Protein-protein interactions as targets for small molecule drug discovery

Protein-protein interactions represent a highly populated class of targets for drug discovery. However, such systems present a number of unique challenges. This review presents an analysis of individual protein-protein interaction systems which have recently yielded success in discovering drug-like inhibitors. The structural characteristics of the protein binding sites and the attributes of the small molecule ligands are focused upon, in an attempt to derive commonly shared principles that may be of general usefulness in future drug discovery efforts within this target class.     

Ultra trace analysis of small molecule by label-free impedimetric immunosensor using multilayer modified electrode

A multilayer electrode modified with a self-assembled thiourea monolayer (SATUM) followed by gold nanoparticles (AuNPs), mercaptosuccinic acid (MSA) and antibody was investigated for the detection of ultra trace amount of a small molecule (chloramphenicol) in an impedimetric system. The formation of the antibody-antigen complex at the electrode surface caused the impedance to increase. Under optimum conditions three modified electrodes were compared the SATUM/AuNPs/MSA electrode provided a wide linear range (0.50-10) × 10?1? M, and a very low determination limit of 1.0 × 10?1? M. This determination limit was much lower than the SATUM/AuNPs electrode, 1.0 × 10?1? M, and SATUM electrode, 4.7 × 10?1? M. The modified electrode provided good selectivity for chloramphenicol detection and can be reused up to 45 times with a relative standard deviation of lower than 4%. When applied to determine chloramphenicol in shrimp samples, the results agreed well with those obtained by the high-performance liquid chromatography coupled with a photo diode array detector (P > 0.05). The developed system can be applied to detect other small molecules using appropriate affinity binding pairs.     

A small molecule microarray platform to select RNA internal loop-ligand interactions.

Herein, we report the development of a microarray platform to select RNA motif-ligand interactions that allows simultaneous screening of both RNA and chemical space. We used this platform to identify the RNA internal loops that bind 6'- N-5-hexynoate kanamycin A ( 1). Selected internal loops that bind 1 were studied in detail and commonly display an adenine across from a cytosine independent of the size of the loop. Additional preferences are also observed. For 3 x 3 nucleotide loops, there is a preference for purines, and for 2 x 2 nucleotide loops there is a preference for pyrimidines neighbored by an adenine across from a cytosine. This technique has several advantageous features for selecting RNA motif-ligand interactions: (1) higher affinity RNA motif-ligand interactions are identified by harvesting bound RNAs from lower ligand loadings; (2) bound RNAs are harvested from the array via gel extraction, mitigating kinetic biases in selections; and (3) multiple selections are completed on a single array surface. To further demonstrate that multiple selections can be completed in parallel on the same array surface, we selected the RNA internal loops from a 4096-member RNA internal loop library that bound a four-member aminoglycoside library. These experiments probed 16,384 (4 aminoglycoside x 4096-member RNA library) interactions in a single experiment. These studies allow for parallel screening of both chemical and RNA space to improve our understanding of RNA-ligand interactions. This information may facilitate the rational and modular design of small molecules targeting RNA.     

A novel evolution-based method for detecting gene-gene interactions

Background

The rapid advance in large-scale SNP-chip technologies offers us great opportunities in elucidating the genetic basis of complex diseases. Methods for large-scale interactions analysis have been under development from several sources. Due to several difficult issues (e.g., sparseness of data in high dimensions and low replication or validation rate), development of fast, powerful and robust methods for detecting various forms of gene-gene interactions continues to be a challenging task.

