Multi‐Objective Molecular De Novo Design by Adaptive Fragment Prioritization

We present the development and application of a computational molecular de novo design method for obtaining bioactive compounds with desired on‐ and off‐target binding. The approach translates the nature‐inspired concept of ant colony optimization to combinatorial building block selection. By relying on publicly available structure–activity data, we developed a predictive quantitative polypharmacology model for 640 human drug targets. By taking reductive amination as an example of a privileged reaction, we obtained novel subtype‐selective and multitarget‐modulating dopamine D₄ antagonists, as well as ligands selective for the sigma‐1 receptor with accurately predicted affinities. The nanomolar potencies of the hits obtained, their high ligand efficiencies, and an overall success rate of 90 % demonstrate that this ligand‐based computer‐aided molecular design method may guide target‐focused combinatorial chemistry.     

Binary quantitative structure-activity relationship (QSAR) analysis of estrogen receptor ligands

The use of high throughput screening (HTS) to identify lead compounds has greatly challenged conventional quantitative structure-activity relationship (QSAR) techniques that typically correlate structural variations in similar compounds with continuous changes in biological activity. A new QSAR-like methodology that can correlate less quantitative assay data (i.e., "active" versus "inactive"), as initially generated by HTS, has been introduced. In the present study, we have, for the first time, applied this approach to a drug discovery problem; that is, the study of the estrogen receptor ligands. The binding affinities of 463 estrogen analogues were transformed into a binary data format, and a predictive binary QSAR model was derived using 410 estrogen analogues as a training set. The model was applied to predict the activity of 53 estrogen analogues not included in the training set. An overall accuracy of 94% was obtained.     

Biosensor-based small molecule fragment screening with biolayer interferometry

Biosensor-based fragment screening is a valuable tool in the drug discovery process. This method is advantageous over many biochemical methods because primary hits can be distinguished from non-specific or non-ideal interactions by examining binding profiles and responses, resulting in reduced false-positive rates. Biolayer interferometry (BLI), a technique that measures changes in an interference pattern generated from visible light reflected from an optical layer and a biolayer containing proteins of interest, is a relatively new method for monitoring small molecule interactions. The BLI format is based on a disposable sensor that is immersed in 96-well or 384-well plates. BLI has been validated for small molecule detection and fragment screening with model systems and well-characterized targets where affinity constants and binding profiles are generally similar to those obtained with surface plasmon resonsance (SPR). Screens with challenging targets involved in protein–protein interactions including BCL-2, JNK1, and eIF4E were performed with a fragment library of 6,500 compounds, and hit rates were compared for these targets. For eIF4E, a protein containing a PPI site and a nucleotide binding site, results from a BLI fragment screen were compared to results obtained in biochemical HTS screens. Overlapping hits were observed for the PPI site, and hits unique to the BLI screen were identified. Hit assessments with SPR and BLI are described.     

Development and validation of a fluorescence-based HTS assay for the identification of P/Q-type calcium channel blockers

Dysfunction of P/Q-type calcium channels is thought to underlie a variety of neurological diseases. There is evidence that migraine, Alzheimer's disease, and epilepsy involve a gain-of-function of the channel, leading to abnormal presynaptic vesicle release. P/Q-channel blockers may normalize current flow and consequently lead to an alleviation of disease symptoms. Although the medical need is high, there are no such compounds on the market. Here we describe a high throughput screen (HTS) for P/Q-type calcium channel blockers and the confirmation of hits by automated electrophysiology. We generated a HEK293 cell line stably expressing the α1A subunit of the P/Q-type calcium channel under control of a tetracycline (Tet) promoter. The accessory β1.1 and α2δ1 subunits were co-expressed constitutively. The cell line was pharmacologically characterized by ion channel specific modulators, and revealed functional P/Q-type calcium currents. Using a fluorescence imaging plate reader (FLIPR), an assay for P/Q-type calcium channels was established based on a calcium sensitive dye. HTS of a 150,000 compound-containing sub-library led to the identification of 3262 hits that inhibited the fluorescence signal with potencies below 10 μM. Hit-to-lead (HTL) efforts identified 12,400 analogues. Compounds were clustered into 37 series, and 8 series of interest were prioritized. An electrophysiological secondary screen, providing a more direct measure of channel function, was implemented into the HTL process. 27 selected exemplars of different chemotypes were validated by automated whole-cell patch clamp analysis at inactivated channel state. The discovery of P/Q-channel blockers may foster the development of new therapeutics for a variety of neurological diseases.     

