蛋白质-蛋白质相互作用及其抑制剂研究进展

蛋白质-蛋白质相互作用在细胞活动和生命过程中扮演着非常重要的角色。基因调节、免疫应答、信号转导、细胞组装等等都离不开蛋白质-蛋白质的相互作用。近几年,靶向蛋白质-蛋白质相互作用及其抑制剂研究也逐渐成为研究的热点;但是蛋白质复合物相互作用界面的一些特点和性质,如相互作用界面较大、结合界面较为平坦等,使蛋白质-蛋白质相互作用及其抑制剂研究充满了挑战。本文主要总结了蛋白质-蛋白质相互作用界面的一些性质和特点,分析了界面特性与其抑制剂设计的关系,并讨论了蛋白质-蛋白质相互作用的理论预测方法及其抑制剂的类型和特点,最后又通过实例说明了如何进行蛋白质-蛋白质相互作用抑制剂的设计。     

beta-Peptides as inhibitors of protein-protein interactions

We became interested several years ago in exploring whether 14-helical beta-peptide foldamers could bind protein surfaces and inhibit protein-protein interactions, and if so, whether their affinities and specificities would compare favorably with those of natural or miniature proteins. This exploration was complicated initially by the absence of a suitable beta-peptide scaffold, one that possessed a well-defined 14-helical structure in water and tolerated the diverse sequence variation required to generate high-affinity protein surface ligands. In this perspective, we describe our approach to the design of adaptable beta-peptide scaffolds with high levels of 14-helix structure in water, track the subsequent development of 14-helical beta-peptide protein-protein interaction inhibitors, and examine the potential of this strategy for targeting other therapeutically important proteins.     

Structural flexibility of small GTPases. Can it explain their functional versatility?

Multiple interactions with many different partners are responsible for the amazing functional versatility of proteins, especially those participating in cellular regulation. The structural properties that could facilitate multiple interactions are examined for small GTPases. The role of cellular constraints, compartmentation and scaffolds on protein-protein interactions is considered.     

发现靶向蛋白质间相互作用的小分子药物研究进展

蛋白质-蛋白质相互作用在多种细胞功能中具有重要的作用。靶向蛋白质-蛋白质相互作用已经成为新药发现的重要策略,但发现能阻断蛋白质-蛋白质相互作用的小分子药物是一个巨大的挑战。尽管如此,近年来人们还是发现了许多能调控蛋白质-蛋白质相互作用的小分子。该文主要总结了在病毒进入、细胞凋亡通路和神经退行性疾病等方面的蛋白质-蛋白质相互作用小分子抑制剂的研究进展。     

Structure- and Ligand-Based Virtual Screening Identifies New Scaffolds for Inhibitors of the Oncoprotein MDM2

A major challenge in the field of ligand discovery is to identify chemically useful fragments that can be developed into inhibitors of specific protein-protein interactions. Low molecular weight fragments (with molecular weight less than 250 Da) are likely to bind weakly to a protein’s surface. Here we use a new virtual screening procedure which uses a combination of similarity searching and docking to identify chemically tractable scaffolds that bind to the p53-interaction site of MDM2. The binding has been verified using capillary electrophoresis which has proven to be an excellent screening method for such small, weakly binding ligands.     

Signaling protein inhibitors via the combinatorial modification of peptide scaffolds

Compounds that selectively interfere with protein-protein interactions are not only invaluable as biological reagents, but may ultimately serve as therapeutically useful drugs for the treatment of a wide variety of disease states. However, unlike active site directed inhibitors that bind to a relatively small, well-defined, hydrophobic pocket, reagents that disrupt protein-protein interactions must contend with a protein surface that is comparatively large, ill defined, and solvent exposed. We have developed a straightforward method for the acquisition of protein-protein interaction inhibitors. The library-based strategy starts with low affinity consensus sequence peptides, which are then transformed in a stepwise fashion into high affinity inhibitors. The approach has been used to create potent ligands for SH2 and SH3 domains, as well as powerful and highly selective inhibitors for protein kinases and phosphatases. The protocol is easily automated and therefore has the potential to be routinely applied, in a high throughput fashion.     

Peptides that inhibit HIV-1 integrase by blocking its protein-protein interactions

HIV-1 integrase (IN) is one of the key enzymes in the viral replication cycle. It mediates the integration of viral cDNA into the host cell genome. IN activity requires interactions with several viral and cellular proteins, as well as IN oligomerization. Inhibition of IN is an important target for the development of anti-HIV therapies, but there is currently only one anti-HIV drug used in the clinic that targets IN. Several other small-molecule anti-IN drug leads are either undergoing clinical trials or in earlier stages of development. These molecules specifically inhibit one of the IN-mediated reactions necessary for successful integration. However, small-molecule inhibitors of protein-protein interactions are difficult to develop. In this review, we focus on peptides that inhibit IN. Peptides have advantages over small-molecule inhibitors of protein-protein interactions: they can mimic the structures of the binding domains within proteins, and are large enough to competitively inhibit protein-protein interactions. The development of peptides that bind IN and inhibit its protein-protein interactions will increase our understanding of the IN mode of action, and lead to the development of new drug leads, such as small molecules derived from these peptides, for better anti-HIV therapy.     

