Natural Products as Zinc‐Dependent Histone Deacetylase Inhibitors

Zinc‐dependent histone deacetylases (HDACs), a family of hydrolases that remove acetyl groups from lysine residues, play an important role in the regulation of multiple processes, from gene expression to protein activity. The dysregulation of HDACs is associated with many diseases including cancer, neurological disorders, cellular metabolism disorders, and inflammation. Molecules that act as HDAC inhibitors (HDACi) exhibit a variety of related bioactivities. In particular, HDACi have been applied clinically for the treatment of cancers. Inhibition through competitive binding of the catalytic domain of these enzymes has been achieved by a diverse array of small‐molecule chemotypes, including a number of natural products. This review provides a systematic introduction of natural HDACi, with an emphasis on their enzyme inhibitory potency, selectivity, and biological activities, highlighting their various binding modes with HDACs.     

Investigation into the Feasibility of Thioditaloside as a Novel Scaffold for Galectin‐3‐Specific Inhibitors

Galectin‐3 is extensively involved in metabolic and disease processes, such as cancer metastasis, thus giving impetus for the design of specific inhibitors targeting this β‐galactose‐binding protein. Thiodigalactoside (TDG) presents a scaffold for construction of galectin inhibitors, and its inhibition of galectin‐1 has already demonstrated beneficial effects as an adjuvant with vaccine immunotherapy, thereby improving the survival outcome of tumour‐challenged mice. A novel approach—replacing galactose with its C2 epimer, talose—offers an alternative framework, as extensions at C2 permit exploitation of a galectin‐3‐specific binding groove, thereby facilitating the design of selective inhibitors. We report the synthesis of thioditaloside (TDT) and crystal structures of the galectin‐3 carbohydrate recognition domain in complexes with TDT and TDG. The different abilities of galactose and talose to anchor to the protein correlate with molecular dynamics studies, likely explaining the relative disaccharide binding affinities. The feasibility of a TDT scaffold to enable access to a particular galectin‐3 binding groove and the need for modifications to optimise such a scaffold for use in the design of potent and selective inhibitors are assessed.     

Design and Synthesis of Tricyclic JAK3 Inhibitors with Picomolar Affinities as Novel Molecular Probes

The Janus kinase (JAK) signaling pathway is of particular importance in the pathology of inflammatory diseases and oncological disorders, and the inhibition of Janus kinase 3 (JAK3) with small molecules has proven to provide therapeutic immunosuppression. A novel class of tricyclic JAK inhibitors derived from the 3‐methyl‐1,6‐dihydrodipyrrolo[2,3‐b:2′,3′‐d]pyridine scaffold was designed based on the tofacitinib–JAK3 crystal structure by applying a rigidization approach. A convenient synthetic strategy to access the scaffold via an intramolecular Heck reaction was developed, and a small library of inhibitors was prepared and characterized using in vitro biochemical as well as cellular assays. IC₅₀ values as low as 220 pM could be achieved with selectivity for JAK3 over other JAK family members. Both activity and selectivity were confirmed in a cellular STAT phosphorylation assay, providing also first‐time data for tofacitinib. Our novel inhibitors may serve as tool compounds and useful probes to explore the role of JAK3 inhibition in pharmacodynamics studies.     

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.     

Progress in the Development of Eukaryotic Elongation Factor 2 Kinase (eEF2K) Natural Product and Synthetic Small Molecule Inhibitors for Cancer Chemotherapy

Eukaryotic elongation factor 2 kinase (eEF2K or Ca²⁺/calmodulin-dependent protein kinase, CAMKIII) is a new member of an atypical α-kinase family different from conventional protein kinases that is now considered as a potential target for the treatment of cancer. This protein regulates the phosphorylation of eukaryotic elongation factor 2 (eEF2) to restrain activity and inhibit the elongation stage of protein synthesis. Mounting evidence shows that eEF2K regulates the cell cycle, autophagy, apoptosis, angiogenesis, invasion, and metastasis in several types of cancers. The expression of eEF2K promotes survival of cancer cells, and the level of this protein is increased in many cancer cells to adapt them to the microenvironment conditions including hypoxia, nutrient depletion, and acidosis. The physiological function of eEF2K and its role in the development and progression of cancer are here reviewed in detail. In addition, a summary of progress for in vitro eEF2K inhibitors from anti-cancer drug discovery research in recent years, along with their structure–activity relationships (SARs) and synthetic routes or natural sources, is also described. Special attention is given to those inhibitors that have been already validated in vivo, with the overall aim to provide reference context for the further development of new first-in-class anti-cancer drugs that target eEF2K.     

