Modulating protein-protein interactions with small molecules: the importance of binding hotspots

The modulation of protein-protein interactions (PPIs) by small drug-like molecules is a relatively new area of research and has opened up new opportunities in drug discovery. However, the progress made in this area is limited to a handful of known cases of small molecules that target specific diseases. With the increasing availability of protein structure complexes, it is highly important to devise strategies exploiting homologous structure space on a large scale for discovering putative PPIs that could be attractive drug targets. Here, we propose a scheme that allows performing large-scale screening of all protein complexes and finding putative small-molecule and/or peptide binding sites overlapping with protein-protein binding sites (so-called "multibinding sites"). We find more than 600 nonredundant proteins from 60 protein families with multibinding sites. Moreover, we show that the multibinding sites are mostly observed in transient complexes, largely overlap with the binding hotspots and are more evolutionarily conserved than other interface sites. We investigate possible mechanisms of how small molecules may modulate protein-protein binding and discuss examples of new candidates for drug design.     

High-throughput fluorescence assay for membrane-protein interaction

Membrane-protein interaction plays key roles in a wide variety of biological processes. Although various methods have been employed to measure membrane binding of soluble proteins, a robust high-throughput assay that is universally applicable to all proteins is lacking at present. Here we report a new fluorescence quenching assay utilizing enhanced green fluorescence protein (EGFP)-fusion proteins and a lipid containing a dark quencher, N-dimethylaminoazobenzenesulfonyl-phosphatidylethanolamine (dabsyl-PE). The EGFP fluorescence emission intensity showed a large decrease (i.e., >50%) when EGFP-fusion proteins bound the vesicles containing 5 mol% dabsyl-PE. This simple assay, which can be performed using either a cuvette-based spectrofluorometer or a fluorescence plate reader, allowed rapid, sensitive, and accurate determination of lipid specificity and affinity for various lipid binding domains, including two pleckstrin homology domains, an epsin N-terminal homology domain, and a phox homology domain. The assay can also be applied to high-throughput screening of small molecules that modulate membrane binding of proteins.     

Different combinations of atomic interactions predict protein‐small molecule and protein‐DNA/RNA affinities with similar accuracy

Interactions between proteins and other molecules play essential roles in all biological processes. Although it is widely held that a protein's ligand specificity is determined primarily by its three‐dimensional structure, the general principles by which structure determines ligand binding remain poorly understood. Here we use statistical analyses of a large number of protein?ligand complexes with associated binding‐affinity measurements to quantitatively characterize how combinations of atomic interactions contribute to ligand affinity. We find that there are significant differences in how atomic interactions determine ligand affinity for proteins that bind small chemical ligands, those that bind DNA/RNA and those that interact with other proteins. Although protein‐small molecule and protein‐DNA/RNA binding affinities can be accurately predicted from structural data, models predicting one type of interaction perform poorly on the others. Additionally, the particular combinations of atomic interactions required to predict binding affinity differed between small‐molecule and DNA/RNA data sets, consistent with the conclusion that the structural bases determining ligand affinity differ among interaction types. In contrast to what we observed for small‐molecule and DNA/RNA interactions, no statistical models were capable of predicting protein?protein affinity with >60% correlation. We demonstrate the potential usefulness of protein‐DNA/RNA binding prediction as a possible tool for high‐throughput virtual screening to guide laboratory investigations, suggesting that quantitative characterization of diverse molecular interactions may have practical applications as well as fundamentally advancing our understanding of how molecular structure translates into function. Proteins 2015; 83:2100–2114. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Oligonucleotide inhibition of the interaction of HIV-1 Tat protein with the trans-activation responsive region (TAR) of HIV RNA

The interaction of HIV-1 Tat protein with its recognition sequence, the trans-activation responsive region TAR is a potential target for drug discovery against HIV infection. We show by use of an in vitro competition filter binding interference assay that synthetic oligodeoxyribonucleotides complementary to the HIV-1 TAR RNA apical stem-loop and bulge region inhibit the binding of Tat protein or a Tat peptide (residues 37-72) better than two small molecules that have been shown to bind TAR RNA, Hoechst 33258 and neomycin B. The inhibition is not sensitive to length between 13 and 16 residues or precise positioning but shorter oligonucleotides are less effective. Enhanced inhibition was obtained for a 16-mer 2'-O-methyl oligoribonucleotide but not for C5-propyne pyrimidine-substituted oligonucleotides. Control non-antisense oligonucleotides were occasionally also effective in filter binding interference but only the complementary antisense 2'-O-methyl oligoribonucleotide was effective in gel mobility shift assays in direct TAR binding or in interference with Tat peptide binding to the TAR stem-loop. This is the first demonstration of effective inhibition of the Tat-TAR interaction by nuclease-stabilized oligonucleotide analogues.     

