Crossing borders to bind proteins—a new concept in protein recognition based on the conjugation of small organic molecules or short peptides to polypeptides from a designed set

A new concept for protein recognition and binding is highlighted. The conjugation of small organic molecules or short peptides to polypeptides from a designed set provides binder molecules that bind proteins with high affinities, and with selectivities that are equal to those of antibodies. The small organic molecules or peptides need to bind the protein targets but only with modest affinities and selectivities, because conjugation to the polypeptides results in molecules with dramatically improved binder performance. The polypeptides are selected from a set of only sixteen sequences designed to bind, in principle, any protein. The small number of polypeptides used to prepare high-affinity binders contrasts sharply with the huge libraries used in binder technologies based on selection or immunization. Also, unlike antibodies and engineered proteins, the polypeptides have unordered three-dimensional structures and adapt to the proteins to which they bind. Binder molecules for the C-reactive protein, human carbonic anhydrase II, acetylcholine esterase, thymidine kinase 1, phosphorylated proteins, the D-dimer, and a number of antibodies are used as examples to demonstrate that affinities are achieved that are higher than those of the small molecules or peptides by as much as four orders of magnitude. Evaluation by pull-down experiments and ELISA-based tests in human serum show selectivities to be equal to those of antibodies. Small organic molecules and peptides are readily available from pools of endogenous ligands, enzyme substrates, inhibitors or products, from screened small molecule libraries, from phage display, and from mRNA display. The technology is an alternative to established binder concepts for applications in drug development, diagnostics, medical imaging, and protein separation.     

Substantial improvements in large-scale redocking and screening using the novel HYDE scoring function

The HYDE scoring function consistently describes hydrogen bonding, the hydrophobic effect and desolvation. It relies on HYdration and DEsolvation terms which are calibrated using octanol/water partition coefficients of small molecules. We do not use affinity data for calibration, therefore HYDE is generally applicable to all protein targets. HYDE reflects the Gibbs free energy of binding while only considering the essential interactions of protein-ligand complexes. The greatest benefit of HYDE is that it yields a very intuitive atom-based score, which can be mapped onto the ligand and protein atoms. This allows the direct visualization of the score and consequently facilitates analysis of protein-ligand complexes during the lead optimization process. In this study, we validated our new scoring function by applying it in large-scale docking experiments. We could successfully predict the correct binding mode in 93% of complexes in redocking calculations on the Astex diverse set, while our performance in virtual screening experiments using the DUD dataset showed significant enrichment values with a mean AUC of 0.77 across all protein targets with little or no structural defects. As part of these studies, we also carried out a very detailed analysis of the data that revealed interesting pitfalls, which we highlight here and which should be addressed in future benchmark datasets.     

Hydrolysis mechanisms for indomethacin and acemethacin in perchloric acid

The acid-catalyzed hydrolysis reactions of the antiinflammatory drugs indomethacin and acemethacin were investigated at 25.0 degrees C in a number of strongly concentrated perchloric acid media. The reaction rates were evaluated by UV measurements, and the intermediate species were detected by UV-vis, 1H NMR, 13C NMR, and mass spectroscopy measurements. A switchover from an A-2 to an A-1 mechanism as a function of the medium acidity is reported for the acid-catalyzed hydrolyses of the amide group of both indomethacin and acemethacin. In the A-2 hydrolysis, two water molecules are involved in the rate-determining step. An analysis of the kinetic data collected for acemethacin by the different techniques used reveals a complex mechanism, indomethacin being a metabolite intermediate species in the hydrolysis of acemethacin. The rate constants for the hydrolysis of the acemethacin ester group were considerably larger compared to those of the amide group.     

Using supramolecular hydrogels to discover the interactions between proteins and molecular nanofibers of small molecules

Here we report the first example of the use of supramolecular hydrogels to discover the protein targets of aggregates of small molecules.     