Development of a Sensitive Microarray Platform for the Ranking of Galectin Inhibitors: Identification of a Selective Galectin‐3 Inhibitor

Glycan microarrays are useful tools for lectin glycan profiling. The use of a glycan microarray based on evanescent‐field fluorescence detection was herein further extended to the screening of lectin inhibitors in competitive experiments. The efficacy of this approach was tested with 2/3′‐mono‐ and 2,3′‐diaromatic type II lactosamine derivatives and galectins as targets and was validated by comparison with fluorescence anisotropy proposed as an orthogonal protein interaction measurement technique. We showed that subtle differences in the architecture of the inhibitor could be sensed that pointed out the preference of galectin‐3 for 2′‐arylamido derivatives over ureas, thioureas, and amines and that of galectin‐7 for derivatives bearing an α substituent at the anomeric position of glucosamine. We eventually identified a diaromatic oxazoline as a highly specific inhibitor of galectin‐3 versus galectin‐1 and galectin‐7.     

Development of Computational Approaches with a Fragment-Based Drug Design Strategy: In Silico Hsp90 Inhibitors Discovery

A semi-exhaustive approach and a heuristic search algorithm use a fragment-based drug design (FBDD) strategy for designing new inhibitors in an in silico process. A deconstruction reconstruction process uses a set of known Hsp90 ligands for generating new ones. The deconstruction process consists of cutting off a known ligand in fragments. The reconstruction process consists of coupling fragments to develop a new set of ligands. For evaluating the approaches, we compare the binding energy of the new ligands with the known ligands.     

Screening of Inhibitors Targeting Heat Shock Protein 47 Involved in the Development of Idiopathic Pulmonary Fibrosis

Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is causally related to fibrotic diseases, including idiopathic pulmonary fibrosis. The identification of Compounds that interfere with the HSP47-collagen interaction is essential for the development of relevant therapeutics. Herein, we prepared human HSP47 as a soluble fusion protein expressed in E. coli and established an assay system for HSP47 inhibitor screening. We screened a natural and synthetic Compound library established at Nagasaki University. Among 1023 Compounds, 13 exhibited inhibitory activity against human HSP47, of which three inhibited its function in a dose-dependent manner. Epigallocatechin-3-O-gallate, one of these three Compounds, is a typical polyphenol Compound derived from tea leaves. Structurally related Compounds were synthesized and examined for their activity, revealing a hydroxyl group at A-ring position 5 as important for its activity. The present findings provide valuable insight for the development of natural product-derived therapeutics for fibrotic diseases, including idiopathic pulmonary fibrosis.     

Discovery of Flavonoids as Novel Inhibitors of ATP Citrate Lyase: Structure–Activity Relationship and Inhibition Profiles

ATP citrate lyase (ACLY) is a key enzyme in glucolipid metabolism and its aberrantly high expression is closely associated with various cancers, hyperlipemia and atherosclerotic cardiovascular diseases. Prospects of ACLY inhibitors as treatments of these diseases are excellent. To date, flavonoids have not been extensively reported as ACLY inhibitors. In our study, 138 flavonoids were screened and 21 of them were subjected to concentration–response curves. A remarkable structure–activity relationship (SAR) trend was found: ortho-dihydroxyphenyl and a conjugated system maintained by a pyrone ring were critical for inhibitory activity. Among these flavonoids, herbacetin had a typical structure and showed a non–aggregated state in solution and a high inhibition potency (IC₅₀ = 0.50 ± 0.08 μM), and therefore was selected as a representative for the ligand–protein interaction study. In thermal shift assays, herbacetin improved the thermal stability of ACLY, suggesting a direct interaction with ACLY. Kinetic studies determined that herbacetin was a noncompetitive inhibitor of ACLY, as illustrated by molecular docking and dynamics simulation. Together, this work demonstrated flavonoids as novel and potent ACLY inhibitors with a remarkable SAR trend, which may help design high–potency ACLY inhibitors. In–depth studies of herbacetin deepened our understanding of the interactions between flavonoids and ACLY.     

