Discovery of protein-protein binding disruptors using multi-component condensations small molecules

A series of small molecule compounds interfering with the binding process of VEGF and NRP1 has been identified and further optimized. Full synthetic details as well as SAR are reported which demonstrate that expeditious MCC-based syntheses may lead to valuable molecules addressing challenging targets such as protein-protein interactions. Preliminary functional assay data confirm that these compounds may be further developed toward drug candidates.     

Studying synthetic lethal interactions in the zebrafish system: insight into disease genes and mechanisms

The post-genomic era is marked by a pressing need to functionally characterize genes through understanding gene-gene interactions, as well as interactions between biological pathways. Exploiting a phenomenon known as synthetic lethality, in which simultaneous loss of two interacting genes leads to loss of viability, aids in the investigation of these interactions. Although synthetic lethal screening is a powerful technique that has been used with great success in many model organisms, including Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans, this approach has not yet been applied in the zebrafish, Danio rerio. Recently, the zebrafish has emerged as a valuable system to model many human disease conditions; thus, the ability to conduct synthetic lethal screening using zebrafish should help to uncover many unknown disease-gene interactions. In this article, we discuss the concept of synthetic lethality and provide examples of its use in other model systems. We further discuss experimental approaches by which the concept of synthetic lethality can be applied to the zebrafish to understand the functions of specific genes.     

dSLAM analysis of genome-wide genetic interactions in Saccharomyces cerevisiae

Analysis of genetic interactions has been extensively exploited to study gene functions and to dissect pathway structures. One such genetic interaction is synthetic lethality, in which the combination of two non-lethal mutations leads to loss of organism viability. We have developed a dSLAM (heterozygote diploid-based synthetic lethality analysis with microarrays) technology that effectively studies synthetic lethality interactions on a genome-wide scale in the budding yeast Saccharomyces cerevisiae. Typically, a query mutation is introduced en masse into a population of approximately 6000 haploid-convertible heterozygote diploid Yeast Knockout (YKO) mutants via integrative transformation. Haploid pools of single and double mutants are freshly generated from the resultant heterozygote diploid double mutant pool after meiosis and haploid selection and studied for potential growth defects of each double mutant combination by microarray analysis of the "molecular barcodes" representing each YKO. This technology has been effectively adapted to study other types of genome-wide genetic interactions including gene-compound synthetic lethality, secondary mutation suppression, dosage-dependent synthetic lethality and suppression.     

Using synthetic peptides and recombinant collagen to understand DDR–collagen interactions

The discoidin domain receptors, DDR1 and DDR2, are a subfamily of receptor tyrosine kinases that are activated upon binding to collagen. DDR–collagen interactions play an important role in cell proliferation and migration. Over the past few decades, synthetic peptides and recombinant collagen have been developed as tools to study the biophysical characteristics of collagen and various protein–collagen interactions. Herein we review how these techniques have been used to understand DDR–collagen interactions. Using synthetic collagen-like peptides, the GVM-GFO motif has been found to be the major binding site on collagens II and III for DDR1 and DDR2. An X-ray co-crystal structure of the DDR2 DS domain bound to a synthetic collagen-like peptide containing the GVM-GFO motif further provides molecular details of the DDR–collagen interactions. Recombinant collagen has also been used to provide further validation of the GVM-GFO binding motif. Although GVM-GFO has been defined as the minimal binding site, in synthetic peptide studies at least two triplets N-terminal to the essential GVM-GFO binding motif in collagen III sequence are needed for DDR2 activation at high peptide concentrations.     

Focus: A Multifaceted Battle Against Cancer: Approaches to Identifying Synthetic Lethal Interactions in Cancer

Targeting synthetic lethal interactions is a promising new therapeutic approach to exploit specific changes that occur within cancer cells. Multiple approaches to investigate these interactions have been developed and successfully implemented, including chemical, siRNA, shRNA, and CRISPR library screens. Genome-wide computational approaches, such as DAISY, also have been successful in predicting synthetic lethal interactions from both cancer cell lines and patient samples. Each approach has its advantages and disadvantages that need to be considered depending on the cancer type and its molecular alterations. This review discusses these approaches and examines case studies that highlight their use.     