On‐Bead Screens Sample Narrower Affinity Ranges of Protein–Ligand Interactions Compared to Equivalent Solution Assays

Conceptually, on‐bead screening is one of the most efficient high‐throughput screening (HTS) methods. One of its inherent advantages is that the solid support has a dual function: it serves as a synthesis platform and as a screening compartment. Compound purification, cleavage and storage and extensive liquid handling are not necessary in bead‐based HTS. Since the establishment of one‐bead one‐compound library synthesis, the properties of polymer beads in chemical reactions have been thoroughly investigated. However, the characterization of the kinetics and thermodynamics of protein–ligand interactions on the beads used for screening has received much less attention. Consequently, the majority of reported on‐bead screens are based on empirically derived procedures, independent of measured equilibrium constants and rate constants of protein binding to ligands on beads. More often than not, on‐bead screens reveal apparent high affinity binders through strong protein complexation on the matrix of the solid support. After decoding, resynthesis, and solution testing the primary hits turn out to be unexpectedly weak binders, or may even fall out of the detection limit of the solution assay. Only a quantitative comparison of on‐bead binding and solution binding events will allow systematically investigating affinity differences as function of protein and small molecule properties. This will open up routes for optimized bead materials, blocking conditions and other improved assay procedures. By making use of the unique features of our previously introduced confocal nanoscanning (CONA) method, we investigated the kinetic and thermodynamic properties of protein–ligand interactions on TentaGel beads, a popular solid support for on‐bead screening. The data obtained from these experiments allowed us to determine dissociation constants for the interaction of bead‐immobilized ligands with soluble proteins. Our results therefore provide, for the first time, a comparison of on‐bead versus solution binding thermodynamics. Our data indicate that affinity ranges found in on‐bead screening are indeed narrower compared to equivalent interactions in homogeneous solution. A thorough physico‐chemical understanding of the molecular recognition between proteins and surface bound ligands will further strengthen the role of on‐bead screening as an ultimately cost‐effective method in hit and lead finding.     

ADLOC: An Aptamer‐Displacement Assay Based on Luminescent Oxygen Channeling

Functional nucleic acids, such as aptamers and allosteric ribozymes, can sense their ligands specifically, thereby undergoing structural alterations that can be converted into a detectable signal. The direct coupling of molecular recognition to signal generation enables the production of versatile reporters that can be applied as molecular probes for various purposes, including high‐throughput screening. Here we describe an unprecedented type of a nucleic acid‐based sensor system and show that it is amenable to high‐throughput screening (HTS) applications. The approach detects the displacement of an aptamer from its bound protein partner by means of luminescent oxygen channeling. In a proof‐of‐principle study we demonstrate that the format is feasible for efficient identification of small drug‐like molecules that bind to a protein target, in this case to the Sec7 domain of cytohesin. We extended the approach to a new cytohesin‐specific single chain DNA aptamer, C10.41, which exhibits a similar binding behavior to cytohesins but has the advantage of being more stable and easier to synthesize and to modify than the RNA‐aptamer M69. The results obtained with both aptamers indicate the general suitability of the aptamer‐displacement assay based on luminescent oxygen channelling (ADLOC) for HTS. We also analyzed the potential for false positive hits and identified from a library of 18 000 drug‐like small molecules two compounds as strong singlet‐oxygen quenchers. With full automation and the use of commercially available plate readers, we estimate that the ADLOC‐based assay described here could be used to screen at least 100 000 compounds per day.     

Fragment-based drug design: computational & experimental state of the art

Fragment-based screening is an emerging technology which is used as an alternative to high-throughput screening (HTS), and often in parallel. Fragment screening focuses on very small compounds. Because of their small size and simplicity, fragments exhibit a low to medium binding affinity (mM to μM) and must therefore be screened at high concentration in order to detect binding events. Since some issues are associated with high-concentration screening in biochemical assays, biophysical methods are generally employed in fragment screening campaigns. Moreover, these techniques are very sensitive and some of them can give precise information about the binding mode of fragments, which facilitates the mandatory hit-to-lead optimization. One of the main advantages of fragment-based screening is that fragment hits generally exhibit a strong binding with respect to their size, and their subsequent optimization should lead to compounds with better pharmacokinetic properties compared to molecules evolved from HTS hits. In other words, fragments are interesting starting points for drug discovery projects. Besides, the chemical space of low-complexity compounds is very limited in comparison to that of drug-like molecules, and thus easier to explore with a screening library of limited size. Furthermore, the "combinatorial explosion" effect ensures that the resulting combinations of interlinked binding fragments may cover a significant part of "drug-like" chemical space. In parallel to experimental screening, virtual screening techniques, dedicated to fragments or wider compounds, are gaining momentum in order to further reduce the number of compounds to test. This article is a review of the latest news in both experimental and in silico virtual screening in the fragment-based discovery field. Given the specificity of this journal, special attention will be given to fragment library design.     