Methodology/Principal Findings

In this article, we have developed an evolution-based method to search for genome-wide epistasis in a case-control design. From an evolutionary perspective, we view that human diseases originate from ancient mutations and consider that the underlying genetic variants play a role in differentiating human population into the healthy and the diseased. Based on this concept, traditional evolutionary measure, fixation index (Fst) for two unlinked loci, which measures the genetic distance between populations, should be able to reveal the responsible genetic interplays for disease traits. To validate our proposal, we first investigated the theoretical distribution of Fst by using extensive simulations. Then, we explored its power for detecting gene-gene interactions via SNP markers, and compared it with the conventional Pearson Chi-square test, mutual information based test and linkage disequilibrium based test under several disease models. The proposed evolution-based method outperformed these compared methods in dominant and additive models, no matter what the disease allele frequencies were. However, its performance was relatively poor in a recessive model. Finally, we applied the proposed evolution-based method to analysis of a published dataset. Our results showed that the P value of the Fst -based statistic is smaller than those obtained by the LD-based statistic or Poisson regression models.

Conclusions/Significance

With rapidly growing large-scale genetic association studies, the proposed evolution-based method can be a promising tool in the identification of epistatic effects.     

A model-based approach for detecting coevolving positions in a molecule

We present a new method for detecting coevolving sites in molecules. The method relies on a set of aligned sequences (nucleic acid or protein) and uses Markov models of evolution to map the substitutions that occurred at each site onto the branches of the underlying phylogenetic tree. This mapping takes into account the uncertainty over ancestral states and among-site rate variation. We then build, for each site, a "substitution vector" containing the posterior estimates of the number of substitutions in each branch. The amount of coevolution for a pair of sites is then measured as the Pearson correlation coefficient between the two corresponding substitution vectors and compared to the expectation under the null hypothesis of independence. We applied the method to a 79-species bacterial ribosomal RNA data set, for which extensive structural characterization has been done over the last 30 years. More than 95% of the intramolecular predicted pairs of sites correspond to known interacting site pairs.     

Polyamines: Naturally occurring small molecule modulators of electrostatic protein-protein interactions

Modulations of protein-protein interactions are a key step in regulating protein function, especially in networks. Modulators of these interactions are supposed to be candidates for the development of novel drugs. Here, we describe the role of the small, polycationic and highly abundant natural polyamines that could efficiently bind to charged spots at protein interfaces as modulators of such protein-protein interactions. Using the mitochondrial cytochrome P45011A1 (CYP11A1) electron transfer system as a model, we have analyzed the capability of putrescine, spermidine, and spermine at physiologically relevant concentrations to affect the protein-protein interactions between adrenodoxin reductase (AdR), adrenodoxin (Adx), and CYP11A1. The actions of polyamines on the individual components, on their association/dissociation, on electron transfer, and on substrate conversion were examined. These studies revealed modulating effects of polyamines on distinct interactions and on the entire system in a complex way. Modulation via changed protein-protein interactions appeared plausible from docking experiments that suggested favourable high-affinity binding sites of polyamines (spermine > spermidine > putrescine) at the AdR-Adx interface. Our findings imply for the first time that small endogenous compounds are capable of interfering with distinct components of transient protein complexes and might control protein functions by modulating electrostatic protein-protein interactions.     

Studies of small molecule interactions with protein phosphatases using biosensor technology

Reversible protein phosphorylation of serine, threonine, and tyrosine residues by protein kinases and phosphatases is important for the regulation of cellular signal transduction and controls many cellular functions. Disturbances in this regulation have been implicated in a growing number of diseases, making kinases and phosphatases useful targets for therapeutic intervention. The suitability of surface plasmon resonance (SPR) technology has been widely demonstrated in many drug discovery applications. A novel and straightforward methodology is presented for analyzing small molecule binding to two serine/threonine phosphatases, PP1 and PP2B (calcineurin), and to the prototypic tyrosine phosphatase, PTP1B. Emphasis was placed on investigating the immobilization conditions of the phosphatases by using reducing conditions, inhibitors and metal ions. A comparison of inhibitor binding, either to phosphatase (PP2B) alone or in complex with the regulatory protein subunit calmodulin, revealed different kinetics. The methodology was also used to test inhibitor specificity toward different phosphatases. Inhibition of regulatory protein PP-inhibitor-2 binding to PP1 by a small molecule inhibitor was demonstrated. This type of information, together with data on compound binding that is independent of enzyme activity and in which affinities are resolved into kinetic rate constants, may be of great significance for the development of highly specific and high-affinity phosphatase inhibitors.     