The effects of network neighbours on protein evolution

Interacting proteins may often experience similar selection pressures. Thus, we may expect that neighbouring proteins in biological interaction networks evolve at similar rates. This has been previously shown for protein-protein interaction networks. Similarly, we find correlated rates of evolution of neighbours in networks based on co-expression, metabolism, and synthetic lethal genetic interactions. While the correlations are statistically significant, their magnitude is small, with network effects explaining only between 2% and 7% of the variation. The strongest known predictor of the rate of protein evolution remains expression level. We confirmed the previous observation that similar expression levels of neighbours indeed explain their similar evolution rates in protein-protein networks, and showed that the same is true for metabolic networks. In co-expression and synthetic lethal genetic interaction networks, however, neighbouring genes still show somewhat similar evolutionary rates even after simultaneously controlling for expression level, gene essentiality and gene length. Thus, similar expression levels and related functions (as inferred from co-expression and synthetic lethal interactions) seem to explain correlated evolutionary rates of network neighbours across all currently available types of biological networks.     

Identification of synthetic lethality based on a functional network by using machine learning algorithms

Synthetic lethality is the synthesis of mutations leading to cell death. Tumor-specific synthetic lethality has been targeted in research to improve cancer therapy. With the advances of techniques in molecular biology, such as RNAi and CRISPR/Cas9 gene editing, efforts have been made to systematically identify synthetic lethal interactions, especially for frequently mutated genes in cancers. However, elucidating the mechanism of synthetic lethality remains a challenge because of the complexity of its influencing conditions. In this study, we proposed a new computational method to identify critical functional features that can accurately predict synthetic lethal interactions. This method incorporates several machine learning algorithms and encodes protein-coding genes by an enrichment system derived from gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways to represent their functional features. We built a random forest-based prediction engine by using 2120 selected features and obtained a Matthews correlation coefficient of 0.532. We examined the top 15 features and found that most of them have potential roles in synthetic lethality according to previous studies. These results demonstrate the ability of our proposed method to predict synthetic lethal interactions and provide a basis for further characterization of these particular genetic combinations.     

Solid Phase Synthesis and Application of Labeled Peptide Derivatives: Probes of Receptor-Opioid Peptide Interactions

Solid phase synthetic methodology has been developed in our laboratory to incorporate an affinity label (a reactive functionality such as isothiocyanate or bromoacetamide) into peptides (Leelasvatanakij and Aldrich J Peptide Res 56, 80, 2000), and we have used this synthetic strategy to prepare affinity label derivatives of a variety of opioid peptides. To date side reactions have been detected only in two cases, both involving intramolecular cyclization. We have identified several peptide-based affinity labels for δ opioid receptors that exhibit wash-resistant inhibition of binding to these receptors and are valuable pharmacological tools to study opioid receptors. Even in cases where the peptide derivatives do not bind covalently to their target receptor, studying their binding has revealed subtle differences in receptor interactions with particular opioid peptide residues, especially Phe residues in the N-terminal “message” sequences. Solid phase synthetic methodology for the incorporation of other labels (e.g. biotin) into the C-terminus of peptides has also been developed in our laboratory (Kumar and Aldrich Org Lett 5, 613, 2003). These two synthetic approaches have been combined to prepare peptides containing multiple labels that can be used as tools to study peptide ligand-receptor interactions. These solid phase synthetic methodologies are versatile strategies that are applicable to the preparation of labeled peptides for a variety of targets in addition to opioid receptors.     

Mapping genetically compensatory pathways from synthetic lethal interactions in yeast

Background

Synthetic lethal genetic interaction analysis has been successfully applied to predicting the functions of genes and their pathway identities. In the context of synthetic lethal interaction data alone, the global similarity of synthetic lethal interaction patterns between two genes is used to predict gene function. With physical interaction data, such as protein-protein interactions, the enrichment of physical interactions within subsets of genes and the enrichment of synthetic lethal interactions between those subsets of genes are used as an indication of compensatory pathways.

