α‐Amylase Modulation: Discovery of Inhibitors Using a Multi‐Pharmacophore Approach for Virtual Screening

Better control of postprandial hyperglycemia can be achieved by delaying the absorption of glucose resulting from carbohydrate digestion. Because α‐amylase initiates the hydrolysis of polysaccharides, the design of α‐amylase inhibitors can lead to the development of new treatments for metabolic disorders such as type II diabetes and obesity. In this study, a rational computer‐aided approach was developed to identify novel α‐amylase inhibitors. Three‐dimensional pharmacophores were developed based on the binding mode analysis of six different families of compounds that bind to this enzyme. In a stepwise virtual screening workflow, seven molecules were selected from a library of 1.4 million. Five out of seven biologically tested compounds showed α‐amylase inhibition, and the two most potent compounds inhibited α‐amylase with IC₅₀ values of 17 and 27 μm . The scaffold benzylideneacetohydrazide was shared by four of the discovered inhibitors, emerging as a novel drug‐like non‐carbohydrate fragment and constituting a promising lead scaffold for α‐amylase inhibition.     

Tertiary‐Amine‐Based Inhibitors of the Astacin Protease Meprin α

Metalloproteinases of the astacin family are drawing ever increasing attention as potential drug targets. However, knowledge regarding inhibitors thereof is limited in most cases. Crucial for the development of metalloprotease inhibitors is high selectivity, to avoid side effects brought about by inhibition of off‐target proteases and interference with physiological pathways. In this study we aimed at the design of novel selective inhibitors for the astacin proteinase meprin α. Based on a recently identified tertiary amine scaffold, a series of compounds was synthesized and evaluated. The compounds exhibit reasonable inhibitory activity with high selectivity over other metalloproteases. The isoenzyme meprin β is only slightly inhibited. Hence, the present study revealed a novel class of selective meprin α inhibitors with improved selectivity over known compounds.     

para‐Substituted 2‐Phenyl‐3,4‐dihydroquinazolin‐4‐ones As Potent and Selective Tankyrase Inhibitors

Human tankyrases are attractive drug targets, especially for the treatment of cancer. We identified a set of highly potent tankyrase inhibitors based on a 2‐phenyl‐3,4‐dihydroquinazolin‐4‐one scaffold. Substitutions at the para position of the scaffold′s phenyl group were evaluated as a strategy to increase potency and improve selectivity. The best compounds displayed single‐digit nanomolar potencies, and profiling against several human diphtheria‐toxin‐like ADP‐ribosyltransferases revealed that a subset of these compounds are highly selective tankyrase inhibitors. The compounds also effectively inhibit Wnt signaling in HEK293 cells. The binding mode of all inhibitors was studied by protein X‐ray crystallography. This allowed us to establish a structural basis for the development of highly potent and selective tankyrase inhibitors based on the 2‐phenyl‐3,4‐dihydroquinazolin‐4‐one scaffold and outline a rational approach to the modification of other inhibitor scaffolds that bind to the nicotinamide site of the catalytic domain.     

Structural Investigation of the Binding of 5‐Substituted Swainsonine Analogues to Golgi α‐Mannosidase II

Golgi α‐mannosidase II (GMII) is a key enzyme in the N‐glycosylation pathway and is a potential target for cancer chemotherapy. The natural product swainsonine is a potent inhibitor of GMII. In this paper we characterize the binding of 5α‐substituted swainsonine analogues to the soluble catalytic domain of Drosophila GMII by X‐ray crystallography. These inhibitors enjoy an advantage over previously reported GMII inhibitors in that they did not significantly decrease the inhibitory potential of the swainsonine head‐group. The phenyl groups of these analogues occupy a portion of the binding site not previously seen to be populated with either substrate analogues or other inhibitors and they form novel hydrophobic interactions. They displace a well‐organized water cluster, but the presence of a C(10) carbonyl allows the reestablishment of important hydrogen bonds. Already approximately tenfold more active against the Golgi enzyme than the lysosomal enzyme, these inhibitors offer the potential of being extended into the N‐acetylglucosamine binding site of GMII for the creation of even more potent and selective GMII inhibitors.     