Solid-phase synthesis of short α-helices stabilized by the hydrogen bond surrogate approach

Stabilized α-helices and nonpeptidic helix mimetics have emerged as powerful molecular scaffolds for the discovery of protein-protein interaction inhibitors. Protein-protein interactions often involve large contact areas, which are often difficult for small molecules to target with high specificity. The hypothesis behind the design of stabilized helices and helix mimetics is that these medium-sized molecules may pursue their targets with higher specificity because of a larger number of contacts. This protocol describes an optimized synthetic strategy for the preparation of stabilized α-helices that feature a carbon-carbon linkage in place of the characteristic N-terminal main-chain hydrogen bond of canonical helices. Formation of the carbon-carbon bond is enabled by a microwave-assisted ring-closing metathesis reaction between two terminal olefins on the peptide chain. The outlined strategy allows the synthesis and purification of a hydrogen bond surrogate (HBS) α-helix in ～ 1 week.     

DARPins and other repeat protein scaffolds: advances in engineering and applications

Antibodies have long been regarded as the only class of binding proteins. With the emergence of protein engineering techniques, new binding proteins based on alternative scaffolds have been designed. Additionally, modern technologies for selection and evolution from libraries are independent of the antibody scaffold and could thus be readily used for obtaining specific binding proteins. One important group of alternative scaffolds is based on repeat proteins. Nature is widely using these proteins to modulate protein-protein interactions, and even in the adaptive immune system of jawless vertebrates; the step to their application as an alternative to antibodies seems therefore logical. In this review, progress on DARPins and other repeat protein scaffolds will be discussed. Advances in their design as well as novel applications will be highlighted.     

Biphenyls as potential mimetics of protein alpha-helix

Based on theoretical arguments, 2,6,3',5'-substituted biphenyl analogues are proposed as protein alpha-helix mimetics superimposing the side chains of the residues i, i+1, i+3 and i+4. Knowing that many protein-protein interactions of therapeutical relevance involve alpha-helix contacts, the communication outlines how this novel category of scaffolds might potentially open access to such targets.     

TPRpred: a tool for prediction of TPR-, PPR- and SEL1-like repeats from protein sequences

Background  

Solenoid repeat proteins of the Tetratrico Peptide Repeat (TPR) family are involved as scaffolds in a broad range of protein-protein interactions. Several resources are available for the prediction of TPRs, however, they often fail to detect divergent repeat units.     

Protein-protein interactions in human disease

Many human diseases are the result of abnormal protein-protein interactions involving endogenous proteins, proteins from pathogens or both. The inhibition of these aberrant associations is of obvious clinical significance. Because of the diverse nature of protein-protein interactions, however, the successful design of therapeutics requires detailed knowledge of each system at a molecular and atomic level. Several recent studies have identified and/or characterised specific interactions from various disease systems, including cervical cancer, bacterial infection, leukaemia and neurodegenerative disease. A range of approaches are being developed to generate inhibitors of protein-protein interactions that may form useful therapeutics for human disease.     

X-ray and molecular modelling in fragment-based design of three small quinoline scaffolds for HIV integrase inhibitors

Crystal structures of three small molecular scaffolds based on quinoline, 2-methylquinoline-5,8-dione, 5-hydroxy-quinaldine-6-carboxylic acid and 8-hydroxy-quinaldine-7-carboxylic acid, were characterised. 5-Hydroxy-quinaldine-6-carboxylic acid was co-crystallized with cobalt(II) chloride to form a model of divalent metal cation-ligand interactions for potential HIV integrase inhibitors. Molecular docking into active site of HIV IN was also performed on 1WKN PDB file. Selected ligand-protein interactions have been found specific for active compounds. Studied structures can be used as scaffolds in fragment-based design of new potent drugs.     