A homogeneous 384-well high-throughput binding assay for a TNF receptor using alphascreen technology

To take advantage of the growing knowledge of cellular signaling pathways, modern-day drug discovery faces an increasing challenge to develop assays to screen for compounds that modulate protein-protein interactions. One bottleneck in achieving this goal is a lack of suitable and robust assay technologies amenable to a high-throughput format. In this report, we describe how we utilized Alphascreen trade mark technology to develop a high-throughput assay to monitor ligand binding to a member of the tumor necrosis factor receptor superfamily. We expressed a fusion protein consisting of the extracellular domain of the OX40 receptor with the constant domains of human IgG. In the presence of OX40 ligand, we determined a binding affinity constant consistent with reported values and optimized the protocol to develop a simple, homogeneous, and sensitive binding assay in a 384-well format. Finally, we assessed if this system could identify small peptides capable of inhibiting the OX40 receptor and ligand interaction. The results showed that the assay was able to detect such peptides and could be used to launch a high-throughput screening campaign for small molecules able to prevent OX40 receptor activation.     

Probing protein-protein interactions in real time

We have used a prototype small cantilever atomic force microscope to observe, in real time, the interactions between individual protein molecules. In particular, we have observed individual molecules of the chaperonin protein GroES binding to and then dissociating from individual GroEL proteins, which were immobilized on a mica support. This work suggests that the small cantilever atomic force microscope is a useful tool for studying protein dynamics at the single molecule level.     

Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays.

Packaging of retroviral genomic RNA during virion assembly is thought to be mediated by specific interactions between the gag polyprotein and RNA sequences (often termed the psi or E region) near the 5' end of the genome. For many retroviruses, including human immunodeficiency virus type 1 (HIV-1), the portions of the gag protein and the RNA that are required for this interaction remain poorly defined. We have used an RNA gel mobility shift assay to measure the in vitro binding of purified glutathione S-transferase-HIV-1 gag fusion proteins to RNA riboprobes. Both the complete gag polyprotein and the nucleocapsid (NC) protein alone were found to bind specifically to an HIV-1 riboprobe. Either Cys-His box of NC could be removed without eliminating specific binding to the psi riboprobe, but portions of gag containing only the MA and CA proteins without NC did not bind to RNA. There were at least two binding sites in HIV-1 genomic RNA that bound to the gag polyprotein: one entirely 5' to gag and one entirely within gag. The HIV-1 NC protein bound to riboprobes containing other retroviral psi sequences almost as well as to the HIV-1 psi riboprobe.     

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.     

Studies on ligand binding to histidine triad nucleotide binding protein 1

Histidine triad nucleotide binding protein (HINT1) is an intracellular protein that binds purine mononucleotides. Strong sequence conservation suggests that these proteins play a fundamental role in cell biology, however its exact cellular function continues to remain elusive. nuclear magnetic resonance (NMR) studies using STD and HSQC were conducted to observe ligand binding to HINT1. These studies were confirmed using fluorescence spectroscopy titrations. We found that AICAR, the first non-phosphate containing ligand, binds to mouse histidine triad nucleotide binding protein 1 (HINT1). Chemical shift perturbations are mapped onto the X-ray structure showing AICAR binds at the same site as GMP. The NMR results demonstrated that this method will be valuable for the future screening of small molecules that can be used to modulate the function of HINT1.     

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin. In recent years, small molecules have been heavily analyzed as potential LC inhibitors based on their increased cellular permeability relative to larger therapeutics (peptides, aptamers, etc.). Lead identification often involves high-throughput screening (HTS), where large libraries of small molecules are screened based on their ability to modulate therapeutic target function. Here we describe a FRET-based assay with a commercial BoNT/A LC substrate and recombinant LC that can be automated for HTS of potential BoNT inhibitors. Moreover, we describe a manual technique that can be used for follow-up secondary screening, or for comparing the potency of several candidate compounds.     