The Discovery of a Highly Selective 5,6,7,8‐Tetrahydrobenzo[4,5]thieno[2,3‐d]pyrimidin‐4(3H)‐one SIRT2 Inhibitor that is Neuroprotective in an in vitro Parkinson’s Disease Model

Sirtuins, NAD⁺‐dependent histone deacetylases (HDACs), have recently emerged as potential therapeutic targets for the treatment of a variety of diseases. The discovery of potent and isoform‐selective inhibitors of this enzyme family should provide chemical tools to help determine the roles of these targets and validate their therapeutic value. Herein, we report the discovery of a novel class of highly selective SIRT2 inhibitors, identified by pharmacophore screening. We report the identification and validation of 3‐((2‐methoxynaphthalen‐1‐yl)methyl)‐7‐((pyridin‐3‐ylmethyl)amino)‐5,6,7,8‐tetrahydrobenzo[4,5]thieno[2,3‐d]pyrimidin‐4(3H)‐one (ICL‐SIRT078), a substrate‐competitive SIRT2 inhibitor with a K_i value of 0.62±0.15 μM and more than 50‐fold selectivity against SIRT1, 3 and 5. Treatment of MCF‐7 breast cancer cells with ICL‐SIRT078 results in hyperacetylation of α‐tubulin, an established SIRT2 biomarker, at doses comparable with the biochemical IC₅₀ data, while suppressing MCF‐7 proliferation at higher concentrations. In concordance with the recent reports that suggest SIRT2 inhibition is a potential strategy for the treatment of Parkinson’s disease, we find that compound ICL‐SIRT078 has a significant neuroprotective effect in a lactacystin‐induced model of Parkinsonian neuronal cell death in the N27 cell line. These results encourage further investigation into the effects of ICL‐SIRT078, or an optimised derivative thereof, as a candidate neuroprotective agent in in vivo models of Parkinson’s disease.     

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.     

A Highly Potent and Selective Caspase 1 Inhibitor that Utilizes a Key 3‐Cyanopropanoic Acid Moiety

Herein, we examine the potential of a nitrile‐containing propionic acid moiety as an electrophile for covalent attack by the active‐site cysteine residue of caspase 1. The syntheses of several cyanopropanate‐containing small molecules based on the optimized peptidic scaffold of prodrug VX‐765 were accomplished. These compounds were found to be potent inhibitors of caspase 1 (IC₅₀ values ≤1 nM ). Examination of these novel small molecules against a caspase panel demonstrated an impressive degree of selectivity for caspase 1 inhibition over other caspase isozymes. Assessment of hydrolytic stability and selected ADME properties highlighted these agents as potentially useful tools for studying caspase 1 down‐regulation in various settings, including in vivo analyses.     

C3‐Symmetrical π‐Scaffolds: Useful Building Blocks to Construct Helical Supramolecular Polymers

In this review, we highlight some relevant examples of C₃‐symmetrical molecules that have been reported to form supramolecular polymers and helical aggregates. In particular, the number and type of non‐covalent forces are key to bias the supramolecular polymerization leading from a simple isodesmic or cooperative mechanism to a more complex self‐assembly process, i. e. pathway complexity. Furthermore, the attachment of stereogenic centres at the peripheral side chains of the C₃‐systems provokes efficient transfer and amplification of chirality phenomena when directional and specific non‐covalent interactions operate. Interestingly, the incorporation of hydrophilic side chains induces the formation of organized aggregates in aqueous media with potential biomedical applications. Overall, the examples shown in this review on C₃‐symmetrical scaffolds illustrate the relevance of this molecular shape in the development of functional supramolecular structures.     

Clickable 4‐Oxo‐β‐lactam‐Based Selective Probing for Human Neutrophil Elastase Related Proteomes

Human neutrophil elastase (HNE) is a serine protease associated with several inflammatory processes such as chronic obstructive pulmonary disease (COPD). The precise involvement of HNE in COPD and other inflammatory disease mechanisms has yet to be clarified. Herein we report a copper‐catalyzed alkyne–azide 1,3‐dipolar cycloaddition (CuAAC, or ′click′ chemistry) approach based on the 4‐oxo‐β‐lactam warhead that yielded potent HNE inhibitors containing a triazole moiety. The resulting structure–activity relationships set the basis to develop fluorescent and biotinylated activity‐based probes as tools for molecular functional analysis. Attaching the tags to the 4‐oxo‐β‐lactam scaffold did not affect HNE inhibitory activity, as revealed by the IC₅₀ values in the nanomolar range (56–118 nm ) displayed by the probes. The nitrobenzoxadiazole (NBD)‐based probe presented the best binding properties (ligand efficiency (LE)=0.31) combined with an excellent lipophilic ligand efficiency (LLE=4.7). Moreover, the probes showed adequate fluorescence properties, internalization in human neutrophils, and suitable detection of HNE in the presence of a large excess of cell lysate proteins. This allows the development of activity‐based probes with promising applications in target validation and identification, as well as diagnostic tools.     

