Small Molecule Binding Sites Research Articles

K2P2.1 (TREK-1)-activator complexes reveal a cryptic selectivity filter binding site.

Polymodal K2P (KCNK) thermo- and mechanosensitive TREK1 potassium channels, generate ‘leak’ currents that regulate neuronal excitability, respond to lipids, temperature, and mechanical stretch, and influence pain, temperature perception, and anesthetic responses1–3. These dimeric voltage-gated ion channel (VGIC) superfamily members have a unique topology comprising two pore forming regions per subunit4–6. Contrasting other potassium channels, K2Ps use a selectivity filter ‘C-type’ gate7–10 as the principal gating site. Despite recent advances3,11,12, K2Ps suffer from a poor pharmacologic profile limiting mechanistic and biological studies. Here, we describe a new small molecule TREK activator class that directly stimulates the C-type gate by acting as molecular wedges that restrict interdomain interface movement behind the selectivity filter. Structures of K2P2.1(TREK-1) alone with two selective K2P2.1(TREK-1) and K2P10.1(TREK-2) activators, an N-aryl-sulfonamide, ML335, and a thiophene-carboxamide, ML402, define a cryptic binding pocket unlike other ion channel small molecule binding sites and, together with functional studies, identify a cation-π interaction that controls selectivity. Together, our data unveil a previously unknown, druggable K2P site that stabilizes the C-type gate ‘leak mode’ and provide direct evidence for K2P selectivity filter gating.

The potential use of single-particle electron microscopy as a tool for structure-based inhibitor design.

Recent developments in electron microscopy (EM) have led to a step change in our ability to solve the structures of previously intractable systems, especially membrane proteins and large protein complexes. This has provided new opportunities in the field of structure-based drug design, with a number of high-profile publications resolving the binding sites of small molecules and peptide inhibitors. There are a number of advantages of EM over the more traditional X-ray crystallographic approach, such as resolving different conformational states and permitting the dynamics of a system to be better resolved when not constrained by a crystal lattice. There are still significant challenges to be overcome using an EM approach, not least the speed of structure determination, difficulties with low-occupancy ligands and the modest resolution that is available. However, with the anticipated developments in the field of EM, the potential of EM to become a key tool for structure-based drug design, often complementing X-ray and NMR studies, seems promising.

Two-Way Gold Nanoparticle Label-Free Sensing of Specific Sequence and Small Molecule Targets Using Switchable Concatemers.

A two-way colorimetric biosensor based on unmodified gold nanoparticles (GNPs) and a switchable double-stranded DNA (dsDNA) concatemer have been demonstrated. Two hairpin probes (H1 and H2) were first designed that provided the fuels to assemble the dsDNA concatemers via hybridization chain reaction (HCR). A functional hairpin (FH) was rationally designed to recognize the target sequences. All the hairpins contained a single-stranded DNA (ssDNA) loop and sticky end to prevent GNPs from salt-induced aggregation. In the presence of target sequence, the capture probe blocked in the FH recognizes the target to form a duplex DNA, which causes the release of the initiator probe by FH conformational change. This process then starts the alternate-opening of H1 and H2 through HCR, and dsDNA concatemers grow from the target sequence. As a result, unmodified GNPs undergo salt-induced aggregation because the formed dsDNA concatemers are stiffer and provide less stabilization. A light purple-to-blue color variation was observed in the bulk solution, termed the light-off sensing way. Furthermore, H1 ingeniously inserted an aptamer sequence to generate dsDNA concatemers with multiple small molecule binding sites. In the presence of small molecule targets, concatemers can be disassembled into mixtures with ssDNA sticky ends. A blue-to-purple reverse color variation was observed due to the regeneration of the ssDNA, termed the light-on way. The two-way biosensor can detect both nucleic acids and small molecule targets with one sensing device. This switchable sensing element is label-free, enzyme-free, and sophisticated-instrumentation-free. The detection limits of both targets were below nanomolar.

