N-Sulfonyl dipeptide nitriles as inhibitors of human cathepsin S: In silico design, synthesis and biochemical characterization.

A library of cathepsin S inhibitors of the dipeptide nitrile chemotype, bearing a bioisosteric sulfonamide moiety, was synthesized. Kinetic investigations were performed at four human cysteine proteases, i.e. cathepsins S, B, K and L. Compound 12 with a terminal 3-biphenyl sulfonamide substituent was the most potent (Ki = 4.02 nM; selectivity ratio cathepsin S/K = 5.8; S/L = 67) and 24 with a 4'-fluoro-4-biphenyl sulfonamide substituent the most selective cathepsin S inhibitor (Ki = 35.5 nM; selectivity ratio cathepsin S/K = 57; S/L = 31). In silico design and biochemical evaluation emphasized the impact of the sulfonamide linkage on selectivity and a possible switch of P2 and P3 substituents with respect to the occupation of the corresponding binding sites of cathepsin S.

Mapping the S1 and S1' subsites of cysteine proteases with new dipeptidyl nitrile inhibitors as trypanocidal agents.

The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. A series of 26 new compounds were designed, synthesized, and tested against the recombinant cruzain (Cz) to map its S1/S1´ subsites. The same series was evaluated on a panel of four human cysteine proteases (CatB, CatK, CatL, CatS) and Leishmania mexicana CPB, which is a potential target for the treatment of cutaneous leishmaniasis. The synthesized compounds are dipeptidyl nitriles designed based on the most promising combinations of different moieties in P1 (ten), P2 (six), and P3 (four different building blocks). Eight compounds exhibited a Ki smaller than 20.0 nM for Cz, whereas three compounds met these criteria for LmCPB. Three inhibitors had an EC50 value of ca. 4.0 µM, thus being equipotent to benznidazole according to the antitrypanosomal effects. Our mapping approach and the respective structure-activity relationships provide insights into the specific ligand-target interactions for therapeutically relevant cysteine proteases.

How Significant Are Unusual Protein-Ligand Interactions? Insights from Database Mining.

We present a new approach to derive interaction propensities of protein-ligand atom pairs from mining of the Protein Data Bank. To ensure solid statistics, we use a line-of-sight contact filter and normalize the observed frequency of hits by a statistical null model based on exposed surface areas of atom types in the protein-ligand binding site. This allows us to investigate which intermolecular interactions and geometries are found more often than expected by chance in protein-ligand complexes. We focus our study on some of the unusual interactions that were postulated to be favorable, including σ-hole bonding of halogen and sulfur atoms, weak hydrogen bonding with fluorine as acceptor, and different types of dipolar interactions. Our results confirm some and challenge other common assumptions on these interactions and highlight other contact types that are yet underexplored in structure-based drug design.

Promiscuous Ligands from Experimentally Determined Structures, Binding Conformations, and Protein Family-Dependent Interaction Hotspots.

Compound promiscuity is often attributed to nonspecific binding or assay artifacts. On the other hand, it is well-known that many pharmaceutically relevant compounds are capable of engaging multiple targets in vivo, giving rise to polypharmacology. To explore and better understand promiscuous binding characteristics of small molecules, we have searched X-ray structures (and very few qualifying solution structures) for ligands that bind to multiple distantly related or unrelated target proteins. Experimental structures of a given ligand bound to different targets represent high-confidence data for exploring promiscuous binding events. A total of 192 ligands were identified that formed crystallographic complexes with proteins from different families and for which activity data were available. These "multifamily" compounds included endogenous ligands and were often more polar than other bound compounds and active in the submicromolar range. Unexpectedly, many promiscuous ligands displayed conserved or similar binding conformations in different active sites. Others were found to conformationally adjust to binding sites of different architectures. A comprehensive analysis of ligand-target interactions revealed that multifamily ligands frequently formed different interaction hotspots in binding sites, even if their bound conformations were similar, thus providing a rationale for promiscuous binding events at the molecular level of detail. As a part of this work, all multifamily ligands we have identified and associated activity data are made freely available.

Can Cysteine Protease Cross-Class Inhibitors Achieve Selectivity?