3‐Amidocoumarins as Potential Multifunctional Agents against Neurodegenerative Diseases

Monoamine oxidase (MAO) generates reactive oxygen species (ROS), which cause neuronal cell death, causing neurodegeneration. Agents that are able to concurrently inhibit MAO and scavenge free radicals represent promising multifunctional neuroprotective agents that could be used to delay or slow the progression of neurodegenerative diseases. In this work, variously substituted 3‐amidocoumarins are described that exert neuroprotection in vitro against hydrogen peroxide in rat cortical neurons, as well as antioxidant activity in a 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH?) radical scavenging assay. Selective and reversible inhibitors of the MAO‐B isoform were identified. Interestingly, in the case of the 3‐benzamidocoumarins, substitution at position 4 with a hydroxy group abolishes MAO‐B activity, but the compounds remain active in the neuroprotection model. Further evaluation of 3‐heteroarylamide derivatives indicates that it is the nature of the heterocycle that determines the neuroprotective effects. Evaluation in a parallel artificial membrane permeability assay (PAMPA) highlighted the need to further improve the blood–brain barrier permeability of this compound class. However, the compounds described herein adhere to Lipinski′s rule of five, suggesting that this novel scaffold has desirable properties for the development of potential drug candidates.     

New Palladium‐Catalyzed Domino Reaction with Intramolecular Ring Closure of an N‐(2‐Chloro‐3‐heteroaryl)arylamide: First Synthesis of Oxazolo[4,5‐b]pyrazines

The synthesis of novel planar heterocycles is at the heart of basic research as such scaffolds constitute key building blocks in important diverse areas of research: drug discovery, material sciences, and pesticides. The well‐known benzoxazole is often contained in drug candidates but tweaking its lipophilicity and target interaction points are often desired. In this respect, the oxazolo[4,5‐b]pyrazine is an attractive heterocyclic scaffold as it possesses increased water solubility as well as two additional hydrogen bonding acceptors. We here report a new Pd(II)‐catalyzed domino reaction comprising the first Pd(II)‐assisted intramolecular cyclization of an N‐(2‐chloro‐3‐heteroaryl)arylamide and validate its value by application to the first synthesis of 2‐substituted oxazolo[4,5‐b]pyrazines. We demonstrate that a bidentate phosphorus ligand as well as the presence of an aromatic nitrogen atom is required for the domino reaction to proceed. The robustness of the methodology is confirmed by the synthesis of 23 2‐substituted oxazolo[4,5‐b]pyrazine analogues in good‐to‐high yields and containing both electron‐withdrawing as well as electron‐donating substituents on the reacting arylamide.

     

Development of Rhodesain Inhibitors with a 3‐Bromoisoxazoline Warhead

Novel rhodesain inhibitors were obtained by combining an enantiomerically pure 3‐bromoisoxazoline warhead with a specific peptidomimetic recognition moiety. All derivatives behaved as inhibitors of rhodesain, with low micromolar K_i values. Their activity against the enzyme was found to be paralleled by an in vitro antitrypanosomal activity, with IC₅₀ values in the mid‐micromolar range. Notably, a preference for parasitic over human proteases, specifically cathepsins B and L, was observed.     

Synthesis and Structure–Activity Relationship Studies of 2‐(1,3,4‐Oxadiazole‐2(3H)‐thione)‐3‐amino‐5‐arylthieno[2,3‐b]pyridines as Inhibitors of DRAK2

In recent years, DAPK‐related apoptosis‐inducing protein kinase 2 (DRAK2) has emerged as a promising target for the treatment of a variety of autoimmune diseases and for the prevention of graft rejection after organ transplantation. However, medicinal chemistry optimization campaigns for the discovery of novel small‐molecule inhibitors of DRAK2 have not yet been published. Screening of a proprietary compound library led to the discovery of a benzothiophene analogue that displays an affinity constant (K_d) value of 0.25 μM . Variation of the core scaffold and of the substitution pattern afforded a series of 5‐arylthieno[2,3‐b]pyridines with strong binding affinity (K_d=0.008 μM for the most potent representative). These compounds also show promising activity in a functional biochemical DRAK2 enzyme assay, with an IC₅₀ value of 0.029 μM for the most potent congener. Selectivity profiling of the most potent compounds revealed that they lack selectivity within the DAPK family of kinases. However, one of the less potent analogues is a selective ligand for DRAK2 and can be used as starting point for the synthesis of selective and potent DRAK2 inhibitors.     