Reactivity differences of indomethacin solid forms with ammonia gas

The present study deals with the acid-base reaction of three solid-state forms of the nonsteroidal antiinflammatory drug indomethacin with ammonia gas. X-ray powder diffraction, optical microscopy, gravimetry, and spectroscopic methods were employed to establish the extent of the reaction as well as the lattice changes of the crystal forms. The glassy amorphous form readily reacts with ammonia gas to yield a corresponding amorphous ammonium salt. In addition, the metastable crystal form of indomethacin (the alpha-form) also reacts with ammonia gas, but produces the corresponding microcrystalline ammonium salt. This reaction is anisotropic and propagates along the a-axis of the crystals. The stable crystal form (the gamma-form), however, is inert to ammonia gas. Amorphous indomethacin can react with ammonia gas because it has more molecular mobility and free volume. The reactivity differences between the alpha- and gamma-forms are dictated by the arrangement of the molecules within the respective crystal lattices. The recently determined crystal structure of the metastable alpha-form of indomethacin (monoclinic P2(1) with Z = 6, V = 2501.8 A(3), D(c) = 1.42 g.cm(-3)) has three molecules of indomethacin in the asymmetric unit. Two molecules form a mutually hydrogen-bonded carboxylic acid dimer, while the carboxylic acid of the third molecule is hydrogen bonded to one of the amide carbonyls of the dimer. The carboxylic acid groups of the alpha-form are exposed on the [100] faces and are accessible to attack by ammonia gas. After one layer of molecules reacts, the reactive groups in the subsequent layer are accessible to the ammonia gas. This process proceeds along the a-axis until the ammonia gas has penetrated the entire crystal. In contrast to the alpha-form, the gamma-form has a centrosymmetric crystal structure in which the hydrogen-bonded carboxylic acid dimers are not accessible to ammonia gas because they are caged inside a hydrophobic shield comprising the remainder of the indomethacin molecule. In view of the significantly lower density of the stable gamma-form as compared to the metastable alpha-form (1.37 and 1.42 g cm(-3), respectively), it became apparent that the reactivity of the crystal forms depends exclusively on the molecular arrangement and not on the packing density of the indomethacin crystals.     

Genetic and genomic approaches to identify and study the targets of bioactive small molecules

Natural and synthetic bioactive small molecules form the backbone of modern therapeutics. These drugs primarily exert their effect by targeting cellular host or foreign proteins that are critical for the progression of disease. Therefore, a crucial step in the process of recognizing valuable new drug leads is identification of their protein targets; this is often a time consuming and difficult task. This report is intended to provide a comprehensive review of recent developments in genetic and genomic approaches to overcome the hurdle of discovering the protein targets of bioactive small molecules.     

Fully functionalized small-molecule probes for integrated phenotypic screening and target identification

Phenotypic screening offers a powerful approach to identify small molecules that perturb complex biological processes in cells and organisms. The tendency of small molecules, however, to interact with multiple protein targets, often with moderate to weak affinities, along with the lack of straightforward technologies to characterize these interactions in living systems, has hindered efforts to understand the mechanistic basis for pharmacological activity. Here we address this challenge by creating a fully functionalized small-molecule library whose membership is endowed with: (1) one or more diversity elements to promote interactions with different protein targets in cells, (2) a photoreactive group for UV light-induced covalent cross-linking to interacting proteins, and (3) an alkyne handle for reporter tag conjugation to visualize and identify cross-linked proteins. A library member was found to inhibit cancer cell proliferation selectively under nutrient-limiting (low glucose) conditions. Quantitative chemoproteomics identified MT-ND1, an integral membrane subunit of the ～1 MDa NADH:ubiquinone oxidoreductase (complex 1) involved in oxidative phosphorylation, as a specific target of the active probe. We further demonstrated that the active probe inhibits complex 1 activity in vitro (IC(50) = 720 nM), an effect that is known to induce cell death in low-glucose conditions. Based on this proof of principle study, we anticipate that the generation and integration of fully functionalized compound libraries into phenotypic screening programs should facilitate the discovery of bioactive probes that are amenable to accelerated target identification and mechanistic characterization using advanced chemoproteomic technologies.     