Structural basis for ELL2 and AFF4 activation of HIV-1 proviral transcription

The intrinsically disordered scaffold proteins AFF1/4 and the transcription elongation factors ELL1/2 are core components of the super elongation complex required for HIV-1 proviral transcription. Here we report the 2.0-Å resolution crystal structure of the human ELL2 C-terminal domain bound to its 50-residue binding site on AFF4, the ELLBow. The ELL2 domain has the same arch-shaped fold as the tight junction protein occludin. The ELLBow consists of an N-terminal helix followed by an extended hairpin that we refer to as the elbow joint, and occupies most of the concave surface of ELL2. This surface is important for the ability of ELL2 to promote HIV-1 Tat-mediated proviral transcription. The AFF4–ELL2 interface is imperfectly packed, leaving a cavity suggestive of a potential binding site for transcription-promoting small molecules.

Mapping platinum adducts on yeast ribosomal RNA using high-throughput sequencing.

Methods to map small-molecule binding sites on cellular RNAs are important for understanding interactions with both endogenous and exogenous compounds. Pt(ii) reagents are well-known DNA and RNA crosslinking agents, but sequence-specific and genome-wide identification of Pt targets following in-cell treatment is challenging. Here we describe application of high-throughput 'Pt-Seq' to identify Pt-rRNA adducts following treatment of S. cerevisiae with cisplatin.

Computational Design of Ligand Binding Proteins.

The ability to design novel small-molecule binding sites in proteins is a stringent test of our understanding of the principles of molecular recognition, and would have many practical applications, in synthetic biology and medicine. Here, we describe a computational method in the context of the macromolecular modeling suite Rosetta to designing proteins with sites featuring predetermined interactions to ligands of choice. The required inputs for the method are a model of the small molecule and the desired interactions (e.g., hydrogen bonding, electrostatics, steric packing), and a set of crystallographic structures of proteins containing existing or predicted binding pockets. Constellations of backbones surrounding the putative pocket are searched for compatibility with the desired binding site conception using RosettaMatch and surrounding amino acid side chain identities are optimized using RosettaDesign. Validation of the design is performed using metrics that evaluate the interface energy of the predicted binding pose, the preformation of key binding site features in the apo-state, and the local compatibility of the designed sequence changes with the wild type backbone structure, and top-ranking candidate designs are generated for experimental validation. This approach can allow for the creation of novel binding sites and for the rational tuning of specificity for congeneric ligands by altering the programmed interactions by design, thus offering a general computational protocol for construction and modulation of protein-small molecule interfaces.

Molecular Mechanism of Action for Allosteric Modulators and Agonists in CC-chemokine Receptor 5 (CCR5)

The small molecule metal ion chelators bipyridine and terpyridine complexed with Zn2+ (ZnBip and ZnTerp) act as CCR5 agonists and strong positive allosteric modulators of CCL3 binding to CCR5, weak modulators of CCL4 binding, and competitors for CCL5 binding. Here we describe their binding site using computational modeling, binding, and functional studies on WT and mutated CCR5. The metal ion Zn2+ is anchored to the chemokine receptor-conserved Glu-283VII:06/7.39 Both chelators interact with aromatic residues in the transmembrane receptor domain. The additional pyridine ring of ZnTerp binds deeply in the major binding pocket and, in contrast to ZnBip, interacts directly with the Trp-248VI:13/6.48 microswitch, contributing to its 8-fold higher potency. The impact of Trp-248 was further confirmed by ZnClTerp, a chloro-substituted version of ZnTerp that showed no inherent agonism but maintained positive allosteric modulation of CCL3 binding. Despite a similar overall binding mode of all three metal ion chelator complexes, the pyridine ring of ZnClTerp blocks the conformational switch of Trp-248 required for receptor activation, thereby explaining its lack of activity. Importantly, ZnClTerp becomes agonist to the same extent as ZnTerp upon Ala mutation of Ile-116III:16/3.40, a residue that constrains the Trp-248 microswitch in its inactive conformation. Binding studies with 125I-CCL3 revealed an allosteric interface between the chemokine and the small molecule binding site, including residues Tyr-37I:07/1.39, Trp-86II:20/2.60, and Phe-109III:09/3.33 The small molecules and CCL3 approach this interface from opposite directions, with some residues being mutually exploited. This study provides new insight into the molecular mechanism of CCR5 activation and paves the way for future allosteric drugs for chemokine receptors.