6‐Hydroxybenzothiophene Ketones: Potent Inhibitors of 17β‐Hydroxysteroid Dehydrogenase Type 1 (17β‐HSD1) Owing to Favorable Molecule Geometry and Conformational Preorganization

The inhibition of 17β‐hydroxysteroid dehydrogenase type 1 (17β‐HSD1), which catalyzes the conversion of estrone into the potent estrogen receptor agonist estradiol (E2), is discussed as a novel therapeutic approach for the treatment of estrogen‐dependent diseases. Because the reduction of E2 would be basically limited to the target tissues, this approach is expected to cause fewer side effects than the currently employed antihormonal therapies. Recently, we reported on 6‐hydroxybenzothiazole ketones as a new class of 17β‐HSD1 inhibitors with a notable activity/selectivity profile. In an attempt to further optimize these parameters, we modified the benzothiazole core by a systematic bioisosteric replacement. Thus, we were able to identify a new 6‐hydroxybenzothiophene derivative that displayed stronger inhibition of 17β‐HSD1 (IC₅₀=13 nM ) and that was also more selective than a benzothiazole analog. Using ab initio calculations, we found that the higher potency of the 6‐hydroxybenzothiophene derivative was probably due to more favorable conformational preorganization of the scaffold for binding to the enzyme.     

Thrombin‐Inhibiting Anticoagulant Liposomes: Development and Characterization

Many peptides and peptidomimetic drugs suffer from rapid clearance in vivo; this can be reduced by increasing their size through oligomerization or covalent conjugation with polymers. As proof of principle, an alternative strategy for drug oligomerization is described, in which peptidomimetic thrombin inhibitors are incorporated into the liposome surface. For this purpose, the inhibitor moieties were covalently coupled to a palmitic acid residue through a short bifunctionalized ethylene glycol spacer. These molecules were directly added to the lipid mixture used for liposome preparation. The obtained liposomes possess strong thrombin inhibitory potency in enzyme kinetic measurements and anticoagulant activity in plasma. Their strong potency and positive ζ potential indicate that large amounts of the benzamidine‐derived inhibitors are located on the surface of the liposomes. This concept should be applicable to other drug molecules that suffer from rapid elimination and allow covalent modification with a suitable fatty acid residue.     

Identification of a Small‐Molecule Inhibitor of HIV‐1 Assembly that Targets the Phosphatidylinositol (4,5)‐bisphosphate Binding Site of the HIV‐1 Matrix Protein

The development of drug resistance remains a critical problem for current HIV‐1 antiviral therapies, creating a need for new inhibitors of HIV‐1 replication. We previously reported on a novel anti‐HIV‐1 compound, N²‐(phenoxyacetyl)‐N‐[4‐(1‐piperidinylcarbonyl)benzyl]glycinamide ( 14 ), that binds to the highly conserved phosphatidylinositol (4,5)‐bisphosphate (PI(4,5)P₂) binding pocket of the HIV‐1 matrix (MA) protein. In this study, we re‐evaluate the hits from the virtual screen used to identify compound 14 and test them directly in an HIV‐1 replication assay using primary human peripheral blood mononuclear cells. This study resulted in the identification of three new compounds with antiviral activity; 2‐(4‐{[3‐(4‐fluorophenyl)‐1,2,4‐oxadiazol‐5‐yl]methyl})‐1‐piperazinyl)‐N‐(4‐methylphenyl)acetamide ( 7 ), 3‐(2‐ethoxyphenyl)‐5‐[[4‐(4‐nitrophenyl)piperazin‐1‐yl]methyl]‐1,2,4‐oxadiazole ( 17 ), and N‐[4‐ethoxy‐3‐(1‐piperidinylsulfonyl)phenyl]‐2‐(imidazo[2,1‐b][1,3]thiazol‐6‐yl)acetamide ( 18 ), with compound 7 being the most potent of these hits. Mechanistic studies on 7 demonstrated that it directly interacts with and functions through HIV‐1 MA. In accordance with our drug target, compound 7 competes with PI(4,5)P₂ for MA binding and, as a result, diminishes the production of new virus. Mutation of residues within the PI(4,5)P₂ binding site of MA decreased the antiviral effect of compound 7 . Additionally, compound 7 displays a broadly neutralizing anti‐HIV activity, with IC₅₀ values of 7.5–15.6 μM for the group M isolates tested. Taken together, these results point towards a novel chemical probe that can be used to more closely study the biological role of MA and could, through further optimization, lead to a new class of anti‐HIV‐1 therapeutics.     