Conditional glycosylation in eukaryotic cells using a biocompatible chemical inducer of dimerization

Chemical inducers of dimerization (CIDs) are cell-permeable small molecules capable of dimerizing two protein targets. The most widely used CID, the natural product rapamycin and its relatives, is immunosuppressive due to interactions with endogenous targets and thus has limited utility in vivo. Here we report a new biocompatible CID, Tmp-SLF, which dimerizes E. coli DHFR and FKBP and has no endogenous mammalian targets that would lead to unwanted in vivo side effects. We employed Tmp-SLF to modulate gene expression in a yeast three-hybrid assay. Finally, we engineered the Golgi-resident glycosyltransferase FucT7 for tunable control by Tmp-SLF in mammalian cells.     

Sulfonated molecules that bind a partially structured species of beta2-microglobulin also influence refolding and fibrillogenesis

Human beta2-microglobulin (beta2-m) is a small amyloidogenic protein responsible for dialysis-related amyloidosis, which represents a severe complication of long-term hemodialysis. A therapeutic approach for this amyloidosis could be based on the stabilization of beta2-m through the binding to a small molecule, to possibly inhibit protein misfolding and amyloid fibril formation. The search of a strong ligand of this protein is extremely challenging: by using CE in affinity and refolding experiments we study the effect that previously selected sulfonated molecules have on the equilibrium between the native form and an ensemble of conformers populating the slow phase of beta2-m folding. These data are correlated with the effect that the same molecules exert on in vitro fibrillogenesis experiments.     

Chemo-genetic optimization of DNA recognition by metallodrugs using a presenter-protein strategy

The mode of action of precious metal anticancer metallodrugs is generally believed to involve DNA as a target. However, the poor specificity of such drugs often requires high doses and leads to undesirable side-effects. With the aim of improving the specificity of a ruthenium piano-stool complex towards DNA, we employed a presenter protein strategy based on the biotin-avidin technology. Guided by the X-ray structure of the assembly of streptavidin and a biotinylated piano-stool, we explored the formation of metallodrug-mediated ternary complexes with the presenter protein and DNA. The assemblies bound more strongly to telomere G-quadruplexes than to double-stranded DNA; chemo-genetic modifications (varying the complex or mutating the protein) modulated binding to these targets. We suggest that rational targeting of small molecules by presenter proteins could be exploited to bind metallodrugs to preferred macromolecular targets.     

RNA and the Small Molecule World

A key role in essential cellular processes is played by RNA molecules, and these are attractive targets for drug design. The functional diversity of RNA can be attributed to the sophisticated three-dimensional structures it assumes. These intricate folds create potential binding pockets for ions, low molecular weight ligands, and proteins. Recent experiments have demonstrated that small molecules such as tobramycin ( 1 ) can regulate gene expression in living cells through specific interactions with a messenger RNA (mRNA).     

Characterisation of indomethacin and nifedipine using variable-temperature solid-state NMR

We have characterised the stable polymorphic forms of two drug molecules, indomethacin (1) and nifedipine (2) by 13C CPMAS NMR and the resonances have been assigned. The signal for the C-Cl carbon of indomethacin has been studied as a function of applied magnetic field, and the observed bandshapes have been simulated. Variable-temperature 1H relaxation measurements of static samples have revealed a T1rho minimum for indomethacin at 17.8 degrees C. The associated activation energy is 38 kJ mol(-1). The relevant motion is probably an internal rotation and it is suggested that this involves the C-OCH3 group. Since the two drug compounds are potential candidates for formulation in the amorphous state, we have examined quench-cooled melts in detail by variable-temperature 13C and 1H NMR. There is a change in slope for T1H and T1rhoH at the glass transition temperature (Tg) for indomethacin, but this occurs a few degrees below Tg for nifedipine, which is perhaps relevant to the lower real-time stability of the amorphous form for the latter compound. Comparison of relaxation time data for the crystalline and amorphous forms of each compound reveals a greater difference for nifedipine than for indomethacin, which again probably relates to real-time stabilities. Recrystallisation of the two drugs has been followed by proton bandshape measurements at higher temperatures. It is shown that, under the conditions of the experiments, recrystallisation of nifedipine can be detected already at 70 degrees C, whereas this does not occur until 110 degrees C for indomethacin. The effect of crushing the amorphous samples has been studied by 13C NMR; nifedipine recrystallises but indomethacin does not. The results were supported by DSC, powder XRD, FTIR and solution-state NMR measurements.     