Mapping the 3D structures of small molecule binding sites

BackgroundAnalysis of the 3D structures of protein–ligand binding sites can provide valuable insights for drug discovery. Binding site comparison (BSC) studies can be employed to elucidate the function of orphan proteins or to predict the potential for polypharmacology. Many previous binding site analyses only consider binding sites surrounding an experimentally observed bound ligand.ResultsTo encompass potential protein–ligand binding sites that do not have ligands known to bind, we have incorporated fpocket cavity detection software and assessed the impact of this inclusion on BSC performance. Using fpocket, we generated a database of ligand-independent potential binding sites and applied the BSC tool, SiteHopper, to analyze similarity relationships between protein binding sites. We developed a method for clustering potential binding sites using a curated dataset of structures for six therapeutically relevant proteins from diverse protein classes in the protein data bank. Two clustering methods were explored; hierarchical clustering and a density-based method adept at excluding noise and outliers from a dataset. We introduce circular plots to visualize binding site structure space. From the datasets analyzed in this study, we highlight a structural relationship between binding sites of cationic trypsin and prothrombin, protein targets known to bind structurally similar small molecules, exemplifying the potential utility of objectively and holistically mapping binding site space from the structural proteome.ConclusionsWe present a workflow for the objective mapping of potential protein–ligand binding sites derived from the currently available structural proteome. We show that ligand-independent binding site detection tools can be introduced without excessive penalty on BSC performance. Clustering combined with intuitive visualization tools can be applied to map relationships between the 3D structures of protein binding sites.Graphical abstractMapping binding site space. Electronic supplementary materialThe online version of this article (doi:10.1186/s13321-016-0180-0) contains supplementary material, which is available to authorized users.

Comparative electrostatic analysis of adenylyl cyclase for isoform dependent regulation properties

The enzyme adenylyl cyclase (AC) plays a pivotal role in a variety of signal transduction pathways inside the cell, where it catalyzes the cyclization of adenosine triphosphate (ATP) into the second-messenger cyclic adenosine monophosphate (cAMP). Among other roles, AC regulates processes involved in neural plasticity, innervation of smooth muscles of the heart and the endocrine system of the pancreas. The functional diversity of AC is manifested in its different isoforms, each having a specific regulation pattern. There is an increasing amount of data available concerning the regulatory properties of AC isoforms, however little is known about the interactions on a structural level. Here, we conducted a comparative electrostatic analysis of the catalytic domains of all nine transmembrane AC isoforms with the aim of detecting, verifying and predicting the binding sites of molecular regulators on AC. The results provide support for the positioning of the binding site of the inhibitory protein Gi α at a pseudo-symmetric position to the stimulatory Gs α binding site. They also provide a structural interpretation of the Gβγ interaction with ACs 2, 4, and 7 and suggest a new binding site for RGS2. Comparison of the small molecule binding sites on AC shows that overall they have high electrostatic similarity, but regions of electrostatic differences are identified. These could provide a basis for the development of novel compounds with isoform-specific modulatory effects on AC. Proteins 2016; 84:1844-1858. © 2016 Wiley Periodicals, Inc.

Computational Methods Applied to Rational Drug Design.

Due to the synergic relationship between medical chemistry, bioinformatics and molecular simulation, the development of new accurate computational tools for small molecules drug design has been rising over the last years. The main result is the increased number of publications where computational techniques such as molecular docking, de novo design as well as virtual screening have been used to estimate the binding mode, site and energy of novel small molecules. In this work I review some tools, which enable the study of biological systems at the atomistic level, providing relevant information and thereby, enhancing the process of rational drug design.

