Sheraz Gul

Identification of Small-Molecule Frequent Hitters from AlphaScreen High-Throughput Screens

Although small-molecule drug discovery efforts have focused largely on enzyme, receptor, and ion-channel targets, there has been an increase in such activities to search for protein-protein interaction (PPI) disruptors by applying high-throughout screening (HTS)–compatible protein-binding assays. However, a disadvantage of these assays is that many primary hits are frequent hitters regardless of the PPI being investigated. We have used the AlphaScreen technology to screen four different robust PPI assays each against 25,000 compounds. These activities led to the identification of 137 compounds that demonstrated repeated activity in all PPI assays. These compounds were subsequently evaluated in two AlphaScreen counter assays, leading to classification of compounds that either interfered with the AlphaScreen chemistry (60 compounds) or prevented the binding of the protein His-tag moiety to nickel chelate (Ni2+-NTA) beads of the AlphaScreen detection system (77 compounds). To further triage the 137 frequent hitters, we subsequently confirmed by a time-resolved fluorescence resonance energy transfer assay that most of these compounds were only frequent hitters in AlphaScreen assays. A chemoinformatics analysis of the apparent hits provided details of the compounds that can be flagged as frequent hitters of the AlphaScreen technology, and these data have broad applicability for users of these detection technologies.

A Bioluminogenic HDAC Activity Assay: Validation and Screening

Histone deacetylase (HDAC) enzymes modify the acetylation state of histones and other important proteins. Aberrant HDAC enzyme function has been implicated in many diseases, and the discovery and development of drugs targeting these enzymes is becoming increasingly important. In this article, the authors report the evaluation of homogeneous, single-addition, bioluminogenic HDAC enzyme activity assays that offer less assay interference by compounds in comparison to fluorescence-based formats. The authors assessed the key operational assay properties including sensitivity, scalability, reproducibility, signal stability, robustness (Z′), DMSO tolerance, and pharmacological response to standard inhibitors against HDAC-1, HDAC-3/NcoR2, HDAC-6, and SIRT-1 enzymes. These assays were successfully miniaturized to a 10 µL assay volume, and their suitability for high-throughput screening was tested in validation experiments using 640 drugs approved by the Food and Drug Administration and the Hypha Discovery MycoDiverse natural products library, which is a collection of 10 049 extracts and fractions from fermentations of higher fungi and contains compounds that are of low molecular weight and wide chemical diversity. Both of these screening campaigns confirmed that the bioluminogenic assay was high-throughput screening compatible and yielded acceptable performance in confirmation, counter, and compound/extract and fraction concentration-response assays.

Nonyloxytryptamine mimics polysialic acid and modulates neuronal and glial functions in cell culture.

Polysialic acid (PSA) is a major regulator of cell–cell interactions in the developing nervous system and in neural plasticity in the adult. As a polyanionic molecule with high water‐binding capacity, PSA increases the intercellular space generating permissive conditions for cell motility. PSA enhances stem cell migration and axon path finding and promotes repair in the lesioned peripheral and central nervous systems, thus contributing to regeneration. As a next step in developing an improved PSA‐based approach to treat nervous system injuries, we searched for small organic compounds that mimic PSA and identified as a PSA mimetic 5‐nonyloxytryptamine oxalate, described as a selective 5‐hydroxytryptamine receptor 1B (5‐HT1B) agonist. Similar to PSA, 5‐nonyloxytryptamine binds to the PSA‐specific monoclonal antibody 735, enhances neurite outgrowth of cultured primary neurons and process formation of Schwann cells, protects neurons from oxidative stress, reduces migration of astrocytes and enhances myelination in vitro. Furthermore, nonyloxytryptamine treatment enhances expression of the neural cell adhesion molecule (NCAM) and its polysialylated form PSA‐NCAM and reduces expression of the microtubule‐associated protein MAP2 in cultured neuroblastoma cells. These results demonstrate that 5‐nonyloxytryptamine mimics PSA and triggers PSA‐mediated functions, thus contributing to the repertoire of molecules with the potential to improve recovery in acute and chronic injuries of the mammalian peripheral and central nervous systems.