Peptide Targeting of PDZ-Dependent Interactions as Pharmacological Intervention in Immune-Related Diseases

PDZ (postsynaptic density (PSD95), discs large (Dlg), and zonula occludens (ZO-1)-dependent interactions are widely distributed within different cell types and regulate a variety of cellular processes. To date, some of these interactions have been identified as targets of small molecules or peptides, mainly related to central nervous system disorders and cancer. Recently, the knowledge of PDZ proteins and their interactions has been extended to various cell types of the immune system, suggesting that their targeting by viral pathogens may constitute an immune evasion mechanism that favors viral replication and dissemination. Thus, the pharmacological modulation of these interactions, either with small molecules or peptides, could help in the control of some immune-related diseases. Deeper structural and functional knowledge of this kind of protein–protein interactions, especially in immune cells, will uncover novel pharmacological targets for a diversity of clinical conditions.     

Defining the RNA internal loops preferred by benzimidazole derivatives via 2D combinatorial screening and computational analysis

RNA is an important therapeutic target; however, RNA targets are generally underexploited due to a lack of understanding of the small molecules that bind RNA and the RNA motifs that bind small molecules. Herein, we describe the identification of the RNA internal loops derived from a 4096 member 3 × 3 nucleotide loop library that are the most specific and highest affinity binders to a series of four designer, druglike benzimidazoles. These studies establish a potentially general protocol to define the highest affinity and most specific RNA motif targets for heterocyclic small molecules. Such information could be used to target functionally important RNAs in genomic sequence.     

A Whole Proteome Inventory of Background Photocrosslinker Binding

Affinity‐based protein profiling (AfBPP) is a widely applied method for the target identification of bioactive molecules. Probes containing photocrosslinkers, such as benzophenones, diazirines, and aryl azides, irreversibly link the molecule of interest to its target protein upon irradiation with UV light. Despite their prevalent application, little is known about photocrosslinker‐specific off‐targets, affecting the reliability of results. Herein, we investigated background protein labeling by gel‐free quantitative proteomics. Characteristic off‐targets were identified for each photoreactive group and compiled in a comprehensive inventory. In a proof‐of‐principle study, H8 , a protein kinase A inhibitor, was equipped with a diazirine moiety. Application of this photoprobe revealed, by alignment with the diazirine background, unprecedented insight into its in situ proteome targets. Taken together, our findings guide the identification of biologically relevant binders in photoprobe experiments.     

Computer-aided design and discovery of protein–protein interaction inhibitors as agents for anti-HIV therapy

Protein–protein interactions (PPI) are involved in most of the essential processes that occur in organisms. In recent years, PPI have become the object of increasing attention in drug discovery, particularly for anti-HIV drugs. Although the use of combinations of existing drugs, termed highly active antiretroviral therapy (HAART), has revolutionized the treatment of HIV/AIDS, problems with these agents, such as the rapid emergence of drug-resistant HIV-1 mutants and serious adverse effects, have highlighted the need for further discovery of new drugs and new targets. Numerous investigations have shown that PPI play a key role in the virus’s life cycle and that blocking or modulating them has a significant therapeutic potential. Here we summarize the recent progress in computer-aided design of PPI inhibitors, mainly focusing on the selection of the drug targets (HIV enzymes and virus entry machinery) and the utilization of peptides and small molecules to prevent a variety of protein–protein interactions (viral–viral or viral–host) that play a vital role in the progression of HIV infection.     