Cysteine proteases are important targets for the discovery of novel therapeutics for many human diseases. From parasitic diseases to cancer, cysteine proteases follow a common mechanism, the formation of an encounter complex with subsequent nucleophilic reactivity of the catalytic cysteine thiol group toward the carbonyl carbon of a peptide bond or an electrophilic group of an inhibitor. Modulation of target enzymes occurs preferably by covalent modification, which imposes challenges in balancing cross-reactivity and selectivity. Given the resurgence of irreversible covalent inhibitors, can they impair off-target effects or are reversible covalent inhibitors a better route to selectivity? This Perspective addresses how small molecule inhibitors may achieve selectivity for different cathepsins, cruzain, rhodesain, and falcipain-2. We discuss target- and ligand-based designs emphasizing repurposing inhibitors from one cysteine protease to others.

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.

Machine Learning Distinguishes with High Accuracy between Pan-Assay Interference Compounds That Are Promiscuous or Represent Dark Chemical Matter.

Assay interference compounds give rise to false-positives and cause substantial problems in medicinal chemistry. Nearly 500 compound classes have been designated as pan-assay interference compounds (PAINS), which typically occur as substructures in other molecules. The structural environment of PAINS substructures is likely to play an important role for their potential reactivity. Given the large number of PAINS and their highly variable structural contexts, it is difficult to study context dependence on the basis of expert knowledge. Hence, we applied machine learning to predict PAINS that are promiscuous and distinguish them from others that are mostly inactive. Surprisingly accurate models can be derived using different methods such as support vector machines, random forests, or deep neural networks. Moreover, structural features that favor correct predictions have been identified, mapped, and categorized, shedding light on the structural context dependence of PAINS effects. The machine learning models presented herein further extend the capacity of PAINS filters.

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.

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.

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.

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.

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.

A Fluorescent-Labeled Phosphono Bisbenzguanidine As an Activity-Based Probe for Matriptase.

Activity-based probes are compounds that exclusively form covalent bonds with active enzymes. They can be utilized to profile enzyme activities in vivo, to identify target enzymes and to characterize their function. The design of a new activity-based probe for matriptase, a member of the type II transmembrane serine proteases, is based on linker-connected bis-benzguanidines. An amino acid, introduced as linker, bears the coumarin fluorophore. Moreover, an incorporated phosphonate allows for a covalent interaction with the active-site serine. The resulting irreversible mode of action was demonstrated, leading to enzyme inactivation and, simultaneously, to a fluorescence labeling of matriptase. The ten-step synthetic approach to a coumarin-labeled bis-benzguanidine and its evaluation as activity-based probe for matriptase based on in-gel fluorescence and fluorescence HPLC is reported. HPLC fluorescence detection as a new application for activity-based probes for proteases is demonstrated herein for the first time.

Privileged Structural Motif Detection and Analysis Using Generative Topographic Maps.

Identification of "privileged structural motifs" associated with specific target families is of particular importance for designing novel bioactive compounds. Here, we demonstrate that they can be extracted from a data distribution represented on a two-dimensional map obtained by Generative Topographic Mapping (GTM). In GTM, structurally related molecules are grouped together on the map. Zones of the map preferentially populated by target-specific compounds were delineated, which helped to capture common substructures on the basis of which these compounds were grouped together by GTM. Such privileged structural motifs were identified across three major target superfamilies including proteases, kinases, and G protein coupled receptors. Traditionally, the search for privileged structural motifs focused on scaffolds, whereas motifs were detected here without prior knowledge of compound classification in GTMs. This alternative way of navigating medicinal chemistry space further extends the classical, scaffold-centric approach. Importantly, detected motifs might also comprise fuzzy sets of similar scaffolds, pharmacophore-like patterns, or, by contrast, well-defined scaffolds with specific substituent patterns.

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.

Highly Promiscuous Small Molecules from Biological Screening Assays Include Many Pan-Assay Interference Compounds but Also Candidates for Polypharmacology.

In PubChem screening assays, 466 highly promiscuous compounds were identified that were examined for known pan-assay interference compounds (PAINS) and aggregators using publicly available filters. These filters detected 210 PAINS and 67 aggregators. Compounds passing the filters included additional PAINS that were not detected, mostly due to tautomerism, and a variety of other potentially reactive compounds currently not encoded as PAINS. For a subset of compounds passing the filters, there was no evidence of potential artifacts. These compounds are considered candidates for further exploring multitarget activities and the molecular basis of polypharmacology.

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.