Functional classification of protein kinase binding sites using Cavbase

Increasingly, drug-discovery processes focus on complete gene families. Tools for analyzing similarities and differences across protein families are important for the understanding of key functional features of proteins. Herein we present a method for classifying protein families on the basis of the properties of their active sites. We have developed Cavbase, a method for describing and comparing protein binding pockets, and show its application to the functional classification of the binding pockets of the protein family of protein kinases. A diverse set of kinase cavities is mutually compared and analyzed in terms of recurring functional recognition patterns in the active sites. We are able to propose a relevant classification based on the binding motifs in the active sites. The obtained classification provides a novel perspective on functional properties across protein space. The classification of the MAP and the c-Abl kinases is analyzed in detail, showing a clear separation of the respective kinase subfamilies. Remarkable cross-relations among protein kinases are detected, in contrast to sequence-based classifications, which are not able to detect these relations. Furthermore, our classification is able to highlight features important in the optimization of protein kinase inhibitors. Using small-molecule inhibition data we could rationalize cross-reactivities between unrelated kinases which become apparent in the structural comparison of their binding sites. This procedure helps in the identification of other possible kinase targets that behave similarly in "binding pocket space" to the kinase under consideration.     

Discovery of a Novel Class of Covalent Dual Inhibitors Targeting the Protein Kinases BMX and BTK

The nonreceptor tyrosine TEC kinases are key regulators of the immune system and play a crucial role in the pathogenesis of diverse hematological malignancies. In contrast to the substantial efforts in inhibitor development for Bruton’s tyrosine kinase (BTK), specific inhibitors of the other TEC kinases, including the bone marrow tyrosine kinase on chromosome X (BMX), remain sparse. Here we present a novel class of dual BMX/BTK inhibitors, which were designed from irreversible inhibitors of Janus kinase (JAK) 3 targeting a cysteine located within the solvent-exposed front region of the ATP binding pocket. Structure-guided design exploiting the differences in the gatekeeper residues enabled the achievement of high selectivity over JAK3 and certain other kinases harboring a sterically demanding residue at this position. The most active compounds inhibited BMX and BTK with apparent IC₅₀ values in the single digit nanomolar range or below showing moderate selectivity within the TEC family and potent cellular target engagement. These compounds represent an important first step towards selective chemical probes for the protein kinase BMX.     

Simulations Studies of Protein Kinases that are Molecular Targets in Cancer

Protein kinases are enzymes with partially overlapping specificities, many of which are important clinical targets. In this article, we give some background on protein kinases and discuss in more depth four such enzymes that have been studied in our labs using computer simulations. The combination of molecular dynamics simulations and enzyme or cell growth experiments was instrumental to explain why certain mutations lead (or do not lead) to resistance to targeted therapy aimed at these proteins. Stochastic network simulations were used to study protein-protein interactions and suggest points for intervention against tumour growth.     

Exploiting bioorthogonal chemistry to elucidate protein-lipid binding interactions and other biological roles of phospholipids

Lipids play critical roles in a litany of physiological and pathophysiological events, often through the regulation of protein function. These activities are generally difficult to characterize, however, because the membrane environment in which lipids operate is very complex. Moreover, lipids have a diverse range of biological functions, including the recruitment of proteins to membrane surfaces, actions as small-molecule ligands, and covalent protein modification through lipidation. Advancements in the development of bioorthogonal reactions have facilitated the study of lipid activities by providing the ability to selectively label probes bearing bioorthogonal tags within complex biological samples. In this Account, we discuss recent efforts to harness the beneficial properties of bioorthogonal labeling strategies in elucidating lipid function. Initially, we summarize strategies for the design and synthesis of lipid probes bearing bioorthogonal tags. This discussion includes issues to be considered when deciding where to incorporate the tag, particularly the presentation within a membrane environment. We then present examples of the application of these probes to the study of lipid activities, with a particular emphasis on the elucidation of protein-lipid binding interactions. One such application involves the development of lipid and membrane microarray analysis as a high-throughput platform for characterizing protein-binding interactions. Here we discuss separate strategies for binding analysis involving the immobilization of either whole liposomes or simplified isolated lipid structures. In addition, we present the different strategies that have been used to derivatize membrane surfaces via bioorthogonal reactions, either by using this chemistry to produce functionalized lipid scaffolds that can be incorporated into membranes or through direct modification of intact membrane surfaces. We then provide an overview of the development of lipid activity probes to label and identify proteins that bind to a particular lipid from complex biological samples. This process involves the strategy of activity-based proteomics, in which proteins are collectively labeled on the basis of function (in this case, ligand binding) rather than abundance. We summarize strategies for designing and applying lipid activity probes that allow for the selective labeling and characterization of protein targets. Additionally, we briefly comment on applications other than studying protein-lipid binding. These include the generation of new lipid structures with beneficial properties, labeling of tagged lipids in live cells for studies involving fluorescence imaging, elucidation of covalent protein lipidation, and identification of biosynthetic lipid intermediates. These applications illustrate the early phase of the promising field of applying bioorthogonal chemistry to the study of lipid function.     