Strategies to search and design stabilizers of protein-protein interactions: a feasibility study

Since protein-protein interactions play a pivotal role in the communication on the molecular level in virtually every biological system and process, the search and design for modulators of such interactions is of utmost importance. In recent years many inhibitors for specific protein-protein interactions have been developed, however, in only a few cases, small and druglike molecules are able to interfere in the complex formation of proteins. On the other hand, there are several small molecules known to modulate protein-protein interactions by means of stabilizing an already assembled complex. To achieve this goal, a ligand is binding to a pocket, which is located rim-exposed at the interface of the interacting proteins, for example as the phytotoxin Fusicoccin, which stabilizes the interaction of plant H+-ATPase and 14-3-3 protein by nearly a factor of 100. To suggest alternative leads, we performed a virtual screening campaign to discover new molecules putatively stabilizing this complex. Furthermore, we screen a dataset of 198 transient recognition protein-protein complexes for cavities, which are located rim-exposed at their interfaces. We provide evidence for high similarity between such rim-exposed cavities and usual ligands accommodating active sites of enzymes. This analysis suggests that rim-exposed cavities at protein-protein interfaces are druggable binding sites. Therefore, the principle of stabilizing protein-protein interactions seems to be a promising alternative to the approach of the competitive inhibition of such interactions by small molecules.     

Protein-protein interactions and cancer: small molecules going in for the kill

There has been much progress in the discovery of small, organic molecules that inhibit protein-protein interactions, particularly in the field of cancer. Tubulin polymerization represents a classic target whose function can be allosterically modulated by small molecules. Several protein-protein complexes that regulate apoptosis, or programmed cell death, appear to be particularly amenable to inhibition by small molecules, and recently described compounds have helped to characterize Bcl-2, MDM2 and XIAP as drug targets. Additionally, small-molecule antagonists have recently been described for several new targets, including Rac1-Tiam1, beta-catenin-T cell factor (Tcf), and Sur-2-ESX. Not only is the list of protein-protein inhibitors growing, but the inhibitors themselves are moving closer to treating disease.     

Bromodomains：表观遗传医学新靶点及其抑制剂

Bromodomains（BRDs）是一类能够特异性识别乙酰化赖氨酸残基的保守蛋白结构域,存在于染色质及与转录相关的蛋白 中,其功能包括染色质重塑和转录调控,并在细胞内由乙酰化介导的蛋白-蛋白相互作用中发挥极为重要的作用,是多种疾病（包括癌 症、炎症和自身免疫病）的表观遗传医学靶点。介绍BRDs 的生物学功能、结构及分类,主要从BET bromodomain 抑制剂和非BET bromodomain 抑制剂两个方面对BRDs 抑制剂的研究进展作一综述,为高活性和选择性的BRDs 抑制剂研发提供参考。     

AutoMatch: target-binding protein design and enzyme design by automatic pinpointing potential active sites in available protein scaffolds

Proteins perform their functions mainly via active sites, whereas other parts of the proteins comprise the scaffolds, which support the active sites. One strategy for protein functional design is transplanting active sites, such as catalytic sites for enzyme or binding hot spots for protein-protein interactions, onto a new scaffold. AutoMatch is a new program designed for efficiently elucidating suitable scaffolds and potential sites on the scaffolds. Backrub motions are used to treat backbone flexibility during the design process. A step-by-step checking strategy and cluster-representation examination strategy were developed to solve the large combinatorial problem for the matching of active-site conformations. In addition, a grid-based binding energy scoring method was used to filter the solutions. An enzyme design benchmark and a protein-protein interaction design benchmark were built to test the algorithm. AutoMatch could identify the hot spots in the nonbinding protein and rank them within the top five results for 8 of 10 target-binding protein design cases. In addition, among the 15 enzymes tested, AutoMatch can identify the catalytic active sites in the apoprotein and rank them within the top five results for 13 cases. AutoMatch was also tested for screening scaffold library in designing binding proteins targeting influenza hemagglutinin, HIV gp120, and epidermal growth factor receptor kinase, respectively. AutoMatch, and the two test sets, ActApo and ActFree, are available for noncommercial applications at http://mdl.ipc.pku.edu.cn/cgi-bin/down.cgi.     

Discovery of selective small-molecule CD80 inhibitors

Protein-protein interactions are widely found in biological systems controlling diverse cellular events. Because these interactions are implicated in many diseases such as autoimmunity and cancer, regulation of protein-protein interactions provides ideal targets for drug intervention. The CD80-CD28 costimulatory pathway plays a critical role in regulation of the immune response and thus constitutes an attractive target for therapeutic manipulation of autoimmune diseases. The objective of this study is to identify small compounds disrupting these pivotal protein-protein interactions. Compounds that specifically blocked binding of CD80 to CD28 were identified using a strategy involving a cell-based scintillation proximity assay as the initial step. Secondary screening (e.g., by analyzing the direct binding of these compounds to the target immobilized on a biosensor surface) revealed that these compounds are highly selective CD80 binders. Screening of structurally related derivatives led to the identification of the chemical features required for inhibition of the CD80-CD28 interaction. In addition, the optimization process led to a 10-fold increase in binding affinity of the CD80 inhibitors. Using this approach, the authors identify low-molecular-weight compounds that specifically and with high potency inhibit the interaction between CD80 and CD28. These compounds serve as promising starting points for further development of CD80 inhibitors as potential immunomodulatory drugs.