residues H323, H449, Y327, Y473, K367 and S289 are key structural elements for the molecular recognition of these ligands and the polar arm of PPARγ binding pocket.     

Molecular analysis of a functional subtype of HLA-B27. A possible evolutionary pathway for HLA-B27 polymorphism

The structure of a new HLA-B27 subtype antigen B27.4(B27D), distinguishable from the HLA-B27.1, B27.2, and B27.3 subtypes by cytolytic T lymphocytes and isoelectric focusing, has been established by comparative peptide mapping and sequence analysis. HLA-B27.4 differs from the main B27.1 subtype in the same two changes of aspartate-77 to serine-77 and valine-152 to glutamate-152, which distinguish the B27.1 and B27.3 subtypes. In addition, there are two other amino acid changes of histidine-114 to aspartate-114 and of aspartate-116 to tyrosine-116, which are unique to B27.4. The close structural relationship between B27.3 and B27.4 explains the similarity of these two subtypes in terms of T cell recognition. The presence of the two single amino acid differences between B27.3 and B27.4 within a span of three residues in the linear sequence provides a new example, suggesting that gene conversion-like mechanisms play a major role in the diversification of HLA-B27. A comparison of the structure of HLA-B27.4 with those of B27.1, B27.2, and B27.3 in the context of their ethnic distribution suggests that the diversification of the HLA-B27 antigens is an ongoing process that has continued after the separation of the major ethnic groups. A tentative evolutionary model for HLA-B27 polymorphism is proposed.     

Immunochemical variants of HLA-B27

Detailed study of HLA-B27 was prompted by the extremely strong associations between this antigen and spondyloarthropathies. Despite the relative homogeneity of this antigen when defined by alloantisera, B27 reactivity with the monoclonal antibody B27M2 suggests previously unrecognized heterogeneity. To define and confirm this heterogeneity on a molecular level, detergent extracts were prepared from B cell lines derived from individuals reactive (+) or unreactive (-) with the B27M2 antibody. Extracts were immunoprecipitated by specific allogeneic or monoclonal antibodies and analyzed by two-dimensional polyacrylamide gel electrophoresis. By this method the B27M2+ and B27M2- variants of HLA-B27 had different isoelectric points (pl) and could be distinguished from each other and from a different (Bw44) control alloantigen. Blockade of glycosylation by pretreatment of cells with tunicamycin did not alter pl but did reduce HLA antigens by approximately 3000 daltons. These data demonstrate that B27 antigens can be subdivided into subsets with different molecular composition. The effects of this heterogeneity upon the associations of B27 and disease are not yet known.     

Structure-based design and discovery of novel inhibitors of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) are important in the regulation of signal transduction processes. Certain enzymes of this class are considered as potential therapeutic targets in the treatment of a variety of diseases such as diabetes, inflammation, and cancer. However, many PTP inhibitors identified to date are peptide-based and contain a highly charged phosphate-mimicking component. These compounds usually lack membrane permeability and this limits their utility in the inhibition of intracellular phosphatases. In the present study, we have used structure-based design and modeling techniques to explore catalytic-site directed, reversible inhibitors of PTPs. Employing a non-charged phosphate mimic and non-peptidyl structural components, we have successfully designed and synthesized a novel series of trifluoromethyl sulfonyl and trifluoromethyl sulfonamido compounds as PTP inhibitors. This is the first time that an uncharged phosphate mimic is reported in the literature for general, reversible, and substrate-competitive inhibition of PTPs. It is an important discovery because the finding may provide a paradigm for the development of phosphatase inhibitors that enter cells and modify signal transduction.     

Structure-Based Design of Nonnatural Ligands for the HLA-B27 Protein

Abstract

X-ray studies as well as structure-activity relationships indicate that the central part of class I MHC-binding nonapeptides represents the main interaction site for a T cell receptor. In order to rationally manipulate T cell epitopes, several nonpeptidic spacer have been designed from the X-ray structure of a MHC-peptide complex and substituted for the T cell receptor-binding part of several antigenic peptides. The binding of the modified epitopes to the HLA-B*2705 protein was studied by an in vitro stabilisation assay and the thermal stability of all complexes examined by circular dichroism spectroscopy. Depending on their chemical nature and length, the introduced spacers may be classified into two categories. Monofunctional spacers (11-amino undecanoate, (R)-3-hydroxybutyrate trimer) simply link two anchoring peptide positions (P3 and P9) but loosely contact the MHC binding groove, and thus decrease more or less the affinity of the altered epitopes to HLA-B*2705. Bifunctional spacers ((R)-3-hydroxybutyrate and β-homoalanine combinations) not only bridges the two distant anchoring amino acids but also strongly interact with the binding cleft and lead to an increase in binding to the MHC protein. The presented modified ligands constitute interesting tools for perturbing the T cell response to the parent antigenic peptide.     

Structure-based design of isoquinoline-5-sulfonamide inhibitors of protein kinase B

Structure-based drug design of novel isoquinoline-5-sulfonamide inhibitors of PKB as potential antitumour agents was investigated. Constrained pyrrolidine analogues that mimicked the bound conformation of linear prototypes were identified and investigated by co-crystal structure determinations with the related protein PKA. Detailed variation in the binding modes between inhibitors with similar overall conformations was observed. Potent PKB inhibitors from this series inhibited GSK3beta phosphorylation in cellular assays, consistent with inhibition of PKB kinase activity in cells.     

Non-antigen presenting effects of HLA-B27

Spondyloarthropathies (SpA) are a group of chronic rheumatic diseases, which show a strong asoociation with human leukocyte antigen (HLA)-B27. Although the association between HLA-B27 and the susceptibility to SpA was discovered thirty years ago, the exact mechanism by which HLA-B27 predisposes to disease development remains unclear. The classical role of MHC class I molecules is to present peptides for CD8+ T cells. Therefore, it has been proposed that the antigen presenting function of HLA-B27 is somehow altered in the patients developing SpA. However, despite extensive research, the attempts to create a comprehensive theory that would explain the role of HLA-B27 as an antigen presenting molecule in the development of SpA have been unsuccessful. Reactive arthritis (ReA) belongs to the group of SpA. It is a joint inflammation developing after certain bacterial infections e.g. Salmonella, Yersinia, and Chlamydia. Several unrelated observations indicate that HLA-B27 modulates the interaction between ReA-triggering bacteria and host cell. These findings suggest that HLA-B27 may possess functions, which are unrelated to antigen presentation. In this paper, we summarize these findings and discuss their potential impact in the development of SpA.     

Molecular typing of HLA-B27 alleles

HLA-B27 represents a family of closely related antigens. Six alleles which differ in a limited number of nucleotide substitutions have been described (B*2701—B*2706). These changes are clustered in 1 and 2 domains. Polymerase chain reaction strategies were designed to amplify specific regions of class I exons 2 and 3. Amplified sequences were tested with eight sequence-specific oligonucleotides to distinguish all B27 subtypes. We also subtyped B27 in 50 healthy Spanish individuals using this procedure. The B*2705 subtype is over-represented in our population (96%). The remaining 4% carried the B*2702 allele. This finding is in agreement with the frequencies described by other techniques (cytotoxic T lymphocytes and isoeletric-focusing) for Caucasian populations. Class I oligotyping is a poorly developed field with significant potential applications. This procedure of genotyping B27 alleles is a reliable method which can be used in transplantation and B27-associated disease studies.