N‐Benzyl‐4‐((heteroaryl)methyl)benzamides: A New Class of Direct NADH‐Dependent 2‐trans Enoyl–Acyl Carrier Protein Reductase (InhA) Inhibitors with Antitubercular Activity

Isoniazid (INH) remains one of the cornerstones of antitubercular chemotherapy for drug‐sensitive strains of M. tuberculosis bacteria. However, the increasing prevalence of multidrug‐resistant (MDR) and extensively drug‐resistant (XDR) strains containing mutations in the KatG enzyme, which is responsible for the activation of INH into its antitubercular form, have rendered this drug of little or no use in many cases of drug‐resistant tuberculosis. Presented herein is a novel family of antitubercular direct NADH‐dependent 2‐trans enoyl–acyl carrier protein reductase (InhA) inhibitors based on an N‐benzyl‐4‐((heteroaryl)methyl)benzamide template; unlike INH, these do not require prior activation by KatG. Given their direct InhA target engagement, these compounds should be able to circumvent KatG‐related resistance in the clinic. The lead molecules were shown to be potent inhibitors of InhA and showed activity against M. tuberculosis bacteria. This new family of inhibitors was found to be chemically tractable, as exemplified by the facile synthesis of analogues and the establishment of structure–activity relationships. Furthermore, a co‐crystal structure of the initial hit with the enzyme is disclosed, providing valuable information toward the design of new InhA inhibitors for the treatment of MDR/XDR tuberculosis.     

α‐Keto Phenylamides as P1′‐Extended Proteasome Inhibitors

The major challenge for proteasome inhibitor design lies in achieving high selectivity for, and activity against, the target, which requires specific interactions with the active site. Novel ligands aim to overcome off‐target‐related side effects such as peripheral neuropathy, which is frequently observed in cancer patients treated with the FDA‐approved proteasome inhibitors bortezomib ( 1 ) or carfilzomib ( 2 ). A systematic comparison of electrophilic headgroups recently identified the class of α‐keto amides as promising for next generation drug development. On the basis of crystallographic knowledge, we were able to develop a structure–activity relationship (SAR)‐based approach for rational ligand design using an electronic parameter (Hammett’s σ) and in silico molecular modeling. This resulted in the tripeptidic α‐keto phenylamide BSc4999 [(S)‐3‐(benzyloxycarbonyl‐(S)‐leucyl‐(S)‐leucylamino)‐5‐methyl‐2‐oxo‐N‐(2,4‐dimethylphenyl)hexanamide, 6 a ], a highly potent (IC₅₀=38 nM ), cell‐permeable, and slowly reversible covalent inhibitor which targets both the primed and non‐primed sites of the proteasome’s substrate binding channel as a special criterion for selectivity. The improved inhibition potency and selectivity of this new α‐keto phenylamide makes it a promising candidate for targeting a wider range of tumor subtypes than commercially available proteasome inhibitors and presents a new candidate for future studies.     

The Chemical Biology of Phosphoinositide 3‐Kinases

Since its discovery in the late 1980s, phosphoinositide 3‐kinase (PI3K), and its isoforms have arguably reached the forefront of signal transduction research. Regulation of this lipid kinase, its functions, its effectors, in short its entire signaling network, has been extensively studied. PI3K inhibitors are frequently used in biochemistry and cell biology. In addition, many pharmaceutical companies have launched drug‐discovery programs to identify modulators of PI3Ks. Despite these efforts and a fairly good knowledge of the PI3K signaling network, we still have only a rudimentary picture of the signaling dynamics of PI3K and its lipid products in space and time. It is therefore essential to create and use novel biological and chemical tools to manipulate the phosphoinositide signaling network with spatial and temporal resolution. In this review, we discuss the current and potential future tools that are available and necessary to unravel the various functions of PI3K and its isoforms.