A New Paradigm for KIM-PTP Drug Discovery: Identification of Allosteric Sites with Potential for Selective Inhibition Using Virtual Screening and LEI Analysis

The kinase interaction motif protein tyrosine phosphatases (KIM-PTPs), HePTP, PTPSL and STEP, are involved in the negative regulation of mitogen-activated protein kinase (MAPK) signalling pathways and are important therapeutic targets for a number of diseases. We have used VSpipe, a virtual screening pipeline, to identify a ligand cluster distribution that is unique to this subfamily of PTPs. Several clusters map onto KIM-PTP specific sequence motifs in contrast to the cluster distribution obtained for PTP1B, a classic PTP that mapped to general PTP motifs. Importantly, the ligand clusters coincide with previously reported functional and substrate binding sites in KIM-PTPs. Assessment of the KIM-PTP specific clusters, using ligand efficiency index (LEI) plots generated by the VSpipe, ascertained that the binders in these clusters reside in a more drug-like chemical–biological space than those at the active site. LEI analysis showed differences between clusters across all KIM-PTPs, highlighting a distinct and specific profile for each phosphatase. The most druggable cluster sites are unexplored allosteric functional sites unique to each target. Exploiting these sites may facilitate the delivery of inhibitors with improved drug-like properties, with selectivity amongst the KIM-PTPs and over other classical PTPs.     

Red-Edge Excitation Shift Spectroscopy (REES): Application to Hidden Bound States of Ligands in Protein–Ligand Complexes

Ligand-protein binding is responsible for the vast majority of bio-molecular functions. Most experimental techniques examine the most populated ligand-bound state. The determination of less populated, intermediate, and transient bound states is experimentally challenging. However, hidden bound states are also important because these can strongly influence ligand binding and unbinding processes. Here, we explored the use of a classical optical spectroscopic technique, red-edge excitation shift spectroscopy (REES) to determine the number, population, and energetics associated with ligand-bound states in protein–ligand complexes. We describe a statistical mechanical model of a two-level fluorescent ligand located amongst a finite number of discrete protein microstates. We relate the progressive emission red shift with red-edge excitation to thermodynamic parameters underlying the protein–ligand free energy landscape and to photo-physical parameters relating to the fluorescent ligand. We applied the theoretical model to published red-edge excitation shift data from small molecule inhibitor–kinase complexes. The derived thermodynamic parameters allowed dissection of the energetic contribution of intermediate bound states to inhibitor–kinase interactions.     

From Type I to Type II: Design,Synthesis, and Characterization of Potent Pyrazin‐2‐ones as DFG‐Out Inhibitors of PDGFRβ

Reversible protein kinase inhibitors that bind in the ATP cleft can be classified as type I or type II binders. Of these, type I inhibitors address the active form, whereas type II inhibitors typically lock the kinase in an inactive form. At the molecular level, the conformation of the flexible activation loop holding the key DFG motif controls access to the ATP site, thereby determining an active or inactive kinase state. Accordingly, type I and type II kinase inhibitors bind to so‐called DFG‐in or DFG‐out conformations, respectively. Based on our former study on highly selective platelet‐derived growth factor receptor β (PDGFRβ) pyrazin‐2‐one type I inhibitors, we expanded this scaffold toward the deep pocket, yielding the highly potent and effective type II inhibitor 5 (4‐[(4‐methylpiperazin‐1‐yl)methyl]‐N‐[3‐[[6‐oxo‐5‐(3,4,5‐trimethoxyphenyl)‐1H‐pyrazin‐3‐yl]methyl]phenyl]benzamide). In vitro characterization, including selectivity panel data from activity‐based assays (300 kinases) and affinity‐based assays (97 kinases) of these PDGFRβ type I ( 1 ; 5‐(4‐hydroxy‐3‐methoxy‐phenyl)‐3‐(3,4,5‐trimethoxyphenyl)‐1H‐pyrazin‐2‐one) and II ( 5 ) inhibitors showing the same pyrazin‐2‐one chemotype are compared. Implications are discussed regarding the data for selectivity and efficacy of type I and type II ligands.     

