De Novo Design,Synthesis and Evaluation of Benzylpiperazine Derivatives as Highly Selective Binders of Mcl‐1

Considerable efforts have been made to the development of small‐molecule inhibitors of antiapoptotic B‐cell lymphoma 2 (Bcl‐2) family proteins (such as Bcl‐2, Bcl‐x_L, and Mcl‐1) as a new class of anticancer therapies. Unlike general inhibitors of the entire family, selective inhibitors of each member protein can hopefully reduce the adverse side effects in chemotherapy treatments of cancers overexpressing different Bcl‐2 family proteins. In this study, we designed four series of benzylpiperazine derivatives as plausible Bcl‐2 inhibitors based on the outcomes of a computational algorithm. A total of 81 compounds were synthesized, and their binding affinities to Bcl‐2, Bcl‐x_L, and Mcl‐1 measured. Encouragingly, 22 compounds exhibited binding affinities in the micromolar range (K_i<20 μM ) to at least one target protein. Moreover, some compounds were observed to be highly selective binders to Mcl‐1 with no detectable binding to Bcl‐2 or Bcl‐x_L, among which the most potent one has a K_i value of 0.18 μM for Mcl‐1. Binding modes of four selected compounds to Mcl‐1 and Bcl‐x_L were derived through molecular docking and molecular dynamics simulations. It seems that the binding affinity and selectivity of these compounds can be reasonably interpreted with these models. Our study demonstrated the possibility for obtaining selective Mcl‐1 inhibitors with relatively simple chemical scaffolds. The active compounds identified by us could be used as lead compounds for developing even more potent selective Mcl‐1 inhibitors with potential pharmaceutical applications.     

Characterization of the Stereochemical Structures of 2H‐Thiazolo[3,2‐a]pyrimidine Compounds and Their Binding Affinities for Anti‐apoptotic Bcl‐2 Family Proteins

In a previous study we reported a class of compounds with a 2H‐thiazolo[3,2‐a]pyrimidine core structure as general inhibitors of anti‐apoptotic Bcl‐2 family proteins. However, the absolute stereochemical configuration of one carbon atom on the core structure remained unsolved, and its potential impact on the binding affinities of compounds in this class was unknown. In this study, we obtained pure R and S enantiomers of four selected compounds by HPLC separation and chiral synthesis. The absolute configurations of these enantiomers were determined by comparing their circular dichroism spectra to that of an appropriate reference compound. In addition, a crystal structure of one selected compound revealed the exocyclic double bond in these compounds to be in the Z configuration. The binding affinities of all four pairs of enantiomers to Bcl‐x_L, Bcl‐2, and Mcl‐1 proteins were measured in a fluorescence‐polarization‐based binding assay, yielding inhibition constants (K_i values) ranging from 0.24 to 2.20 μM . Interestingly, our results indicate that most R and S enantiomers exhibit similar binding affinities for the three tested proteins. A binding mode for this compound class was derived by molecular docking and molecular dynamics simulations to provide a reasonable interpretation of this observation.     

Two‐Face,Two‐Turn α‐Helix Mimetics Based on a Cross‐Acridine Scaffold: Analogues of the Bim BH3 Domain

The design of a cross‐acridine scaffold mimicking the i, i+3, i+5, and i+7 residues distributed over a two‐face, two‐turn α‐helix is described. Docking studies and 2D ¹H,¹⁵N HSQC NMR spectroscopy provide compelling evidence that compound 3 d accurately reproduces the arrangement of four hotspots in the Bim BH3 peptide to permit binding to the Mcl‐1 and Bcl‐2 proteins (K_i 0.079 and 0.056 μM , respectively). Furthermore, the hotspot mutation could also be mimicked by individual or multiple deletions of side chains on the scaffold.     

Structure‐Guided Rational Design of α/β‐Peptide Foldamers with High Affinity for BCL‐2 Family Prosurvival Proteins

We have used computational methods to improve the affinity of a foldamer ligand for its target protein. The effort began with a previously reported α/β‐peptide based on the BH3 domain of the proapoptotic protein Puma; this foldamer binds tightly to Bcl‐x_L but weakly to Mcl‐1. The crystal structure of the Puma‐derived α/β‐peptide complexed to Bcl‐x_L was used as the basis for computational design of variants intended to display improved binding to Mcl‐1. Molecular modelling suggested modification of three α residues of the original α/β backbone. Individually, each substitution caused only a modest (4‐ to 15‐fold) gain in affinity; however, together the three substitutions led to a 250‐fold increase in binding to Mcl‐1. These modifications had very little effect on affinity for Bcl‐x_L. Crystal structures of a number of the new α/β‐peptides bound to either Mcl‐1 or Bcl‐x_L validated the selection of each substitution. Overall, our findings demonstrate that structure‐guided rational design can be used to improve affinity and alter partner selectivity of peptidic ligands with unnatural backbones that bind to specific protein partners.     