New Modalities for Challenging Targets in Drug Discovery

Our ever‐increasing understanding of biological systems is providing a range of exciting novel biological targets, whose modulation may enable novel therapeutic options for many diseases. These targets include protein–protein and protein–nucleic acid interactions, which are, however, often refractory to classical small‐molecule approaches. Other types of molecules, or modalities, are therefore required to address these targets, which has led several academic research groups and pharmaceutical companies to increasingly use the concept of so‐called “new modalities”. This Review defines for the first time the scope of this term, which includes novel peptidic scaffolds, oligonucleotides, hybrids, molecular conjugates, as well as new uses of classical small molecules. We provide the most representative examples of these modalities to target large binding surface areas such as those found in protein–protein interactions and for biological processes at the center of cell regulation.     

Identification of the cellular targets of bioactive small organic molecules using affinity reagents

The elucidation of molecular targets of bioactive small organic molecules remains a significant challenge in modern biomedical research and drug discovery. This tutorial review summarizes strategies for the derivatization of bioactive small molecules and their use as affinity probes to identify cellular binding partners. Special emphasis is placed on logistical concerns as well as common problems encountered during such target identification experiments. The roadmap provided is a guide through the process of affinity probe selection, target identification, and downstream target validation.     

Small‐Molecule Control of Intracellular Protein Levels through Modulation of the Ubiquitin Proteasome System

Traditionally, biological probes and drugs have targeted the activities of proteins (such as enzymes and receptors) that can be readily controlled by small molecules. The remaining majority of the proteome has been deemed “undruggable”. By using small‐molecule modulators of the ubiquitin proteasome, protein levels, rather than protein activity, can be targeted instead, thus increasing the number of druggable targets. Whereas targeting of the proteasome itself can lead to a global increase in protein levels, the targeting of other components of the UPS (e.g., the E3 ubiquitin ligases) can lead to an increase in protein levels in a more targeted fashion. Alternatively, multiple strategies for inducing protein degradation with small‐molecule probes are emerging. With the ability to induce and inhibit the degradation of targeted proteins, small‐molecule modulators of the UPS have the potential to significantly expand the druggable portion of the proteome beyond traditional targets, such as enzymes and receptors.     

Snooker: a structure-based pharmacophore generation tool applied to class A GPCRs

G-protein coupled receptors (GPCRs) are important drug targets for various diseases and of major interest to pharmaceutical companies. The function of individual members of this protein family can be modulated by the binding of small molecules at the extracellular side of the structurally conserved transmembrane (TM) domain. Here, we present Snooker, a structure-based approach to generate pharmacophore hypotheses for compounds binding to this extracellular side of the TM domain. Snooker does not require knowledge of ligands, is therefore suitable for apo-proteins, and can be applied to all receptors of the GPCR protein family. The method comprises the construction of a homology model of the TM domains and prioritization of residues on the probability of being ligand binding. Subsequently, protein properties are converted to ligand space, and pharmacophore features are generated at positions where protein ligand interactions are likely. Using this semiautomated knowledge-driven bioinformatics approach we have created pharmacophore hypotheses for 15 different GPCRs from several different subfamilies. For the beta-2-adrenergic receptor we show that ligand poses predicted by Snooker pharmacophore hypotheses reproduce literature supported binding modes for ～75% of compounds fulfilling pharmacophore constraints. All 15 pharmacophore hypotheses represent interactions with essential residues for ligand binding as observed in mutagenesis experiments and compound selections based on these hypotheses are shown to be target specific. For 8 out of 15 targets enrichment factors above 10-fold are observed in the top 0.5% ranked compounds in a virtual screen. Additionally, prospectively predicted ligand binding poses in the human dopamine D3 receptor based on Snooker pharmacophores were ranked among the best models in the community wide GPCR dock 2010.