Erik Gilberg

Identification and analysis of promiscuity cliffs formed by bioactive compounds and experimental implications

Multi-target activities of small molecules must be distinguished from apparent promiscuity resulting from assay artifacts. The molecular origins of specific multi-target activities are currently poorly understood. Compounds from the medicinal chemistry literature with available high-confidence activity data were systematically searched for ‘promiscuity cliffs’, defined as pairs of structural analogs with large differences between the number of targets they are active against. During the search, compounds with detectable aggregator properties, pan-assay interference characteristics, or other possible chemical liabilities were eliminated. A large number of promiscuity cliffs remained, many of which were centered on a limited number of highly promiscuous compounds, as revealed by network representations. The analysis of promiscuity cliffs often suggested follow-up experiments to further explore the molecular basis of promiscuity and assess the influence of data sparseness. Therefore, promiscuity cliffs identified herein are made freely available to support follow-up investigations.

Activity profiles of analog series containing pan assay interference compounds

Activity artifacts in assays present a major problem for biological screening and medicinal chemistry. Such artifacts are often caused by compounds that form aggregates or are reactive under assay conditions. Many pan assay interference compounds (PAINS) have been proposed to cause false-positive assay readouts. PAINS are typically contained as substructures in larger molecules. They are used as computational filters to detect compounds with potential chemical liabilities. Recent studies have shown that molecules containing the same PAINS substructure often have greatly varying hit rates in screening assays. Even the overall most frequently active PAINS substructures are found in compounds that are only rarely active or consistently inactive in many assays they are tested in. These observations suggest that the structural context in which PAINS are presented may play an important role for eliciting false-positive activities. However, this assumption remains to be investigated. Herein, we report the systematic identification of analog series of screening compounds that contain PAINS or exclusively consist of PAINS and the analysis of their activity profiles. Comparison of analogs or different series of analogs containing the same PAINS substructure provides structural context information. For many PAINS, extensively tested series with distinct activity profiles were detected. Furthermore, analogs within the same series often displayed significant differences in hit rates. The analog series reported herein organize PAINS in different structural contexts. Their activity profiles provide many opportunities for experimental follow-up investigations to better understand PAINS characteristics.

Structure-Promiscuity Relationship Puzzles—Extensively Assayed Analogs with Large Differences in Target Annotations

Publicly available screening data were systematically searched for extensively assayed structural analogs with large differences in the number of targets they were active against. Screening compounds with potential chemical liabilities that may give rise to assay artifacts were identified and excluded from the analysis. “Promiscuity cliffs” were frequently identified, defined here as pairs of structural analogs with a difference of at least 20 target annotations across all assays they were tested in. New assay indices were introduced to prioritize cliffs formed by screening compounds that were extensively tested in comparably large numbers of assays including many shared assays. In these cases, large differences in promiscuity degrees were not attributable to differences in assay frequency and/or lack of assay overlap. Such analog pairs have high priority for further exploring molecular origins of multi-target activities. Therefore, these promiscuity cliffs and associated target annotations are made freely available. The corresponding analogs often represent equally puzzling and interesting examples of structure-promiscuity relationships.

Towards a systematic assessment of assay interference: Identification of extensively tested compounds with high assay promiscuity

A large-scale statistical analysis of hit rates of extensively assayed compounds is presented to provide a basis for a further assessment of assay interference potential and multi-target activities. A special feature of this investigation has been the inclusion of compound series information in activity analysis and the characterization of analog series using different parameters derived from assay statistics. No prior knowledge of compounds or targets was taken into consideration in the data-driven study of analog series. It was anticipated that taking large volumes of activity data, assay frequency, and assay overlap information into account would lead to statistically sound and chemically meaningful results. More than 6000 unique series of analogs with high hit rates were identified, more than 5000 of which did not contain known interference candidates, hence providing ample opportunities for follow-up analyses from a medicinal chemistry perspective.

Evaluation of bisbenzamidines as inhibitors for matriptase-2

The serine protease matriptase-2 has attracted much attention as a potential target for the treatment of iron overload diseases. In this study, a series of 27 symmetric, achiral bisbenzamidines was evaluated for inhibitory activity against human matriptase-2, against the closely related enzyme human matriptase, as well as against human thrombin, bovine factor Xa and human trypsin. The conformationally restricted piperazine derivative 19 and the oxamide-derived bisbenzamidine 1 were identified as the most potent inhibitors of this series for matriptase-2 and matriptase, respectively.