Fluorine-NMR competition binding experiments for high-throughput screening of large compound mixtures

High-throughput ligand-based NMR screening with competition binding experiments is extended to (19)F detection. Fluorine is a favorable nucleus for these experiments because of the significant contribution of the Chemical Shift Anisotropy (CSA) to the (19)F transverse relaxation of the ligand signal when bound to a macromolecular target. A low to moderate affinity ligand containing a fluorine atom is used as a reference molecule for the detection and characterization of new ligands. Titration NMR experiments with the selected reference compound are performed for finding the optimal set-up conditions for HTS and for deriving the binding constants of the identified NMR hits. Rapid HTS of large chemical mixtures and plant or fungi extracts against the receptor of interest is possible due to the high sensitivity of the (19)F nucleus and the absence of overlap with the signals of the mixtures to be screened. Finally, a novel approach for HTS using a reference molecule in combination with a control molecule is presented.     

Thermodynamical studies of designed ligands binding to Cel7A using partial-filling capillary electrophoresis

A convenient experimental method for thermodynamical studies based on partial-filling affinity CE is presented. The advantages of this approach are the possibility to determine binding energies from relatively weak interactions as well as the small amounts of samples consumed. In order to explore the affinity and selectivity of the cellobiohydrolase Cel7A, a number of propranolol analogues were recently designed. The affinities of a selection of these ligands were determined in the temperature interval 15-40 degrees C, and DeltaG degrees , DeltaH degrees and DeltaS degrees were obtained by means of Van't Hoff plots. Through these experiments, the importance of the entropy contribution in the complexation between the ligands and Cel7A has been demonstrated.     

Exploring the active site of human factor Xa protein by NMR screening of small molecule probes

A collection of small molecules (MW < 350 Da) was screened for binding to human factor Xa using saturation transfer difference NMR spectroscopy to detect binding. The NMR screening experiments identified four hits. Binding isotherms constructed from NMR linewidth data showed that the binding affinities of the hits were all in the 30-210 microM range. Competition binding experiments showed that three of the ligands were displaced by a known microM inhibitor of factor Xa. The success of the method for identifying new ligands and the relevance of this information to the design of new factor Xa inhibitors are discussed.     

NIR-mbc94, a fluorescent ligand that binds to endogenous CB(2) receptors and is amenable to high-throughput screening

High-throughput screening (HTS) of chemical libraries is often used for the unbiased identification of compounds interacting with G protein-coupled receptors (GPCRs), the largest family of therapeutic targets. However, current HTS methods require removing GPCRs from their native environment, which modifies their pharmacodynamic properties and biases the screen toward false positive hits. Here, we developed and validated a molecular imaging (MI) agent, NIR-mbc94, which emits near infrared (NIR) light and selectively binds to endogenously expressed cannabinoid CB(2) receptors,?a recognized target for treating autoimmune diseases, chronic pain and cancer. The precision and ease of this assay allows for the HTS of compounds interacting with CB(2) receptors expressed in their native environment.     

PowerMV: a software environment for molecular viewing, descriptor generation, data analysis and hit evaluation

Ideally, a team of biologists, medicinal chemists and information specialists will evaluate the hits from high throughput screening. In practice, it often falls to nonmedicinal chemists to make the initial evaluation of HTS hits. Chemical genetics and high content screening both rely on screening in cells or animals where the biological target may not be known. There is a need to place active compounds into a context to suggest potential biological mechanisms. Our idea is to build an operating environment to help the biologist make the initial evaluation of HTS data. To this end the operating environment provides viewing of compound structure files, computation of basic biologically relevant chemical properties and searching against biologically annotated chemical structure databases. The benefit is to help the nonmedicinal chemist, biologist and statistician put compounds into a potentially informative biological context. Although there are several similar public and private programs used in the pharmaceutical industry to help evaluate hits, these programs are often built for computational chemists. Our program is designed for use by biologists and statisticians.     