Result

In this paper, we propose a method of mapping genetically compensatory pathways from synthetic lethal interactions. Our method is designed to discover pairs of gene-sets in which synthetic lethal interactions are depleted among the genes in an individual set and where such gene-set pairs are connected by many synthetic lethal interactions. By its nature, our method could select compensatory pathway pairs that buffer the deleterious effect of the failure of either one, without the need of physical interaction data. By focusing on compensatory pathway pairs where genes in each individual pathway have a highly homogenous cellular function, we show that many cellular functions have genetically compensatory properties.

Conclusion

We conclude that synthetic lethal interaction data are a powerful source to map genetically compensatory pathways, especially in systems lacking physical interaction information, and that the cellular function network contains abundant compensatory properties.     

Measurement of the affinities of heparins, naturally occurring glycosaminoglycans, and other sulfated polymers for antithrombin III and thrombin

Heparin, other glycosaminoglycans, and synthetic sulfated polymers have antithrombotic and anticoagulant activities, which may be mediated through a range of interactions with different proteins. A simple, quantitative method has been developed for assessing the affinity of interaction between sulfated polymers and proteins in the liquid phase. This has been used to compare the binding of a range of glycosaminoglycans and other sulfated polymers to antithrombin III and thrombin, a major inhibitor of and a central protease in the coagulation system, respectively. The results are consistent with the binding of naturally occurring glycosaminoglycans to antithrombin III solely through the well-defined antithrombin III-binding pentasaccharide found in heparin, the apparent affinity of a preparation depending upon its content of this pentasaccharide. Highly sulfated synthetic polymers will, however, bind antithrombin III by a second mechanism. The affinity of heparin for thrombin decreased with decreasing molecular weight. However, results obtained with heparan sulfate preparations did not indicate any clear relationship between either molecular weight or sulfate content and thrombin binding, but suggested that there may be an oligosaccharide sequence containing N-sulfate residues which confers high affinity for thrombin. In addition, some of the synthetic sulfated polymers bound thrombin with very high affinity.     

Synthetic lethality of cohesins with PARPs and replication fork mediators

Synthetic lethality has been proposed as a way to leverage the genetic differences found in tumor cells to affect their selective killing. Cohesins, which tether sister chromatids together until anaphase onset, are mutated in a variety of tumor types. The elucidation of synthetic lethal interactions with cohesin mutants therefore identifies potential therapeutic targets. We used a cross-species approach to identify robust negative genetic interactions with cohesin mutants. Utilizing essential and non-essential mutant synthetic genetic arrays in Saccharomyces cerevisiae, we screened genome-wide for genetic interactions with hypomorphic mutations in cohesin genes. A somatic cell proliferation assay in Caenorhabditis elegans demonstrated that the majority of interactions were conserved. Analysis of the interactions found that cohesin mutants require the function of genes that mediate replication fork progression. Conservation of these interactions between replication fork mediators and cohesin in both yeast and C. elegans prompted us to test whether other replication fork mediators not found in the yeast were required for viability in cohesin mutants. PARP1 has roles in the DNA damage response but also in the restart of stalled replication forks. We found that a hypomorphic allele of the C. elegans SMC1 orthologue, him-1(e879), genetically interacted with mutations in the orthologues of PAR metabolism genes resulting in a reduced brood size and somatic cell defects. We then demonstrated that this interaction is conserved in human cells by showing that PARP inhibitors reduce the viability of cultured human cells depleted for cohesin components. This work demonstrates that large-scale genetic interaction screening in yeast can identify clinically relevant genetic interactions and suggests that PARP inhibitors, which are currently undergoing clinical trials as a treatment of homologous recombination-deficient cancers, may be effective in treating cancers that harbor cohesin mutations.     