Small Molecule Binding Sites Explored in Variable Heavy Chain Antibodies

Polybrominated diphenyl ethers (PBDEs) are chemical flame retardants that have been used extensively over the last 40 years in furniture, plastics, wiring, and electronics. Some PBDEs have been classified as possible human carcinogens. PBDEs have been widely found in human blood, breast milk, and body fat. While PBDEs have recently been placed on the California Biomonitoring Program and have been banned by the EPA, these PBDEs persist in the environment. 2,2′,4,4′‐tetrabrominated diphenyl ether (BDE‐47) and Tetrabromobisphenol A (TBBPA) are two of these chemical flame retardants that are persistent in the environment and detecting them with an VHH‐based method has been developed. However, it is not well understood how these detection methods work and it is difficult to improve on the methods without this understanding. The variable heavy chain domain antibodies (VHH) from camelids have many advantages compared to conventional monoclonal antibodies including increased stability and ease of recombinant production. Although it is well established how VHH interact with proteins, it is less well understood how these antibodies interact with small molecules and haptens. There are currently four known binding modes available for VHH to interact with small molecules including: combining VHH's three CDR loops, domain swapping utilizing a 2:1 binding stoichiometry, and tunneling under the CDR1 loop. Using VHH specific for BDE‐47 and TBBPA as a model for PBDEs we aim to examine VHH small molecule interactions. We have successfully completed expression tests of seven VHH and purified 2 VHH. Crystallization trails have yielded preliminary crystals. Future work will focus on crystal optimization and structure determination.Support or Funding Information‐BW was supported with funds from the Undergraduate rGrant from The Associated Students, Inc. at California State University, Fresno.

Insights Into the Allosteric Inhibition of the SUMO E2 Enzyme Ubc9.

Conjugation of the small ubiquitin-like modifier (SUMO) to protein substrates is an important disease-associated posttranslational modification, although few inhibitors of this process are known. Herein, we report the discovery of an allosteric small-molecule binding site on Ubc9, the sole SUMO E2 enzyme. An X-ray crystallographic screen was used to identify two distinct small-molecule fragments that bind to Ubc9 at a site distal to its catalytic cysteine. These fragments and related compounds inhibit SUMO conjugation in biochemical assays with potencies of 1.9-5.8 mm. Mechanistic and biophysical analyses, coupled with molecular dynamics simulations, point toward ligand-induced rigidification of Ubc9 as a mechanism of inhibition.

A Binding Site on IL-17A for Inhibitory Macrocycles Revealed by Hydrogen/Deuterium Exchange Mass Spectrometry

Computational assessment of the IL-17A structure identified two distinct binding pockets, the β-hairpin pocket and the α-helix pocket. The β-hairpin pocket was hypothesized to be the site of binding for peptide macrocycles. Support for this hypothesis was obtained using HDX-MS which revealed protection to exchange only within the β-hairpin pocket. This data represents the first direct structural evidence of a small molecule binding site on IL-17A that functions to disrupt the interaction with its receptor.

Local Geometry and Evolutionary Conservation of Protein Surfaces Reveal the Multiple Recognition Patches in Protein-Protein Interactions

Proteins regulate biological processes through a complex network of dynamical interactions. Protein-protein interfaces, e.g., those involved in signaling complexes, constitute increasingly important therapeutic targets. We present a new method, JET2, for accurately predicting protein interfaces at large scale (Laine and Carbone, 2015). Contrary to machine learning approaches, JET2 combines in a rational and very straightforward way three sequence- and structure-based descriptors of protein residues: evolutionary conservation, physico-chemical properties and local geometry. The implemented strategy yields very precise predictions for a wide range of protein-protein interfaces and discriminates them from small-molecule binding sites. Beyond its predictive power, the approach permits to dissect interaction surfaces and unravel their complexity. JET2 is useful to predict and learn about the different partners of a protein. It allows to identify multiple recognition patches within large protein interfaces and characterize their different properties and origins. We show how the analysis of the predicted patches can foster new strategies for protein-potein interaction modulation and interaction surface redesign. JET2 was applied on more than 20 000 protein structures, representing the non-redundant set of non-homologous chains extracted from the Protein Data Bank. The generated knowledge base will soon be made accessible to the community (Ripoche et al., 2015). JET2 is implemented as a fully automated tool, freely available at www.lcqb.upmc.fr/JET2. E. Laine, A. Carbone. The local geometry and evolutionary conservation of protein surfaces reveal the multiple recognition patches in protein-protein interactions, under revision, 2015. H. Ripoche∗, E. Laine∗, N. Ceres, A. Carbone. JET2 Viewer: a web server predicting multiple protein-protein interaction sites for PDB structures, submitted, 2015.

