Assessment of Tractable Cysteines for Covalent Targeting by Screening Covalent Fragments

Targeted covalent inhibition and the use of irreversible chemical probes are important strategies in chemical biology and drug discovery. To date, the availability and reactivity of cysteine residues amenable for covalent targeting have been evaluated by proteomic and computational tools. Herein, we present a toolbox of fragments containing a 3,5-bis(trifluoromethyl)phenyl core that was equipped with chemically diverse electrophilic warheads showing a range of reactivities. We characterized the library members for their reactivity, aqueous stability and specificity for nucleophilic amino acids. By screening this library against a set of enzymes amenable for covalent inhibition, we showed that this approach experimentally characterized the accessibility and reactivity of targeted cysteines. Interesting covalent fragment hits were obtained for all investigated cysteine-containing enzymes.     

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.     

Small Molecules Inspired by the Natural Product Withanolides as Potent Inhibitors of Wnt Signaling

Wnt signaling is a fundamental pathway that drives embryonic development and is essential for stem cell maintenance and tissue homeostasis. Dysregulation of Wnt signaling is linked to various diseases, and a constitutively active Wnt pathway drives tumorigenesis. Thus, disruption of the Wnt response is deemed a promising strategy for cancer drug discovery. However, only few clinical drug candidates that target Wnt signaling are available so far, and new small‐molecule modulators of Wnt‐related processes are in high demand. Here we describe the synthesis of small molecules inspired by withanolide natural products by using a pregnenolone‐derived β‐lactone as the key intermediate that was transformed into a δ‐lactone appended to the D‐ring of the steroidal scaffold. This natural‐product‐inspired compound library contained potent inhibitors of Wnt signaling that act upstream of the destruction complex to stabilize Axin in a tankyrase‐independent manner.     

LEGO‐Inspired Drug Design: Unveiling a Class of Benzo[d]thiazoles Containing a 3,4‐Dihydroxyphenyl Moiety as Plasma Membrane H+‐ATPase Inhibitors

The fungal plasma membrane H⁺‐ATPase (Pma1p) is a potential target for the discovery of new antifungal agents. Surprisingly, no structure–activity relationship studies for small molecules targeting Pma1p have been reported. Herein, we disclose a LEGO‐inspired fragment assembly strategy for the design, synthesis, and discovery of benzo[d]thiazoles containing a 3,4‐dihydroxyphenyl moiety as potential Pma1p inhibitors. A series of 2‐(benzo[d]thiazol‐2‐ylthio)‐1‐(3,4‐dihydroxyphenyl)ethanones was found to inhibit Pma1p, with the most potent IC₅₀ value of 8 μm in an in vitro plasma membrane H⁺‐ATPase assay. These compounds were also found to strongly inhibit the action of proton pumping when Pma1p was reconstituted into liposomes. 1‐(3,4‐Dihydroxyphenyl)‐2‐((6‐(trifluoromethyl)benzo[d]thiazol‐2‐yl)thio)ethan‐1‐one (compound 38 ) showed inhibitory activities on the growth of Candida albicans and Saccharomyces cerevisiae, which could be correlated and substantiated with the ability to inhibit Pma1p in vitro.     

Towards Tuneable Retaining Glycosidase‐Inhibiting Peptides by Mimicry of a Plant Flavonol Warhead

Retaining glycosidases are an important class of enzymes involved in glycan degradation. To study better the role of specific enzymes in deglycosylation processes, and thereby the importance of particular glycosylation patterns, a set of potent inhibitors, each specific to a particular glycosidase, would be an invaluable toolkit. Towards this goal, we detail here a more in‐depth study of a prototypical macrocyclic peptide inhibitor of the model retaining glycosidase human pancreatic α‐amylase (HPA). Notably, incorporation of l ‐DOPA into this peptide affords an inhibitor of HPA with potency that is tenfold higher (K_i=480 pm ) than that of the previously found consensus sequence. This represents a first successful step in converting a recently discovered natural‐product‐derived motif, already specific for the catalytic side‐chain arrangement conserved in the active sites of retaining glycosidases, into a tuneable retaining glycosidase inhibition warhead.     