Identification of Novel Antagonists Targeting Cannabinoid Receptor 2 Using a Multi-Step Virtual Screening Strategy

The endocannabinoid system plays an essential role in the regulation of analgesia and human immunity, and Cannabinoid Receptor 2 (CB2) has been proved to be an ideal target for the treatment of liver diseases and some cancers. In this study, we identified CB2 antagonists using a three-step “deep learning–pharmacophore–molecular docking” virtual screening approach. From the ChemDiv database (1,178,506 compounds), 15 hits were selected and tested by radioligand binding assays and cAMP functional assays. A total of 7 out of the 15 hits were found to exhibit binding affinities in the radioligand binding assays against CB2 receptor, with a pK_i of 5.15–6.66, among which five compounds showed antagonistic activities with pIC₅₀ of 5.25–6.93 in the cAMP functional assays. Among these hits, Compound 8 with the 4H-pyrido[1,2-a]pyrimidin-4-one scaffold showed the best binding affinity and antagonistic activity with a pK_i of 6.66 and pIC₅₀ of 6.93, respectively. The new scaffold could serve as a lead for further development of CB2 drugs. Additionally, we hope that the model in this study could be further utilized to identify more novel CB2 receptor antagonists, and the developed approach could also be used to design potent ligands for other therapeutic targets.     

ALARM NMR: a rapid and robust experimental method to detect reactive false positives in biochemical screens

High-throughput screening (HTS) of large compound collections typically results in numerous small molecule hits that must be carefully evaluated to identify valid drug leads. Although several filtering mechanisms and other tools exist that can assist the chemist in this process, it is often the case that costly synthetic resources are expended in pursuing false positives. We report here a rapid and reliable NMR-based method for identifying reactive false positives including those that oxidize or alkylate a protein target. Importantly, the reactive species need not be the parent compound, as both reactive impurities and breakdown products can be detected. The assay is called ALARM NMR (a La assay to detect reactive molecules by nuclear magnetic resonance) and is based on monitoring DTT-dependent (13)C chemical shift changes of the human La antigen in the presence of a test compound or mixture. Extensive validation has been performed to demonstrate the reliability and utility of using ALARM NMR to assess thiol reactivity. This included comparing ALARM NMR to a glutathione-based fluorescence assay, as well as testing a collection of more than 3500 compounds containing HTS hits from 23 drug targets. The data show that current in silico filtering tools fail to identify more than half of the compounds that can act via reactive mechanisms. Significantly, we show how ALARM NMR data has been critical in identifying reactive compounds that would otherwise have been prioritized for lead optimization. In addition, a new filtering tool has been developed on the basis of the ALARM NMR data that can augment current in silico programs for identifying nuisance compounds and improving the process of hit triage.     

Application of affinity selection-mass spectrometry assays to purification and affinity-based screening of the chemokine receptor CXCR4

Affinity selection-mass spectrometry (AS-MS) is a sensitive technology for identifying small molecules that bind to target proteins, and assays enabled by AS-MS can be used to delineate relative binding affinities of ligands for proteins. 'Indirect' AS-MS assays employ size-exclusion techniques to separate target-ligand complexes from unbound ligands, and target-associated ligands are then specifically detected by liquid chromatography mass spectrometry. We report how indirect AS-MS binding assays with known reference control compounds were used as guideposts for development of an optimized purification method for CXCR4, a G-protein coupled chemokine receptor, for which we sought novel antagonists. The CXCR4 purification method that was developed was amenable to scale-up and enabled the screening of purified recombinant human CXCR4 against a large combinatorial library of small molecules by high throughput indirect AS-MS. The screen resulted in the discovery of new ligands that competed off binding of reference compounds to CXCR4 in AS-MS binding assays and that antagonized SDF1α-triggered responses and CXCR4-mediated HIV1 viral uptake in cell-based assays. This report provides a methodological paradigm whereby indirect AS-MS-based ligand binding assays may be used to guide optimal integral membrane protein purification methods that enable downstream affinity selection-based applications such as high throughput AS-MS screens.     

Search of ligands for the amyloidogenic protein beta2-microglobulin by capillary electrophoresis and other techniques

Beta2-microglobulin (beta2-m) is a small amyloidogenic protein normally present on the surface of most nucleated cells and 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, and consequent inhibition of protein misfolding and amyloid fibril formation. A few compounds have been described to weakly bind beta2-m, including the drug suramin. The lack of a binding site for nonpolypeptidic ligands on the beta2-m structure makes it difficult for both the identification of functional groups responsible for the binding and the search of hits to be optimized. The characterization of the binding properties of suramin for beta2-m by using three different techniques (surface plasmon resonance, affinity CE (ACE), ultrafiltration) is here described and the results obtained are compared. The common features of the chemical structures of the compounds known to bind the protein led us to select 200 sulfonated/suramin-like molecules from a wider chemical library on the basis of similarity rules, so as to possibly single out some interesting hits and to gain more information on the functional groups involved in the binding. The development of screening methods to test the compounds by using ultrafiltration and ACE is described.     