Isolation of protein subpopulations undergoing protein-protein interactions

A new method is described for isolating and identifying proteins participating in protein-protein interactions in a complex mixture. The method uses a cyanogen bromide-activated Sepharose matrix to isolate proteins that are non-covalently bound to other proteins. Because the proteins are accessible to chemical manipulation, mass spectrometric identification of the proteins can yield information on specific classes of interacting proteins, such as calcium-dependent or substrate-dependent protein interactions. This permits selection of a subpopulation of proteins from a complex mixture on the basis of specified interaction criteria. The new method has the advantage of screening the entire proteome simultaneously, unlike the two-hybrid system or phage display, which can only detect proteins binding to a single bait protein at a time. The method was tested by selecting rat brain extract for proteins exhibiting calcium-dependent protein interactions. Of 12 proteins identified by mass spectrometry, eight were either known calcium-binding proteins or proteins with known calcium-dependent protein interactions, indicating that the method is capable of enriching a subpopulation of proteins from a complex mixture on the basis of a specific class of protein interactions. Because only naturally occurring interactions of proteins in their native state are observed, this method will have wide applicability to studies of protein interactions in tissue samples and autopsy specimens, for screening for perturbations of protein-protein interactions by signaling molecules, pharmacological agents or toxins, and screening for differences between cancerous and untransformed cells.     

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.     

Displacement of protein-bound aptamers with small molecules screened by fluorescence polarization

Small molecule inhibitors of proteins are invaluable tools in research and as starting points for drug development. However, their screening can be tedious, as most screening methods have to be tailored to the corresponding drug target. Here, we describe a detailed protocol for a modular and generally applicable assay for the identification of small organic compounds that displace an aptamer complexed to its target protein. The method relies on fluorescence-labeled aptamers and the increase of fluorescence polarization upon their binding to the target protein. The assay has high Z'-factors, making it compatible with high-throughput screening. It allows easy automation, making fluorescence readout the time-limiting step. As aptamers can be generated for virtually any protein target, the assay allows identification of small molecule inhibitors for targets or individual protein domains for which no functional screen is available. We provide the step-by-step protocol to screen for antagonists of the cytohesin class of small guanosine exchange factors.     

Mapping the RNA binding sites for human immunodeficiency virus type-1 gag and NC proteins within the complete HIV-1 and -2 untranslated leader regions.

Encapsidation of HIV-1 genomic RNA is mediated by specific interactions between the RNA packaging signal and the Gag protein. During maturation of the virion, the Gag protein is processed into smaller fragments, including the nucleocapsid (NC) domain which remains associated with the viral genomic RNA. We have investigated the binding of glutathione- S -transferase (GST) Gag and NC fusion proteins from HIV-1, to the entire HIV-1 and -2 leader RNAencompassing the packaging signal. We have mapped the binding sites at conditions where only about two complexes are formed and find that GST-Gag and GST-NC fusion proteins bind specifically to discrete sites within the leader. Analysis of the HIV-1 leader indicated that GST-Gag strongly associates with the PSI stem-loop and to a lesser extent with regions near the primer binding site. GST-NC binds the same regions but with reversed preferences. The HIV-1 proteins also interact specifically with the 5'-leader of HIV-2 and the major site of interaction mapped to a stem-loop, with homology to the HIV-1 PSI stem-loop structure. The different specificities of Gag and NC may reflect functionally distinct roles in the viral replication, and suggest that the RNA binding specificity of NC is modulated by its structural context.     

Universal screening methods and applications of ThermoFluor

The genomics revolution has unveiled a wealth of poorly characterized proteins. Scientists are often able to produce milligram quantities of proteins for which function is unknown or hypothetical, based only on very distant sequence homology. Broadly applicable tools for functional characterization are essential to the illumination of these orphan proteins. An additional challenge is the direct detection of inhibitors of protein-protein interactions (and allosteric effectors). Both of these research problems are relevant to, among other things, the challenge of finding and validating new protein targets for drug action. Screening collections of small molecules has long been used in the pharmaceutical industry as 1 method of discovering drug leads. Screening in this context typically involves a function-based assay. Given a sufficient quantity of a protein of interest, significant effort may still be required for functional characterization, assay development, and assay configuration for screening. Increasingly, techniques are being reported that facilitate screening for specific ligands for a protein of unknown function. Such techniques also allow for function-independent screening with better characterized proteins. ThermoFluor, a screening instrument based on monitoring ligand effects on temperature-dependent protein unfolding, can be applied when protein function is unknown. This technology has proven useful in the decryption of an essential bacterial enzyme and in the discovery of a series of inhibitors of a cancer-related, protein-protein interaction. The authors review some of the tools relevant to these research problems in drug discovery, and describe our experiences with 2 different proteins.