Reversible Covalent Inhibition of eEF‐2K by Carbonitriles

eEF‐2K is a potential target for treating cancer. However, potent specific inhibitors for this enzyme are lacking. Previously, we identified 2,6‐diamino‐4‐(2‐fluorophenyl)‐4H‐thiopyran‐3,5‐dicarbonitrile (DFTD) as an inhibitor of eEF‐2K. Here we describe its mechanism of action against eEF‐2K, on the basis of kinetic, mutational, and docking studies, and use chemoinformatic approaches to identify a similar class of carbonitrile‐containing compounds that exhibit the same mechanism of action. We show that DFTD behaves as a reversible covalent inhibitor of eEF‐2K with a two‐step mechanism of inhibition: a fast initial binding step, followed by a slower reversible inactivation step. Molecular docking suggests that a nitrile group of DFTD binds within 4.5 Å of the active site Cys146 to form a reversible thioimidate adduct. Because Cys146 is not conserved amongst other related kinases, targeting this residue holds promise for the development of selective covalent inhibitors of eEF‐2K.     

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 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.     

A Novel Competitive Class of α‐Glucosidase Inhibitors: (E)‐1‐Phenyl‐3‐(4‐Styrylphenyl)Urea Derivatives

Competitive glycosidase inhibitors are generally sugar mimics that are costly and tedious to obtain because they require challenging and elongated chemical synthesis, which must be stereo‐ and regiocontrolled. Here, we show that readily accessible achiral (E)‐1‐phenyl‐3‐(4‐strylphenyl)ureas are potent competitive α‐glucosidase inhibitors. A systematic synthesis study shows that the 1‐phenyl moiety on the urea is critical for ensuring competitive inhibition, and substituents on both terminal phenyl groups contribute to inhibition potency. The most potent inhibitor, compound 12 (IC₅₀=8.4 μM , K_i=3.2 μM ), manifested a simple slow‐binding inhibition profile for α‐glucosidase with the kinetic parameters k₃=0.005256 μM ^?1 min^?1, k₄=0.003024 min^?1, and ${K{{{\rm app}\hfill \atop {\rm i}\hfill}}}$ =0.5753 μM .     

The Natural Products Beauveriolide I and III: a New Class of β‐Amyloid‐Lowering Compounds

Attacking Alzheimer's by ACAT : The aggregation of β‐amyloid peptides, especially Aβ₄₂, into senile plaques is a hallmark of Alzheimer's disease (AD). We show that the fungal natural products beauveriolides I and III can potently decrease Aβ secretion from cells expressing human amyloid precursor protein; this offers a potential new scaffold for the development of compounds with proven bioavailability for the treatment of AD.

     

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.     

Synthesis and Structure–Activity Correlation of a Brunsvicamide‐Inspired Cyclopeptide Collection

Cyanobacterial cyclopeptides : A series of analogues of the cyanobacterial cyclopeptide brunsvicamide A was prepared by effective solid‐support‐based total synthesis. Variations in stereochemistry revealed the importance of the D ‐lysine and the L ‐isoleucine residues for the substrate‐competitive inhibitory activity of brunsvicamide A against carboxypeptidase A.

     

Towards Dynamic Drug Design: Identification and Optimization of β‐Galactosidase Inhibitors from a Dynamic Hemithioacetal System

A discovery strategy relying on the identification of fragments through resolution of a constitutional dynamic system, coupled to subsequent static ligand design and optimization, is demonstrated. The strategic design and synthesis of the best molecular fragments identified from a dynamic hemithioacetal system into static ligand structures yielded a range of β‐galactosidase inhibitors. Two series of structures mimicking the hemithioacetal motif were envisaged: thioglycosides and C‐glycosides. Inhibition studies provided important structural information for the two groups, and 1‐thiobenzyl‐β‐D ‐galactopyranoside demonstrated the best inhibitory effects.     