Aminoacyl-tRNA Synthetases as Valuable Targets for Antimicrobial Drug Discovery

Aminoacyl-tRNA synthetases (aaRSs) catalyze the esterification of tRNA with a cognate amino acid and are essential enzymes in all three kingdoms of life. Due to their important role in the translation of the genetic code, aaRSs have been recognized as suitable targets for the development of small molecule anti-infectives. In this review, following a concise discussion of aaRS catalytic and proof-reading activities, the various inhibitory mechanisms of reported natural and synthetic aaRS inhibitors are discussed. Using the expanding repository of ligand-bound X-ray crystal structures, we classified these compounds based on their binding sites, focusing on their ability to compete with the association of one, or more of the canonical aaRS substrates. In parallel, we examined the determinants of species-selectivity and discuss potential resistance mechanisms of some of the inhibitor classes. Combined, this structural perspective highlights the opportunities for further exploration of the aaRS enzyme family as antimicrobial targets.     

Pyruvate-Kinase-Coupled Glycosyltransferase Assays: Limitations,Struggles and Problem Resolution

Enzyme assays involving coupled pyruvate kinase (PK) have been used for many years to monitor the activity of major classes of enzymes including glycosyltransferases. Numerous potent inhibitors have been discovered and kinetically characterized thanks to this technology. However, when inhibitors of these important enzymes are screened, PK inhibitors or activators are very often observed. In this study we report solutions to resolve the problems encountered either during the screening or during the kinetic characterization of glycosyltransferase inhibitors by means of PK-coupled assays. The enzyme under study—WaaC—is an important glycosyltransferase involved in the bacterial lipopolysaccharide (LPS) biosynthesis pathway. Firstly we showed that alternative kinases such as nucleoside 5-diphosphate kinase (NDPK), myokinase (MK), and ADPdependent hexokinase that catalyze similar reactions to PK are prone to the same troubles. Moreover, an ADP chemosensor was used as an alternative but the sensitivity was not sufficient to allow a proper screening. Finally, we found that a stepwise PK/luciferase assay resolved the problems encountered with PK inhibitors and that a WaaC HPLC assay allowed the identification of WaaC inhibitors acting as PK activators, thus allowing false positive and false negative results linked to the coupling to PK to be eliminated.     

Identification of Novel GSK-3β Hits Using Competitive Biophysical Assays

Glycogen synthase kinase 3 beta (GSK-3β) is an evolutionarily conserved serine-threonine kinase dysregulated in numerous pathologies, such as Alzheimer’s disease and cancer. Even though GSK-3β is a validated pharmacological target most of its inhibitors have two main limitations: the lack of selectivity due to the high homology that characterizes the ATP binding site of most kinases, and the toxicity that emerges from GSK-3β complete inhibition which translates into the impairment of the plethora of pathways GSK-3β is involved in. Starting from a 1D ¹⁹F NMR fragment screening, we set up several biophysical assays for the identification of GSK-3β inhibitors capable of binding protein hotspots other than the ATP binding pocket or to the ATP binding pocket, but with an affinity able of competing with a reference binder. A phosphorylation activity assay on a panel of several kinases provided selectivity data that were further rationalized and corroborated by structural information on GSK-3β in complex with the hit compounds. In this study, we identified promising fragments, inhibitors of GSK-3β, while proposing an alternative screening workflow that allows facing the flaws that characterize the most common GSK-3β inhibitors through the identification of selective inhibitors and/or inhibitors able to modulate GSK-3β activity without leading to its complete inhibition.     