Tampering with Cell Division by Using Small‐Molecule Inhibitors of CDK–CKS Protein Interactions

Cyclin‐dependent kinases (CDKs) control many cellular processes and are considered important therapeutic targets. Large collections of inhibitors targeting CDK active sites have been discovered, but their use in chemical biology or drug development has been often hampered by their general lack of specificity. An alternative approach to develop more specific inhibitors is targeting protein interactions involving CDKs. CKS proteins interact with some CDKs and play important roles in cell division. We discovered two small‐molecule inhibitors of CDK–CKS interactions. They bind to CDK2, do not inhibit its enzymatic activity, inhibit the proliferation of tumor cell lines, induce an increase in G1 and/or S‐phase cell populations, and cause a decrease in CDK2, cyclin A, and p27^Kip1 levels. These molecules should help decipher the complex contributions of CDK–CKS complexes in the regulation of cell division, and they might present an interesting therapeutic potential.     

Small-Molecule Inhibitors of Shp2 Phosphatase as Potential Chemotherapeutic Agents for Glioblastoma: A Minireview

Glioblastoma multiforme (GBM) is a dreadful cancer characterised by poor prognosis, low survival rate and difficult clinical correlations. Several signalling pathways and molecular mediators are known to precipitate GBM, and small-molecular targets of these mediators have become a favoured thrust area for researchers to develop potent anti-GBM drugs. Shp2, an important phosphatase of the nonreceptor type protein tyrosine phosphatase (PTPN) subfamily is responsible for master regulation of several such signalling pathways in normal and glioma cells. Thus, inhibition of Shp2 is a logical strategy for the design and development of anti-neoplastic drugs against GBM. Though tapping the full potential of Shp2 binding sites has been challenging, nevertheless, many synthetic and natural scaffolds have been documented as possessing potent and selective anti-Shp2 activities in biochemical and cellular assays, through either active-site or allosteric binding. Most of these scaffolds share a few common pharmacophoric features, a thorough study of which is useful in paving the way for the design and development of improved Shp2 inhibitors. This minireview summarizes the current scenario of potent small-molecule Shp2 inhibitors and emphasizes the anti-GBM potential of some important scaffolds that have shown promising GBM-specific activity in in vitro and in vivo models, thus proving their efficacy in GBM therapy. This review could guide researchers to design new and improved anti-Shp2 pharmacophores and develop them as anti-GBM agents by employing GBM-centric drug-discovery protocols.     

Discovery of Affinity-Based Probes for Btk Occupancy Assays

Bruton's tyrosine kinase (Btk) is an attractive target for the treatment of a wide array of B-cell malignancies and autoimmune diseases. Small-molecule covalent irreversible Btk inhibitors targeting Cys481 have been developed for the treatment of such diseases. In clinical trials, probe molecules are required in occupancy studies to measure the level of engagement of the protein by these covalent irreversible inhibitors. The result of this pharmacodynamic (PD) activity provides guidance for appropriate dosage selection to optimize inhibition of the drug target and correlation of target inhibition with disease treatment efficacy. This information is crucial for successful evaluation of drug candidates in clinical trials. Based on the pyridine carboxamide scaffold of a novel solvent-accessible pocket (SAP) series of covalent irreversible Btk inhibitors, we successfully developed a potent and selective affinity-based biotinylated probe 12 (2-[(4-{4-[5-(1-{5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamido}-3,6,9,12-tetraoxapentadecan-15-amido)pentanoyl]piperazine-1-carbonyl}phenyl)amino]-6-[1-(prop-2-enoyl)piperidin-4-yl]pyridine-3-carboxamide). Compound 12 has been used in Btk occupancy assays for preclinical studies to determine the therapeutic efficacy of Btk inhibition in two mouse lupus models driven by TLR7 activation and type I interferon.     