Solution NMR characterization of apical membrane antigen 1 and small molecule interactions as a basis for designing new antimalarials.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) plays an important role in the invasion by merozoites of human red blood cells during a malaria infection. A key region of PfAMA1 is a conserved hydrophobic cleft formed by 12 hydrophobic residues. As anti-apical membrane antigen 1 antibodies and other inhibitory molecules that target this hydrophobic cleft are able to block the invasion process, PfAMA1 is an attractive target for the development of strain-transcending antimalarial agents. As solution nuclear magnetic resonance spectroscopy is a valuable technique for the rapid characterization of protein-ligand interactions, we have determined the sequence-specific backbone assignments for PfAMA1 from two P. falciparum strains, FVO and 3D7. Both selective labelling and unlabelling strategies were used to complement triple-resonance experiments in order to facilitate the assignment process. We have then used these assignments for mapping the binding sites for small molecules, including benzimidazoles, pyrazoles and 2-aminothiazoles, which were selected on the basis of their affinities measured from surface plasmon resonance binding experiments. Among the compounds tested, benzimidazoles showed binding to a similar region on both FVO and 3D7 PfAMA1, suggesting that these compounds are promising scaffolds for the development of novel PfAMA1 inhibitors. Copyright © 2016 John Wiley & Sons, Ltd.

Small-molecule binding sites to explore protein-protein interactions in the cancer proteome.

The Cancer Genome Atlas (TCGA) offers an unprecedented opportunity to identify small-molecule binding sites on proteins with overexpressed mRNA levels that correlate with poor survival. Here, we analyze RNA-seq and clinical data for 10 tumor types to identify genes that are both overexpressed and correlate with patient survival. Protein products of these genes were scanned for binding sites that possess shape and physicochemical properties that can accommodate small-molecule probes or therapeutic agents (druggable). These binding sites were classified as enzyme active sites (ENZ), protein-protein interaction sites (PPI), or other sites whose function is unknown (OTH). Interestingly, the overwhelming majority of binding sites were classified as OTH. We find that ENZ, PPI, and OTH binding sites often occurred on the same structure suggesting that many of these OTH cavities can be used for allosteric modulation of enzyme activity or protein-protein interactions with small molecules. We discovered several ENZ (PYCR1, QPRT, and HSPA6) and PPI (CASC5, ZBTB32, and CSAD) binding sites on proteins that have been seldom explored in cancer. We also found proteins that have been extensively studied in cancer that have not been previously explored with small molecules that harbor ENZ (PKMYT1, STEAP3, and NNMT) and PPI (HNF4A, MEF2B, and CBX2) binding sites. All binding sites were classified by the signaling pathways to which the protein that harbors them belongs using KEGG. In addition, binding sites were mapped onto structural protein-protein interaction networks to identify promising sites for drug discovery. Finally, we identify pockets that harbor missense mutations previously identified from analysis of TCGA data. The occurrence of mutations in these binding sites provides new opportunities to develop small-molecule probes to explore their function in cancer.

Local Geometry and Evolutionary Conservation of Protein Surfaces Reveal the Multiple Recognition Patches in Protein-Protein Interactions.

Protein-protein interactions (PPIs) are essential to all biological processes and they represent increasingly important therapeutic targets. Here, we present a new method for accurately predicting protein-protein interfaces, understanding their properties, origins and binding to multiple partners. Contrary to machine learning approaches, our method combines in a rational and very straightforward way three sequence- and structure-based descriptors of protein residues: evolutionary conservation, physico-chemical properties and local geometry. The implemented strategy yields very precise predictions for a wide range of protein-protein interfaces and discriminates them from small-molecule binding sites. Beyond its predictive power, the approach permits to dissect interaction surfaces and unravel their complexity. We show how the analysis of the predicted patches can foster new strategies for PPIs modulation and interaction surface redesign. The approach is implemented in JET2, an automated tool based on the Joint Evolutionary Trees (JET) method for sequence-based protein interface prediction. JET2 is freely available at www.lcqb.upmc.fr/JET2.