X-ray-Structure-Based Identification of Compounds with Activity against Targets from Different Families and Generation of Templates for Multitarget Ligand Design

Compounds with multitarget activity (promiscuity) are increasingly sought in drug discovery. However, promiscuous compounds are often viewed controversially in light of potential assay artifacts that may give rise to false-positive activity annotations. We have reasoned that the strongest evidence for true multitarget activity of small molecules would be provided by experimentally determined structures of ligand–target complexes. Therefore, we have carried out a systematic search of currently available X-ray structures for compounds forming complexes with different targets. Rather unexpectedly, 1418 such crystallographic ligands were identified, including 702 that formed complexes with targets from different protein families (multifamily ligands). About half of these multifamily ligands originated from the medicinal chemistry literature, making it possible to consider additional target annotations and search for analogues. From 168 distinct series of analogues containing one or more multifamily ligands, 133 unique analogue-series-based scaffolds were isolated that can serve as templates for the design of new compounds with multitarget activity. As a part of our study, all of the multifamily ligands we have identified and the analogue-series-based scaffolds are made freely available.

X-ray Structures of Target–Ligand Complexes Containing Compounds with Assay Interference Potential

Pan assay interference compounds (PAINS) have become a paradigm for compound classes that might cause artifacts in biological assays. PAINS-defining substructures are typically contained in larger compounds. We have systematically examined X-ray structures of protein-ligand complexes for compounds containing PAINS motifs. In 2874 X-ray structures, 1107 unique ligands with PAINS substructures belonging to 70 different classes were identified. PAINS most frequently detected in crystallographic ligands included a number of prominent candidates such as quinones, catechols, or Mannich bases. However, on the basis of X-ray data, the presence of specific ligand-target interactions and reactivity under assay conditions were not mutually exclusive. In some instances, reactivity of ligands was likely responsible for complex formation. Different categories of PAINS-containing ligands were distinguished, which aided in the interpretation of specific interactions versus potential assay artifacts. Careful consideration of structural data adds another dimension to the analysis of interference compounds.

Series of screening compounds with high hit rates for the exploration of multi-target activities and assay interference

Aim: Generation of a database of analog series (ASs) with high assay hit rates for the exploration of assay interference and multi-target activities of compounds. Methodology: ASs were computationally extracted from extensively tested screening compounds with high hit rates. Data: A total of 6941 ASs were assembled comprising 14,646 unique compounds that were tested in a total of 1241 different assays covering 426 specified targets. These ASs were organized and prioritized on the basis of different activity and assay frequency criteria. All ASs and associated information are made available in an open access deposition. Next steps: The large set of ASs will be further analyzed computationally and from a chemical perspective to identify assay interference compounds and candidates for exploring target promiscuity.

Cathepsin B: Active Site Mapping with Peptidic Substrates and Inhibitors

The potential of papain-like cysteine proteases, such as cathepsin B, as drug discovery targets for systemic human diseases has prevailed over the past years. The development of potent and selective low-molecular cathepsin B inhibitors relies on the detailed expertise on preferred amino acid and inhibitor residues interacting with the corresponding specificity pockets of cathepsin B. Such knowledge might be obtained by mapping the active site of the protease with combinatorial libraries of peptidic substrates and peptidomimetic inhibitors. This review, for the first time, summarizes a wide spectrum of active site mapping approaches. It considers relevant X-ray crystallographic data and discloses propensities towards favorable protein-ligand interactions in case of the therapeutically relevant protease cathepsin B.

Design of an Activity-Based Probe for Human Neutrophil Elastase: Implementation of the Lossen Rearrangement To Induce Förster Resonance Energy Transfers

Human neutrophil elastase is an important regulator of the immune response and plays a role in host defense mechanisms and further physiological processes. The uncontrolled activity of this serine protease may cause severe tissue alterations and impair inflammatory states. The design of an activity-based probe for human neutrophil elastase reported herein relies on a sulfonyloxyphthalimide moiety as a new type of warhead that is linker-connected to a coumarin fluorophore. The inhibitory potency of the activity-based probe was assessed against several serine and cysteine proteases, and the selectivity for human neutrophil elastase (Ki = 6.85 nM) was determined. The adequate fluorescent tag of the probe allowed for the in-gel fluorescence detection of human neutrophil elastase in the low nanomolar range. The coumarin moiety and the anthranilic acid function of the probe, produced in the course of a Lossen rearrangement, were part of two different Förster resonance energy transfers.