Identification of a key domain in annexin and 14-3-3 proteins that stimulate calcium-dependent exocytosis in permeabilized adrenal chromaffin cells

Calcium-dependent secretion in digitonin-permeabilized adrenal chromaffin cells is stimulated by exogenous annexin II and 14-3-3 proteins. These proteins share a conserved domain that has been suggested to be involved in specific protein-protein interactions. We examined whether this domain was involved in secretion by using a synthetic peptide (P16) of sequence KGDYQKALLYLCGGDD corresponding to the C-terminus of annexin II. P16, but not truncated peptides, prevented the stimulation of secretion by 14-3-3 proteins and produced a partial inhibition of control secretion. These data suggest that the shared annexin/14-3-3 domain is important in the mechanisms controlling Ca²⁺-dependent secretion and may play a key role in protein-protein interactions during exocytosis.     

Immobilized glycoconjugates for cell recognition studies

Specific cell-cell recognition and adhesion may involve cell surface glycoconjugates on one cell binding the complementary carbohydrate receptors on an apposing cell surface. Such interactions have been modeled by immobilizing simple synthetic glycosides, glycoproteins, glycosaminoglycans, and glycolipids on otherwise inert plastic surfaces and incubating them with intact cells. Using this approach, the ability of several cell types to recognize specific carbohydrates has been demonstrated. This carbohydrate-directed cell adhesion may depend on cell surface carbohydrate receptors which mediate both the initial specific adhesion and complex postrecognition cellular responses. While the relationship of the cell adhesion demonstrated here to cell-cell recognition in vivo has yet to be determined, this well-controlled biochemical approach may reveal new information on the way in which cells analyze and respond to their immediate external environment.     

A high-throughput assay for assessing the cell permeability of combinatorial libraries

There is great interest in the identification of synthetic molecules that are capable of manipulating protein-protein interactions in living cells. Peptides, unlike other classes of small molecules, have binding properties appropriate for this application, but most are poorly cell permeable and sensitive to proteases. Therefore, considerable effort has been expended in the development of libraries of oligomeric peptide-like molecules. However, there are no clear-cut rules to guide the design of libraries rich in cell permeable compounds. Furthermore, currently available empirical methods to assess permeability may not accurately reflect true permeability and/or are capable of only modest throughput. We describe here an assay for assessing the relative cell permeability of synthetic molecules in the context of steroid fusions that is capable of high throughput and can be used in any transfectable cell line.     

Structural and dynamic studies of the gamma-M4 trans-membrane domain of the nicotinic acetylcholine receptor

A structural characterization of a synthetic peptide corresponding to the fourth transmembrane domain (M4-TMD) of the gamma-subunit of the nicotinic acetylcholine receptor from Torpedo californica has been undertaken. Solid-state NMR and CD spectroscopy studies indicate that upon reconstitution into lipid vesicles or magnetically aligned lipid bilayers, the synthetic M4-TMD adopts a linear alpha-helical conformation with the helix aligned within 15 degrees of the membrane normal. Furthermore, analysis of the motional averaging of anisotropic interactions present in the solid-state NMR spectra of the reconstituted peptide, indicate that the dynamics of the peptide within the bilayer are highly sensitive to the phase adopted by the lipid bilayer, providing an insight into how the interaction of lipids with this domain may play a important role in the modulation of this receptor by its lipid environment.     

Charge transfer chromatography used to study the interaction between synthetic phospholipids and nonylphenyl-nonylglycolate

The interaction between 8 synthetic phospholipids and the nonionic tenzide nonylphenyl-nonylglycolate was studied by charge transfer chromatography. The method has been improved by carrying out the determination at different organic phase concentrations of eluent and by extrapolating the interactive strength back to pure water. It was suggested that two types of interaction of commensurable strength may exist between the tenzide and the phospholipids; hydrophil-hydrophil interactions between the polar head groups of phospholipids and the hydrophilic ethylene-oxide chain of tenzide; hydrophob-hydrophob interactions between the fatty acid chains of phospholipids and the alkyl chain of tenzide. This last effect strongly depends on the length of the fatty acid chain, but it is independent of the presence of double bonds in the lipophilic region of phospholipid.     