Discovery of 4-benzylamino-substituted α-carbolines as a novel class of receptor tyrosine kinase inhibitors

Within the last decade, interest in the development of new anticancer drugs increased mainly from emerging resistance against established drugs, which were found to be limited by the multidrug resistance (MDR) phenomenon. Several anticancer targets have been investigated for the development of structurally new drugs which were thought to be unaffected by the MDR phenomenon. Receptor tyrosine kinases (RTKs) make up one interesting group of anticancer targets. The overexpression and mutation of RTKs lead to an ongoing stimulus of cell growth and cancer progression. Early approaches to selective inhibition of single RTKs were generally disappointing in clinical studies, due in part to occurring resistance. Therefore, a new strategy involves the identification of multi‐kinase inhibitors to slow the development of potential resistance. Moreover, the expected side effects of the first nonselective inhibitors were less dramatic than had been expected. We have discovered novel 4‐benzylamino‐α‐carbolines as a new class of RTK inhibitors. Docking studies suggest a binding mode to the addressed target structures of the epidermal growth factor receptor (EGFR) and to the vascular endothelial growth factor receptor 2 (VEGFR2). Selectivity profiling against a panel of kinases and antiproliferative studies have highlighted one inhibitor, active in the nanomolar range, as a highly interesting candidate for further clinical studies.     

Using Chemical Probes to Assess the Feasibility of Targeting SecA for Developing Antimicrobial Agents against Gram‐Negative Bacteria

With the widespread emergence of drug resistance, there is an urgent need to search for new antimicrobials, especially those against Gram‐negative bacteria. Along this line, the identification of viable targets is a critical first step. The protein translocase SecA is commonly believed to be an excellent target for the development of broad‐spectrum antimicrobials. In recent years, we developed three structural classes of SecA inhibitors that have proven to be very effective against Gram‐positive bacteria. However, we have not achieved the same level of success against Gram‐negative bacteria, despite the potent inhibition of SecA in enzyme assays by the same inhibitors. In this study, we use representative inhibitors as chemical probes to gain an understanding as to why these inhibitors were not effective against Gram‐negative bacteria. The results validate our initial postulation that the major difference in effectiveness against Gram‐positive and Gram‐negative bacteria is in the additional permeability barrier posed by the outer membrane of Gram‐negative bacteria. We also found that the expression of efflux pumps, which are responsible for multidrug resistance (MDR), have no effect on the effectiveness of these SecA inhibitors. Identification of an inhibitor‐resistant mutant and complementation tests of the plasmids containing secA in a secAts mutant showed that a single secA‐azi‐9 mutation increased the resistance, providing genetic evidence that SecA is indeed the target of these inhibitors in bacteria. Such results strongly suggest SecA as an excellent target for developing effective antimicrobials against Gram‐negative bacteria with the intrinsic ability to overcome MDR. A key future research direction should be the optimization of membrane permeability.     

Rational Design,Synthesis and Biological Evaluation of Modular Fluorogenic Substrates with High Affinity and Selectivity for PTP1B

Protein‐tyrosine phosphatase 1B (PTP1B) is a key regulatory enzyme in several signal transduction pathways, and its upregulation has been associated with type‐2 diabetes, obesity and cancer. Selective determination of the functional significance of PTP1B remains a major challenge because the activity of this crucial enzyme is currently evaluated through the use of fluorescent probes that lack selectivity and are limited to biochemical assays. Here we describe the rational design, synthesis and biological evaluation of new modular PTP1B fluorogenic substrates. The self‐immolative 4‐hydroxybenzyl alcohol has been used as a key component for the design of phosphotyrosine mimics linked to a latent chromophore, which is released through an enzyme‐initiated domino reaction. Preliminary biological investigations showed that, by optimising the stereoelectronic properties and the binding interactions at the enzyme active site, it is possible to achieve substrates with high affinity and promising selectivity. Due to their modular nature, the synthesised fluorogenic probes represent versatile tools; customisation of the different subunits could widen the scope of these probes to a broader range of in vitro assays. Finally, these studies elucidate the critical role played by Asp181 in the PTP1B‐catalysed dephosphorylation mechanism: disruption of the native conformation of this key amino acid residue on the WDP loop yields fluorogenic inhibitors, rather than substrates. For this reason, our studies also represent a step forward for the development of improved PTP1B noncovalent inhibitors.     