A label-free assay of exonuclease activity using a pyrosequencing technique

Enzymes with 3′-5′ exonuclease activities are important in promoting the accuracy of DNA replication and DNA repair by proofreading. The alteration of the function of these enzymes by endogenous or exogenous effectors could, therefore, have a considerable impact on DNA replication and ultimately on genome integrity. We have developed a label-free high-throughput screening method for quantifying the effects of different reagents on exonuclease activity. The assay is based on a hairpin-forming biotinylated oligonucleotide substrate that contains one or more exonuclease-resistant phosphorothioate nucleotides. The activity and specificity of the selected 3′-5′ exonuclease is determined indirectly using a sensitive pyrosequencing reaction after cleanup of the samples. In this pyrosequencing step, the amount of nucleotides filled into each position of the exonucleolytically degraded 3′ end of the substrate can be recorded quantitatively and equals the amount of the nucleotides removed by the exonuclease. This system allows the estimation of both processivity and efficiency of the exonuclease activity. We have employed compounds reported in the literature to inhibit the exonuclease activities of either exonuclease III or the large fragment of polymerase I (Klenow fragment) to evaluate the assay.     

A non-peptidyl neurotrophic small molecule for midbrain dopaminergic neurons

Abstract A small organic molecule (CUR-162590) that selectively enhances survival of midbrain dopaminergic neurons was identified by screening small molecule compound libraries. In embryonic midbrain cultures, CUR-162590 increased dopamine uptake and the number of dopaminergic neurons without altering the number of total neurons or astroglia or the uptake of GABA or serotonin. CUR-162590 reduced apoptosis of cultured dopaminergic neurons and protected against death induced by toxins such as MPP(+). Several synthetic analogs of CUR-162590 also had similar bioactivities. CUR-162590 thus represents a new class of neurotrophic small molecules that may have utility in the treatment of Parkinson's disease, which is marked by degeneration of midbrain dopaminergic neurons.     

A label-free optical sensor based on nanoporous gold arrays for the detection of oligodeoxynucleotides

Interest in using nanoporous materials for sensing applications has increased. The present study reports a method of preparing well-ordered nanoporous gold arrays using a porous silicon (PSi) template. Gold nanolayer could be electrodeposited on the surface of the PSi template at low electrolysis currents in low concentration of chloroauric acid (HAuCl₄) solution. Surface morphology characterizations and optical measurements revealed that a PSi-templated nanoporous gold (Au–PSi) array well replicated the nanoporous structure and retained the optical properties of PSi. Fourier transform reflectometric interference spectra showed that a characteristic blue-shifted effective optical thickness (EOT) was observed due to the low refractive index of the gold film. An optical DNA biosensor was then fabricated via the self-assembly of single-stranded DNA (ssDNA) with a specific sequence on the surface of Au–PSi. The attachment of ssDNA and its hybridization with target oligonucleotides (ODNs) persistently caused the blue shift of the EOT. Consequently, a relationship between the EOT shift and the ODN concentration was established. The mechanism of the optical response caused by DNA hybridization on the Au–PSi surface was qualitatively explained by the electromagnetic theory and electrochemical impedance spectroscopy (EIS). The lowest detection limit for target ODNs was estimated at around 10⁻¹⁴ mol L⁻¹, when the baseline noise, a variation in the value of EOT is around 5 nm. The fabricated Au–PSi based optical biosensor has potential use in the discovery of new ODN drugs because it will be able to detect the binding event between ODNs and the target DNA.     