Abstract B40: Selective suppression of Myc transcription with a G-quadruplex stabilizing small molecule

Abstract The transcription factor Myc is disregulated in some 70% of human cancers, where it triggers aberrant growth and signaling, and contributes to tumorigenesis. Thus, Myc is a highly sought after drug target. However, targeting the Myc protein directly with small molecules has proven to be a significant challenge due to a lack of small molecule binding sites and rapid protein turnover in the cell. To date, most successes in inhibiting Myc function have come through alternative indirect approaches such as BET bromodomain inhibition. Here, we present a novel class of small molecules that silences Myc expression by stabilization of the G-quadruplex structure present in the NHEIII region of the Myc promoter. We used a small molecule microarray screen to identify molecules that selectively bind to the Myc G-quadruplex. Biophysical studies, including thermal melt and SPR analysis, demonstrated that the compound binds reversibly to the quadruplex structure with low micromolar affinity. Next, we demonstrated that the compound inhibited polymerase progression in an in vitro PCR-stop assay in a dose dependent fashion, while the compound had no effect on amplification of an oligo incapable of forming a quadruplex. Target specificity was demonstrated using a cell-based assay where one copy of the Myc gene has a translocation and is no longer under the control of a quadruplex. In this assay, Myc is only silenced upon treatment when there is a quadruplex in the promoter. Further studies demonstrate dose- and time-dependent effects on cell viability and Myc protein levels in a panel of myeloma cells. The compound also had minimal effects on viability of PBMCs from a healthy donor. Finally, gene expression analysis demonstrated that in contrast to other quadruplex-stabilizing small molecules, the compound reported here does not silence several other genes that are also under the control of quadruplexes. Citation Format: John K. Simmons, Lindsey B. Saunders, Kenneth Felsenstein, Peter Gareiss, Shuling Zhang, Beverly Mock, John S. Schneekloth, Jr.. Selective suppression of Myc transcription with a G-quadruplex stabilizing small molecule. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B40.

Discovery of small molecule inhibitors of MyD88-dependent signaling pathways using a computational screen.

In this study, we used high-throughput computational screening to discover drug-like inhibitors of the host MyD88 protein-protein signaling interaction implicated in the potentially lethal immune response associated with Staphylococcal enterotoxins. We built a protein-protein dimeric docking model of the Toll-interleukin receptor (TIR)-domain of MyD88 and identified a binding site for docking small molecules. Computational screening of 5 million drug-like compounds led to testing of 30 small molecules; one of these molecules inhibits the TIR-TIR domain interaction and attenuates pro-inflammatory cytokine production in human primary cell cultures. Compounds chemically similar to this hit from the PubChem database were observed to be more potent with improved drug-like properties. Most of these 2nd generation compounds inhibit Staphylococcal enterotoxin B (SEB)-induced TNF-α, IFN-γ, IL-6, and IL-1β production at 2–10 μM in human primary cells. Biochemical analysis and a cell-based reporter assay revealed that the most promising compound, T6167923, disrupts MyD88 homodimeric formation, which is critical for its signaling function. Furthermore, we observed that administration of a single dose of T6167923 completely protects mice from lethal SEB-induced toxic shock. In summary, our in silico approach has identified anti-inflammatory inhibitors against in vitro and in vivo toxin exposure with promise to treat other MyD88-related pro-inflammatory diseases.

Identification of A Novel Small-Molecule Binding Site of the Fat Mass and Obesity Associated Protein (FTO).

N-(5-Chloro-2,4-dihydroxyphenyl)-1-phenylcyclobutanecarboxamide (N-CDPCB, 1a) is found to be an inhibitor of the fat mass and obesity associated protein (FTO). The crystal structure of human FTO with 1a reveals a novel binding site for the FTO inhibitor and defines the molecular basis for recognition by FTO of the inhibitor. The identification of the new binding site offers new opportunities for further development of selective and potent inhibitors of FTO, which is expected to provide information concerning novel therapeutic targets for treatment of obesity or obesity-associated diseases.