Protein–protein interaction modulator drug discovery: past efforts and future opportunities using a rich source of low- and high-throughput screening assays

Introduction: Historically, small-molecule drug discovery projects have largely focused on the G-protein-coupled receptor, ion-channel and enzyme target classes. More recently, there have been successes demonstrating that protein–protein interactions (PPIs) can be targeted by small-molecules and that this strategy has the potential to provide appropriate specificity and selectivity. However, a disadvantage is that compounds that modulate PPIs are often associated with relatively weak affinities as the targeted interaction surfaces are often relatively large. Moreover, from a small-molecule screening perspective, a large proportion of the initial screening Hits are often false positives and these need to be identified and excluded in order to focus on genuine modulators of the PPI being investigated. Areas covered: The authors review previous efforts on PPI modulator drug discovery. Furthermore, they review assays that can be employed in small-molecule screening and/or Hit validation. The PPI assays are categorized as: i) low-throughput target-based biochemical assays, which are primarily employed for Hit validation at the post-screening stage; ii) high-throughput target-based biochemical assays that are suitable for screening campaigns; and iii) cell-based assays, which are suitable for high-throughput screening campaigns and/or Hit validation. Expert opinion: Modulating the interaction of PPIs offers the potential to develop novel drugs to treat a wide range of diseases. New assay technologies are continually being developed and it is anticipated that these will be able to be directly used for small-molecule screening campaigns in the future.

Exemplification of the challenges associated with utilising fluorescence intensity based assays in discovery.

Importance of the field: Despite the advances in the understanding of biological processes, significant challenges still face those engaged in small molecule drug discovery. To complicate matters further, researchers are often overwhelmed with a range of off-the-shelf as well as bespoke assay formats to choose from when initiating a drug discovery programme. Although fluorescence intensity based assays have traditionally been adopted in drug discovery programmes for a wide range of target classes, it is essential to fully validate the chosen readouts to confirm that they accurately reflect the underlying biological mechanism under investigation. Areas covered in this review: This review exemplifies the challenges that are often encountered with fluorescence intensity based assays and particular attention is paid to compound interference, the protease, deacetylating enzyme and kinase enzyme target classes. What the reader will gain: Designing a critical path in early stage drug discovery, which combines several diverse and minimally overlapping readout modes, will maximise the chance that compound activities will translate between the primary assay (utilised in the initial screening campaign) and secondary assay (utilised to evaluate the confirmed hits identified in the primary assay, usually a cell based assay) formats in a meaningful way. However, this is not always the case as is amply demonstrated across both academia and the pharmaceutical industry. Paying insufficient attention to these points can lead to the early termination of drug discovery programmes, not for want of resources or confidence in the rationale underlying the target, but instead because decision making has been driven by assay data originating from a different biological mechanism than the one under investigation. Take home message: Although fluorescence intensity based assays are likely to remain popular for many target classes in drug discovery, in particular in small molecule screening campaigns, it is essential that at the outset they are sufficiently well validated so that compounds are likely to exhibit profiles that are confirmed in subsequent assays.

Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs

Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC50 values against T. brucei below 10 μM. Four X-ray crystal structures and docking studies explained the observed structure-activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 μM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability.

Optimisation and validation of a high throughput screening compatible assay to identify inhibitors of the plasma membrane calcium ATPase pump--a novel therapeutic target for contraception and malaria.

PURPOSE ATPases, which constitute a major category of ion transporters in the human body, have a variety of significant biological and pathological roles. However, the lack of high throughput assays for ATPases has significantly limited drug discovery in this area. We have recently found that the genetic deletion of the ATP dependent calcium pump PMCA4 (plasma membrane calcium/calmodulin dependent ATPase, isoform 4) results in infertility in male mice due to a selective defect in sperm motility. In addition, recent discoveries in humans have indicated that a single nucleotide polymorphism (SNP) in the PMCA4 gene determines the susceptibility towards malaria plasmodium infection. Therefore, there is an urgent need to develop specific PMCA4 inhibitors. In the current study, we aim to optimise and validate a high throughput screening compatible assay using recombinantly expressed PMCA4 and the HTRF® Transcreener® ADP (TR-FRET) assay to screen a drug library. METHODS AND RESULTS PMCA4 membrane microsomes were prepared from HEK293 cells overexpressing PMCA4. Western blot quantification revealed nearly nine-fold increased expression of PMCA4 compared to LacZ (control virus)-infected cells. Maximal PMCA4 microsomal activity was achieved in the TR-FRET assay with 15ng/μl microsomal concentration, 30-minute pre-incubation with compounds at 37°C, and calcium buffering with 1mM EGTA providing 1μM free-calcium. Finally a dose-response curve for carboxyeosin (a non-specific PMCA inhibitor) under optimised conditions showed significant PMCA4 inhibition. Upon confirmation that the assay was suitable for high-throughput screening, we have screened the ChemBioNet small molecule library (~21,000 compounds) against the PMCA4 assay to identify those that are its apparent inhibitors. This screening yielded 1,494 primary hits. CONCLUSIONS We have optimised the HTRF® Transcreener® ADP assay for high-throughput screening to identify PMCA4 inhibitors. The output of the screening campaign has provided preliminary chemical starting points that could be further developed to specific PMCA4 inhibitors for non-hormonal contraception or anti-malaria therapy.