Label Transfer Reagents to Probe p38 MAPK Binding Partners

Protein kinases are essential enzymes for cellular signaling, and are often regulated by participation in protein complexes. The mitogen‐activated protein kinase (MAPK) p38 is involved in multiple pathways, and its regulation depends on its interactions with other signaling proteins. However, the identification of p38‐interacting proteins is challenging. For this reason, we have developed label transfer reagents (LTRs) that allow labeling of p38 signaling complexes. These LTRs leverage the potency and selectivity of known p38 inhibitors to place a photo‐crosslinker and tag in the vicinity of p38 and its binding partners. Upon UV irradiation, proteins that are in close proximity to p38 are covalently crosslinked, and labeled proteins are detected and/or purified with an orthogonal chemical handle. Here we demonstrate that p38‐selective LTRs selectively label a diversity of p38 binding partners, including substrates, activators, and inactivators. Furthermore, these LTRs can be used in immunoprecipitations to provide low‐resolution structural information on p38‐containing complexes.     

Design, synthesis and biological evaluation of Trypanosoma brucei trypanothione synthetase inhibitors

Trypanothione synthetase (TryS) is essential for the survival of the protozoan parasite Trypanosoma brucei, which causes human African trypanosomiasis. It is one of only a handful of chemically validated targets for T. brucei in vivo. To identify novel inhibitors of TbTryS we screened our in-house diverse compound library that contains 62,000 compounds. This resulted in the identification of six novel hit series of TbTryS inhibitors. Herein we describe the SAR exploration of these hit series, which gave rise to one common series with potency against the enzyme target. Cellular studies on these inhibitors confirmed on-target activity, and the compounds have proven to be very useful tools for further study of the trypanothione pathway in kinetoplastids.     

Are MAP Kinases Drug Targets? Yes,but Difficult Ones

Pharmaceutical companies are facing an increasing interest in new target identification and validation. In particular, extensive efforts are being made in the field of protein kinase inhibitors research and development, and the past ten years of effort in this field have altered our perception of the potential of kinases as drug targets. Therefore, in the drug discovery process, the selection of relevant, susceptible protein kinase targets combined with searches for leads and candidates have become a crucial approach. The success of recent launches of protein kinase inhibitors (Gleevec, Imatinib, Sutent, Iressa, Nexavar, Sprycel) gave another push to this field. Numerous other kinase inhibitors are currently undergoing clinical trials or clinical development. Some questions are nevertheless unanswered, mostly related to the great number of known kinases in the human genome, to their similarity with each other, to the existence of functionally redundant kinases for specific pathways, and also because the connection between particular pathways and diseases is not always clear. The review is leading the reader through a panoramic view of protein kinase inhibition with a major focus on MAPK, successful examples and clinical candidates.     

Substrate Activity Screening (SAS) and Related Approaches in Medicinal Chemistry

Substrate activity screening (SAS) was presented a decade ago by Ellman and co‐workers as a straightforward methodology for the identification of fragment‐sized building blocks for enzyme inhibitors. Ever since, SAS and variations derived from it have been successfully applied to the discovery of inhibitors of various families of enzymatically active drug targets. This review covers key achievements and challenges of SAS and related methodologies, including the modified substrate activity screening (MSAS) approach. Special attention is given to the kinetic and thermodynamic aspects of these methodologies, as a thorough understanding thereof is crucial for successfully transforming the identified fragment‐sized hits into potent inhibitors.     

In Silico Identification of Potential Druggable Binding Sites on CIN85 SH3 Domain

Protein-protein interactions (PPIs) play a pivotal role in the regulation of many physiological processes. The dysfunction of some PPIs interactions led to the alteration of different biological pathways causing various diseases including cancer. In this context, the inhibition of PPIs represents an attractive strategy for the design of new antitumoral agents. In recent years, computational approaches were successfully used to study the interactions between proteins, providing useful hints for the design of small molecules able to modulate PPIs. Targeting PPIs presents several challenges mainly due to the large and flat binding surface that lack the typical binding pockets of traditional drug targets. Despite these hurdles, substantial progress has been made in the last decade resulting in the identification of PPI modulators where some of them even found clinical use. This study focuses on MUC1-CIN85 PPI which is involved in the migration and invasion of cancer cells. Particularly, we investigated the presence of druggable binding sites on the CIN85 surface which provided new insights for the structure-based design of novel MUC1-CIN85 PPI inhibitors as anti-metastatic agents.     