Unexpected relative aqueous solubilities of a phosphotyrosine analogue and two phosphonate derivatives

Phosphotyrosine (pTyr) is an essential component of biological signaling, often being a determinant of protein-protein interactions. Accordingly, a number of drug discovery efforts targeting signal transduction pathways have included phosphotyrosine and analogues as essential components of the lead compounds. Toward the goal of improved biological efficacy, the phosphonate and difluoro phosphonate analogues of pTyr have been employed in inhibitor design because of their stability to hydrolysis and enhanced binding affinity in certain cases. To quantitate the contribution of aqueous solubility of pTyr, phosphonomethyl phenylalanine (Pmp), and difluorophosphonomethyl phenylalanine (F(2)Pmp) to their relative binding affinities, free energy perturbation calculations were undertaken on the mimetics phenol phosphate (PP), benzyl phosphonate (BP), and difluorobenzyl phosphonate (F(2)BP), including development of empirical force field parameters compatible with the CHARMM all-atom force fields. Notably, it is shown that the most favorably solvated compound of the series is BP, followed by PP, with F(2)BP the least favorably solvated for both the mono- and dianionic forms of the compounds. The molecular origin of this ordering is shown to be due to changes in charge distribution, in the comparatively larger size of the fluorine atoms, as well as in differences of local solvation between PP and BP. The implications of the differences in aqueous solubility toward the relative binding potencies of pTyr-, Pmp-, and F(2)Pmp-containing peptide ligands are discussed. Our results indicate that one general principle explaining the efficacy of selective fluorination to enhance binding affinities may lie in the ability of fluorine atoms to increase the hydrophobicity of a ligand while maintaining its capability to form hydrogen bonds.     

Common and divergent structural features of a series of corticotropin releasing factor-related peptides

Members of the corticoliberin family include the corticotropin releasing factors (CRFs), sauvagine, the urotensins, and urocortin 1 (Ucn1), which bind to both the CRF receptors CRF-R1 and CRF-R2, and the urocortins 2 (Ucn2) and 3 (Ucn3), which are selective agonists of CRF-R2. Structure activity relationship studies led to several potent and long-acting analogues with selective binding to either one of the receptors. NMR structures of six ligands of this family (the antagonists astressin B and astressin2-B, the agonists stressin1, and the natural ligands human Ucn1, Ucn2, and Ucn3) were determined in DMSO. These six peptides show differences in binding affinities, receptor-selectivity, and NMR structure. Overall, their backbones are alpha-helical, with a small kink or a turn around residues 25-27, resulting in a helix-loop-helix motif. The C-terminal helices are of amphipathic nature, whereas the N-terminal helices vary in their amphipathicity. The C-terminal helices thereby assume a conformation very similar to that of astressin bound to the ECD1 of CRF-R2 recently reported by our group.1 On the basis of an analysis of the observed 3D structures and relative potencies of [Ala]-substituted analogues, it is proposed that both helices could play a crucial role in receptor binding and selectivity. In conclusion, the C-terminal helices may interact along their hydrophobic faces with the ECD1, whereas the entire N-terminal helical surface may be involved in receptor activation. On the basis of the common and divergent features observed in the 3D structures of these ligands, multiple binding models are proposed that may explain their plurality of actions.     

Analysis of a high-throughput screening data set using potency-scaled molecular similarity algorithms

Molecular similarity methods for ligand-based virtual screening (VS) generally do not take compound potency as a variable or search parameter into account. We have incorporated a logarithmic potency scaling function into two conceptually distinct VS algorithms to account for relative compound potency during search calculations. A high-throughput screening (HTS) data set containing cathepsin B inhibitors was analyzed to evaluate the effects of potency scaling. Sets of template compounds were randomly selected from the HTS data and used to search for hits having varying potency levels in the presence or absence of potency scaling. Enrichment of potent compounds in small subsets of the HTS data set was observed as a consequence of potency scaling. In part, observed enrichments could be rationalized as a result of recentering chemical reference space on a subspace populated by potent compounds. Our findings suggest that VS calculations using multiple reference compounds can be directed toward the preferential detection of potent database hits by scaling compound contributions according to potency differences.