Phylogenetic methodology for detecting protein interactions

Detecting protein-protein interactions and assigning proteins to functional complexes are key challenges of modern biology. The rise of genomics has lead to evidence that correlated patterns of presence/absence and/or fusing of proteins in any organism suggest these proteins interact. Unfortunately, methods based on such data work best with divergent genomes, whereas major sequencing efforts in vertebrates, for example, are yielding alignments of the same set of proteins sampled from the same set of taxa (species). Using vertebrate mitochondrial genomes to illustrate a novel method, we associate proteins based on vectors of their evolutionary tree edge (branch or internode) lengths. This approach is based on the expectation that molecular coevolution is greatest between proteins that interact in some way. Mitochondrial DNA-encoded proteins are associated into groups largely consistent with the complexes they come from. This association is apparently not due to the tree structure or mutation processes, leaving coevolution as the best explanation. We show that it is important that the tree used to derive the edge-length vector is estimated accurately in terms of both topology and edge lengths. Although more complex substitution models reduce systematic error, they also inflate stochastic error. This makes the use of less complex substitution models preferable in some circumstances. We describe a method to estimate correlations of pairwise evolutionary distances, which adjusts for non-independent correlations due to shared evolutionary history. Associations of proteins based on their edge-length vectors are visualized and assessed using a variety of hierarchical clustering and multidimensional scaling methods. New formula for estimating the fit of data to model, including the average percent standard deviation of distances on least squares trees, are presented. Use of edge-length vectors is compared and contrasted with correlated distance methods, correlated rates methods, and site-specific evidence of coevolution.     

A stereocomplex platform efficiently detecting antigen-antibody interactions

Ultrathin poly(methyl methacrylate) (PMMA) stereocomplex films with macromolecularly double-stranded regular nanostructures were prepared by layer-by-layer assembly of isotactic and syndiotactic PMMAs on solid surfaces. Antibodies were immobilized through the Fc region-capturing protein A, which had been physically adsorbed on the complex film, and the binding of antigens to immobilized antibodies was quantitatively investigated by the quartz crystal microbalance technique. Greater amounts of protein A with native forms were adsorbed on the complex film than those on conventional single-component PMMA films. Antibodies with high target-binding activities were also immobilized on the complex film. A greater amount of antigens could be detected on the complex film. The activity of protein A was maintained on the complex for a long time even within a dried state. The mechanism for the preservation of protein native forms on the complex surface was speculated by analyzing the physical adsorption of proteins with various secondary structures. Stereocomplex films can be utilized as novel coating nanomaterials for efficiently detecting protein-protein interactions.     

Kinetic studies of small molecule interactions with protein kinases using biosensor technology

Protein kinases are among the most commonly targeted groups of molecules in drug discovery today. Despite this, there are few examples of using surface plasmon resonance (SPR) for kinase inhibitor interaction studies, probably reflecting the need for better developed assays for these proteins. In this article, we present a general methodology that uses biosensor technology to study small molecule binding to eight different serine/threonine and tyrosine kinases. Mild immobilization conditions and a carefully composed assay buffer were identified as key success factors. The methodology package consists of direct binding studies of compounds to immobilized kinases, kinase activity assays to confirm inhibitory effects, detailed kinetic analyses of inhibitor binding, and competition assays with ATP for identification of competitive inhibitors. The kinetic assays resolve affinity into the rates of inhibitor binding and dissociation. Therefore, more detailed information on the relation between inhibitor structure and function is obtained. This might be of key importance for the development of effective kinase inhibitors.     