Fluorophore-Labeled Cyclic Nucleotides as Potent Agonists of Cyclic Nucleotide-Regulated Ion Channels

High-affinity fluorescent derivatives of cyclic adenosine and guanosine monophosphate are powerful tools for investigating their natural targets. Cyclic nucleotide-regulated ion channels belong to these targets and are vital for many signal transduction processes, such as vision and olfaction. The relation of ligand binding to activation gating is still challenging, and there is a need for fluorescent probes that enable the process to be broken down to the single-molecule level. This inspired us to prepare fluorophore-labeled cyclic nucleotides, which are composed of a bright dye and a nucleotide derivative with a thiophenol motif at position 8 that has already been shown to enable superior binding affinity. These bioconjugates were prepared by a novel cross-linking strategy that involves substitution of the nucleobase with a modified thiophenolate in good yield. Both fluorescent nucleotides are potent activators of different cyclic nucleotide-regulated ion channels with respect to the natural ligand and previously reported substances. Molecular docking of the probes excluding the fluorophore reveals that the high potency can be attributed to additional hydrophobic and cation-π interactions between the ligand and the protein. Moreover, the introduced substances have the potential to investigate related target proteins, such as cAMP- and cGMP-dependent protein kinases, exchange proteins directly activated by cAMP or phosphodiesterases.     

Inspired by Nature: The 3‐Halo‐4,5‐dihydroisoxazole Moiety as a Novel Molecular Warhead for the Design of Covalent Inhibitors

Over the past few decades, there has been an increasing interest in the development of covalent enzyme inhibitors. As it was recently re‐emphasized, the selective, covalent binding of a drug to the desired target can increase efficiency and lower the inhibitor concentration required to achieve a therapeutic effect. In this context, the naturally occurring antibiotic acivicin, and in particular its 3‐chloro‐4,5‐dihydroisoxazole scaffold, has provided a wealth of inspiration to medicinal chemists and chemical biologists alike. In this Concept, to underline the great potentiality that the 3‐halo‐4,5‐dihydroisoxazole warhead has in drug discovery, we present a number of examples, grouped by their potential biological activity and targets, in which this scaffold has been fruitfully used to develop novel biologically active compounds. Through these examples, we show that the 3‐halo‐4,5‐dihydroisoxazole moiety represents an outstanding warhead with high potential for the design of novel covalent enzyme inhibitors.     

Discovery of Small-Molecule Modulators of Protein–RNA Interactions by Fluorescence Intensity-Based Binding Assay

Protein–RNA interactions mediate various cellular processes, the dysregulation of which has been associated with a list of diseases. Thus, novel experimental tools for monitoring protein–RNA interactions are highly desirable to identify new chemical modulators of these therapeutic targets. In this study, we constructed simple fluorescence intensity-based protein–RNA binding assays by testing multiple environment-sensitive organic fluorophores. We selected the oncogenic interaction between Lin28 and the let-7 microRNA and the important immunomodulatory Roquin–Tnf CDE interaction as representative targets. We adapted this assay to high-throughput screening for the identification of pyrazolyl thiazolidinedione-type molecules as potent small-molecule inhibitors of protein–microRNA interactions. We clearly showed the structure–activity relationships of this new class of Lin28–let-7 interaction inhibitors, and confirmed that cellular mature let-7 microRNAs and their target genes could be modulated upon treatment with the pyrazolyl thiazolidinedione-type inhibitor. We expect that our simple and adaptable screening approach can be applied for the development of various assay systems aimed at the identification of bioactive small molecules targeting protein–RNA interactions.     

Discovery of Diverse Small‐Molecule Inhibitors of Mammalian Sterile20‐like Kinase 3 (MST3)

Increasing evidence suggests key roles for members of the mammalian Sterile20‐like (MST) family of kinases in many aspects of biology. MST3 is a member of the STRIPAK complex, the deregulation of which has recently been associated with cancer cell migration and metastasis. Targeting MST3 with small‐molecule inhibitors may be beneficial for the treatment of certain cancers, but little information exists on the potential of kinase inhibitor scaffolds to engage with MST3. In this study we screened MST3 against a library of 277 kinase inhibitors using differential scanning fluorimetry and confirmed 14 previously unknown MST3 inhibitors by X‐ray crystallography. These compounds, of which eight are in clinical trials or FDA approved, comprise nine distinct chemical scaffolds that inhibit MST3 enzymatic activity with IC₅₀ values between 0.003 and 23 μm . The structure–activity relationships explain the differential inhibitory activity of these compounds against MST3 and the structural basis for high binding potential, the information of which may serve as a framework for the rational design of MST3‐selective inhibitors as potential therapeutics and to interrogate the function of this enzyme in diseased cells.