Temperature-dependences of the kinetics of reactions of papain and actinidin with a series of reactivity probes differing in key molecular recognition features

The temperature-dependences of the second-order rate constants (k) of the reactions of the catalytic site thiol groups of two cysteine peptidases papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) with a series of seven 2-pyridyl disulphide reactivity probes (R-S-S-2-Py, in which R provides variation in recognition features) were determined at pH 6.7 at temperatures in the range 4-30 degrees C by stopped-flow methodology and were used to calculate values of DeltaS++, DeltaH++ and DeltaG++. The marked changes in DeltaS++ from negative to positive in the papain reactions consequent on provision of increase in the opportunities for key non-covalent recognition interactions may implicate microsite desolvation in binding site-catalytic site signalling to provide a catalytically relevant transition state. The substantially different behaviour of actinidin including apparent masking of changes in DeltaH++ by an endothermic conformational change suggests a difference in mechanism involving kinetically significant conformational change.

Identification of Small-Molecule Frequent Hitters of Glutathione S-Transferase–Glutathione Interaction

In high-throughput screening (HTS) campaigns, the binding of glutathione S-transferase (GST) to glutathione (GSH) is used for detection of GST-tagged proteins in protein-protein interactions or enzyme assays. However, many false-positives, so-called frequent hitters (FH), arise that either prevent GST/GSH interaction or interfere with assay signal generation or detection. To identify GST-FH compounds, we analyzed the data of five independent AlphaScreen-based screening campaigns to classify compounds that inhibit the GST/GSH interaction. We identified 53 compounds affecting GST/GSH binding but not influencing His-tag/Ni2+-NTA interaction and general AlphaScreen signals. The structures of these 53 experimentally identified GST-FHs were analyzed in chemoinformatic studies to categorize substructural features that promote interference with GST/GSH binding. Here, we confirmed several existing chemoinformatic filters and more importantly extended them as well as added novel filters that specify compounds with anti–GST/GSH activity. Selected compounds were also tested using different antibody-based GST detection technologies and exhibited no interference clearly demonstrating specificity toward their GST/GSH interaction. Thus, these newly described GST-FH will further contribute to the identification of FH compounds containing promiscuous substructures. The developed filters were uploaded to the OCHEM website (http://ochem.eu) and are publicly accessible for analysis of future HTS results.

Evidence for ‘lock and key’ character in an anti-phosphonate hydrolytic antibody catalytic site augmented by non-reaction centre recognition: variation in substrate selectivity between an anti-phosphonate antibody, an anti-phosphate antibody and two hydrolytic enzymes

The substrate selectivities of an anti-phosphonate and an anti-phosphate kinetically homogeneous polyclonal catalytic antibody preparation and two hydrolytic enzymes were compared by using hapten-analogous and truncated carbonate and ester substrates each containing a 4-nitrophenolate leaving group. Syntheses of the truncated substrates devoid of recognition features in the non-leaving group parts of the substrates are reported. The relatively high kinetic selectivity of the more active anti-phosphonate antibody preparation is considered to depend on a relatively rigid catalytic site with substantial reaction centre specificity together with other important recognition interactions with the extended non-leaving group part of the substrate. In contrast, the less catalytically active, more flexible anti-phosphate antibody exhibits much lower kinetic selectivity for the substrate reaction centre comparable with that of the hydrolytic enzymes with activity much less dependent on recognition interactions with the non-leaving group part of the substrate. The ways in which haptenic flexibility and IgG architecture might contribute to the differential kinetic selectivities are indicated.