Development of Phosphodiesterase–Protein-Kinase Complexes as Novel Targets for Discovery of Inhibitors with Enhanced Specificity

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (protein kinases, PKs) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PKs generates an expanded active site that enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PKs and aid in signal termination. PDEs are important drug targets, and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targeted PDE–PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay was adapted to identify inhibitors that block cyclic nucleotide pockets in PDE–PK complexes in one mode and disrupt protein-protein interactions between PDEs and PKs in a second mode. We tested this approach with three different systems—cAMP-specific PDE8–PKAR, cGMP-specific PDE5–PKG, and dual-specificity RegA–R_D complexes—and ranked inhibitors according to their inhibition potency. Targeting PDE–PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.     

In Silico Drug Repositioning to Target the SARS-CoV-2 Main Protease as Covalent Inhibitors Employing a Combined Structure-Based Virtual Screening Strategy of Pharmacophore Models and Covalent Docking

The epidemic caused by the SARS-CoV-2 coronavirus, which has spread rapidly throughout the world, requires urgent and effective treatments considering that the appearance of viral variants limits the efficacy of vaccines. The main protease of SARS-CoV-2 (M^pro) is a highly conserved cysteine proteinase, fundamental for the replication of the coronavirus and with a specific cleavage mechanism that positions it as an attractive therapeutic target for the proposal of irreversible inhibitors. A structure-based strategy combining 3D pharmacophoric modeling, virtual screening, and covalent docking was employed to identify the interactions required for molecular recognition, as well as the spatial orientation of the electrophilic warhead, of various drugs, to achieve a covalent interaction with Cys145 of M^pro. The virtual screening on the structure-based pharmacophoric map of the SARS-CoV-2 M^pro in complex with an inhibitor N3 (reference compound) provided high efficiency by identifying 53 drugs (FDA and DrugBank databases) with probabilities of covalent binding, including N3 (Michael acceptor) and others with a variety of electrophilic warheads. Adding the energy contributions of affinity for non-covalent and covalent docking, 16 promising drugs were obtained. Our findings suggest that the FDA-approved drugs Vaborbactam, Cimetidine, Ixazomib, Scopolamine, and Bicalutamide, as well as the other investigational peptide-like drugs (DB04234, DB03456, DB07224, DB7252, and CMX-2043) are potential covalent inhibitors of SARS-CoV-2 M^pro.     

Rational Design of Resorcylic Acid Lactone Analogues as Covalent MNK1/2 Kinase Inhibitors by Tuning the Reactivity of an Enamide Michael Acceptor

Recent biological and computational advances in drug design have led to renewed interest in targeted covalent inhibition as an efficient and practical approach for the development of new drugs. As part of our continuing efforts in the exploration of the therapeutic potential of resorcylic acid lactones (RALs), we report herein the design, synthesis, and biological evaluation of conveniently accessible RAL enamide analogues as novel covalent inhibitors of MAP kinase interacting kinases (MNKs). In this study, we have successfully demonstrated that the covalent binding ability of RAL enamides can be tuned by attaching an electron‐withdrawing motif, such as an acyl group, to enhance its reactivity toward the cysteine residues at the MNK1/2 binding sites. We have also shown that ¹H NMR spectroscopy is a convenient and effective tool for screening the covalent binding activities of enamides using cysteamine as a mimic of the key cysteine residue in the enzyme, whereas mass spectrometric analysis confirms covalent modification of the kinases. Preliminary optimization of the initial hit led to the discovery of enamides with low micromolar activity in MNK assays. Cancer cell line assays have identified RAL enamides that inhibit the growth of cancer cells with similar potency to the natural product L‐783 ,277.