Designing synthetic materials to control stem cell phenotype

The micro-environment in which stem cells reside regulates their fate, and synthetic materials have recently been designed to emulate these regulatory processes for various medical applications. Ligands inspired by the natural extracellular matrix, cell-cell contacts, and growth factors have been incorporated into synthetic materials with precisely engineered density and presentation. Furthermore, material architecture and mechanical properties are material design parameters that provide a context for receptor-ligand interactions and thereby contribute to fate determination of uncommitted stem cells. Although significant progress has been made in biomaterials development for cellular control, the design of more sophisticated and robust synthetic materials can address future challenges in achieving spatiotemporal control of cellular phenotype and in implementing histocompatible clinical therapies.     

Spatial organization of enzymes for metabolic engineering

As synthetic pathways built from exogenous enzymes become more complicated, the probability of encountering undesired interactions with host organisms increases, thereby lowering product titer. An emerging strategy to combat this problem is to spatially organize pathway enzymes into multi-protein complexes, where high local concentrations of enzymes and metabolites may enhance flux and limit problematic interactions with the cellular milieu. Co-localizing enzymes using synthetic scaffolds has improved titers for multiple pathways. While lacking physical diffusion barriers, scaffolded systems could concentrate intermediates locally through a mechanism analogous to naturally occurring microdomains. A more direct strategy for compartmentalizing pathway components would be to encapsulate them within protein shells. Several classes of shells have been loaded with exogenous proteins and expressed successfully in industrial hosts. A critical challenge for achieving ideal pathway compartmentalization with protein shells will likely be evolving pores to selectively limit intermediate diffusion. Eventually, these tools should enhance our ability to rationally design metabolic pathways.     

Structure and modeling studies of the carboxy-terminus region of human tropoelastin.

Elastin macromolecular assembly is a highly complex mechanism involving many steps including coacervation, cross-linking, and probably other (not known) phenomena. In past studies, it has been proposed that the C-terminal part of tropoelastin is also involved in this process and may play a key role in tropoelastin interactions with other proteins of the final elastic fibres scaffold. Presented here are the results of the biophysical studies (biospectroscopy, bioinformatics) of the C-terminal domain of tropoelastin. We report the detailed structures adopted by the oxidized (native) and reduced forms of the free synthetic peptide with sequence encoded by exon 36 of human tropoelastin (GGACLGKACGRKRK) and propose a dynamical interpretation of which structures may be involved in interactions with other extra-cellular matrix proteins. We also suggest that these structures may be retrieved in other proteins sharing a consensus sequence; however no definitive conclusion can be drawn here on a possible structure-function relationship.     

Synthetic lethal interactions for the development of cancer therapeutics: biological and methodological advancements

Synthetic lethal interaction is defined as a combination of two mutations that is lethal when present in the same cell; each individual mutation is non-lethal. Synthetic lethal interactions attract attention in cancer research fields since the discovery of synthetic lethal genes with either oncogenes or tumor suppressor genes (TSGs) provides novel cancer therapeutic targets. Due to the selective lethal effect on cancer cells harboring specific genetic alterations, it is expected that targeting synthetic lethal genes would provide wider therapeutic windows compared with cytotoxic chemotherapeutics. Here, we review the current status of the application of synthetic lethal screening in cancer research fields from biological and methodological viewpoints. Very recent studies seeking to identify synthetic lethal genes with K-RAS and p53, which are known to be the most frequently occurring oncogenes and TSGs, respectively, are introduced. Among the accumulating amount of research on synthetic lethal interactions, the synthetic lethality between BRCA1/2 and PARP1 inhibition has been clinically proven. Thus, both preclinical and clinical data showing a preferential anti-tumor effect on BRCA1/2 deficient tumors by a PARP1 inhibitor are the best examples of the synthetic lethal approach of cancer therapeutics. Finally, methodological progress regarding synthetic lethal screening, including barcode shRNA screening and in vivo synthetic lethal screening, is described. Given the fact that an increasing number of synthetic lethal genes for major cancerous genes have been validated in preclinical studies, this intriguing approach awaits clinical verification of preferential benefits for cancer patients with specific genetic alterations as a clear predictive factor for tumor response.