Development of Small‐Molecule Trypanosoma brucei N‐Myristoyltransferase Inhibitors: Discovery and Optimisation of a Novel Binding Mode

The enzyme N‐myristoyltransferase (NMT) from Trypanosoma brucei has been validated both chemically and biologically as a potential drug target for human African trypanosomiasis. We previously reported the development of some very potent compounds based around a pyrazole sulfonamide series, derived from a high‐throughput screen. Herein we describe work around thiazolidinone and benzomorpholine scaffolds that were also identified in the screen. An X‐ray crystal structure of the thiazolidinone hit in Leishmania major NMT showed the compound bound in the previously reported active site, utilising a novel binding mode. This provides potential for further optimisation. The benzomorpholinone was also found to bind in a similar region. Using an X‐ray crystallography/structure‐based design approach, the benzomorpholinone series was further optimised, increasing activity against T. brucei NMT by >1000‐fold. A series of trypanocidal compounds were identified with suitable in vitro DMPK properties, including CNS exposure for further development. Further work is required to increase selectivity over the human NMT isoform and activity against T. brucei.     

Design and Synthesis of A‐Ring Simplified Pyripyropene A Analogues as Potent and Selective Synthetic SOAT2 Inhibitors

Currently, pyripyropene A, which is isolated from the culture broth of Aspergillus fumigatus FO‐1289, is the only compound known to strongly and selectively inhibit the isozyme sterol O‐acyltransferase 2 (SOAT2). To aid in the development of new cholesterol‐lowering or anti‐atherosclerotic agents, new A‐ring simplified pyripyropene A analogues have been designed and synthesized based on total synthesis, and the results of structure–activity relationship studies of pyripyropene A. Among the analogues, two A‐ring simplified pyripyropene A analogues exhibited equally efficient SOAT2 inhibitory activity to that of natural pyripyropene A. These new analogues are the most potent and selective SOAT2 inhibitors to be used as synthetic compounds and attractive seed compounds for the development of drug for dyslipidemia, including atherosclerotic disease and steatosis.     

Development and Evolution of DNA-Dependent Protein Kinase Inhibitors toward Cancer Therapy

DNA double-strand break (DSB) is considered the most deleterious type of DNA damage, which is generated by ionizing radiation (IR) and a subset of anticancer drugs. DNA-dependent protein kinase (DNA-PK), which is composed of a DNA-PK catalytic subunit (DNA-PKcs) and Ku80-Ku70 heterodimer, acts as the molecular sensor for DSB and plays a pivotal role in DSB repair through non-homologous end joining (NHEJ). Cells deficient for DNA-PKcs show hypersensitivity to IR and several DNA-damaging agents. Cellular sensitivity to IR and DNA-damaging agents can be augmented by the inhibition of DNA-PK. A number of small molecules that inhibit DNA-PK have been developed. Here, the development and evolution of inhibitors targeting DNA-PK for cancer therapy is reviewed. Significant parts of the inhibitors were developed based on the structural similarity of DNA-PK to phosphatidylinositol 3-kinases (PI3Ks) and PI3K-related kinases (PIKKs), including Ataxia-telangiectasia mutated (ATM). Some of DNA-PK inhibitors, e.g., NU7026 and NU7441, have been used extensively in the studies for cellular function of DNA-PK. Recently developed inhibitors, e.g., M3814 and AZD7648, are in clinical trials and on the way to be utilized in cancer therapy in combination with radiotherapy and chemotherapy.     

The Journey of DDR1 and DDR2 Kinase Inhibitors as Rising Stars in the Fight Against Cancer

Discoidin domain receptor (DDR) is a collagen-activated receptor tyrosine kinase that plays critical roles in regulating essential cellular processes such as morphogenesis, differentiation, proliferation, adhesion, migration, invasion, and matrix remodeling. As a result, DDR dysregulation has been attributed to a variety of human cancer disorders, for instance, non-small-cell lung carcinoma (NSCLC), ovarian cancer, glioblastoma, and breast cancer, in addition to some inflammatory and neurodegenerative disorders. Since the target identification in the early 1990s to date, a lot of efforts have been devoted to the development of DDR inhibitors. From a medicinal chemistry perspective, we attempted to reveal the progress in the development of the most promising DDR1 and DDR2 small molecule inhibitors covering their design approaches, structure-activity relationship (SAR), biological activity, and selectivity.