Towards a label-free optical porous silicon DNA sensor

We report on the fabrication of an optical silicon-based label-free DNA sensor. n-Type crystalline silicon wafers have been electrochemically etched to form porous silicon layers and characterized in terms of porosity, pore distribution, surface composition and photoluminescence. Samples (0.25 cm(2)) have been cut and properly derivatized using trimethoxy-3-bromoacetamidopropylsilane in order to link single strand DNA (ss-DNA). Such a molecule is not commercially available and has been ad-hoc prepared by reacting hydrobromic acid and 3-aminopropyltrimethoxysilane in presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as coupling agent. Trimethoxy-3-bromoacetamidopropylsilane acts as a bridge anchored to the porous silicon surface through the silane group while immobilizing ss-DNA by means of the bromoacetamido moiety. We have found that derivatized samples exhibit a photoluminescence that is stable in time and is not modified after exposure to non-complementary DNA strand. On the other hand, a sensible enhancement of the light emission has been observed when the derivatized samples react with the complementary strand, showing that the specific ss-DNA/complementary DNA (c-DNA) interaction can be optically sensed without using further labeling steps. This strongly strengthens the possible role of silicon as a material for biosensors.     

A stem cell-based tool for small molecule screening in adipogenesis

Techniques for small molecule screening are widely used in biological mechanism study and drug discovery. Here, we reported a novel adipocyte differentiation assay for small molecule selection, based on human mesenchymal stem cells (hMSCs) transduced with fluorescence reporter gene driven by adipogenic specific promoter--adipocyte Protein 2 (aP2; also namely Fatty Acid Binding Protein 4, FABP4). During normal adipogenic induction as well as adipogenic inhibition by Ly294002, we confirmed that the intensity of green fluorescence protein corresponded well to the expression level of aP2 gene. Furthermore, this variation of green fluorescence protein intensity can be read simply through fluorescence spectrophotometer. By testing another two small molecules in adipogenesis--Troglitazone and CHIR99021, we proved that this is a simple and sensitive method, which could be applied in adipocyte biology, drug discovery and toxicological study in the future.     

A colorimetric technique for detecting trichothecenes and assessing relative potencies

We tested a novel colorimetric toxicity test, based on inhibition of beta-galactosidase activity in the yeast Kluyveromyces marxianus, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B1, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 micrograms/ml, nor could aflatoxins B1 and M1 be detected at concentrations up to 25 micrograms/ml. This test should be useful for trichothecene detection and for studies of relevant interactions-both between trichothecenes themselves and between trichothecenes and other food constituents.     

A rapid computer technique for analysing molecular interactions

A rapid method for analysing enzyme — substrate interactionsusing a discriminant analysis program is described. This technqueidentifies the structural features of substrate molecules whichare important in determining metabolic activity. Two model systems,nucleoside diphosphatase activity of Golgi membranes and theinteraction of yeast hexokinase with a range of D-sugars, areused as illustrations of the technique. The conclusions fromboth models are consistent with those previously obtained fromanalytical techniques. Received on January 21, 1986; accepted on April 1, 1986     

Development and application of microwave-assisted extraction technique in biological sample preparation for small molecule analysis

Microwave-assisted extraction (MAE) has emerged as an efficient extraction technique for various kinds of biological samples due to its low usage of extraction solvents and shorter extraction time. This review will focus on the recent developments and advantages of incorporating MAE in sample preparation protocols for the analysis of small molecules in plant, food and clinical samples in recent years. The operating principles of this technique and the key parameters influencing its extraction efficiency, including the nature of solvent, temperature, power and extraction time and their limitations are first mentioned. This is followed by a discussion on the advantages of applying MAE to extract organic contaminants in food for routine food safety analysis and active ingredients recovery. The successful application of MAE technique to recover bioactive compounds from plants in drug discovery studies and quality control purposes is then described. Additionally, the feasibility of using green solvents such as water, micelle and ionic liquids with MAE for plant metabolite profiling studies is evaluated and the associated challenges discussed. Finally, the application of MAE in clinical samples is highlighted. The use of MAE in this field is currently limited to the targeted detection of small molecules in human samples, due to a lack of knowledge of its effects on thermally labile metabolites. Consequently, the need for additional studies on how MAE impacts the recoveries of different metabolite classes in mammalian samples is discussed. The outcome of these studies can potentially broaden MAE applications in the clinical field.