Discovery and development of thiazolo[3,2-a]pyrimidinone derivatives as general inhibitors of Bcl-2 family proteins

A class of compounds with a common thiazolo[3,2‐a]pyrimidinone motif has been developed as general inhibitors of Bcl‐2 family proteins. The lead compound was originally identified in a random screening of a small compound library using a fluorescence polarization‐based competitive binding assay. Its binding to the Bcl‐x_L protein was further confirmed by ¹⁵N‐HSQC NMR experiments. Structural modifications on the lead compound were guided by the outcomes of molecular modeling studies. Among the 42 compounds obtained, a number of them exhibited much improved binding affinities to Bcl‐2 family proteins as compared to the lead compound. The most potent compound, BCL‐LZH‐ 40 , inhibited the binding of BH3 peptides to Bcl‐x_L, Bcl‐2, and Mcl‐1 with inhibition constants (K_i) of 17, 534, and 200 nM , respectively.     

Discovery of a Small‐Molecule pBcl‐2 Inhibitor that Overcomes pBcl‐2‐Mediated Resistance to Apoptosis

Although the role of Bcl‐2 phosphorylation is still under debate, it has been identified in a resistance mechanism to BH3 mimetics, for example ABT‐737 and S1 . We identified an S1 analogue, S1‐16 , as a small‐molecule inhibitor of pBcl‐2. S1‐16 efficiently kills EEE‐Bcl‐2 (a T69E, S70E, and S87E mutant mimicking phosphorylation)‐expressing HL‐60 cells and high endogenously expressing pBcl‐2 cells, by disrupting EEE‐Bcl‐2 or native pBcl‐2 interactions with Bax and Bak, followed by apoptosis. In vitro binding assays showed that S1‐16 binds to the BH3 binding groove of EEE‐Bcl‐2 (K_d=0.38 μM by ITC; IC₅₀=0.16 μM by ELISA), as well as nonphosphorylated Bcl‐2 (npBcl‐2; K_d=0.38 μM ; IC₅₀=0.12 μM ). However, ABT‐737 and S1 had much weaker affinities to EEE‐Bcl‐2 (IC₅₀=1.43 and >10 μM , respectively), compared with npBcl‐2 (IC₅₀=0.011 and 0.74 μM , respectively). The allosteric effect on BH3 binding groove by Bcl‐2 phosphorylation in the loop region was illustrated for the first time.     

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 .     

Diaryl‐Substituted Azolylthioacetamides: Inhibitor Discovery of New Delhi Metallo‐β‐Lactamase‐1 (NDM‐1)

The emergence and spread of antibiotic‐resistant pathogens is a global public health problem. Metallo‐β‐lactamases (MβLs) such as New Delhi MβL‐1 (NDM‐1) are principle contributors to the emergence of resistance because of their ability to hydrolyze almost all known β‐lactam antibiotics including penicillins, cephalosporins, and carbapenems. A clinical inhibitor of MBLs has not yet been found. In this study we developed eighteen new diaryl‐substituted azolylthioacetamides and found all of them to be inhibitors of the MβL L1 from Stenotrophomonas maltophilia (K_i<2 μM ), thirteen to be mixed inhibitors of NDM‐1 (K_i<7 μM ), and four to be broad‐spectrum inhibitors of all four tested MβLs CcrA from Bacteroides fragilis, NDM‐1 and ImiS from Aeromonas veronii, and L1 (K_i<52 μM ), which are representative of the B1a, B1b, B2, and B3 subclasses, respectively. Docking studies revealed that the azolylthioacetamides, which have the broadest inhibitory activity, coordinate to the Zn^II ion(s) preferentially via the triazole moiety, while other moieties interact mostly with the conserved active site residues Lys224 (CcrA, NDM‐1, and ImiS) or Ser221 (L1).     

Probing the Peptidylglycine α‐Hydroxylating Monooxygenase Active Site with Novel 4‐Phenyl‐3‐butenoic Acid Based Inhibitors

Specific inhibition of the copper‐containing peptidylglycine α‐hydroxylating monooxygenase (PHM), which catalyzes the post‐translational modification of peptides involved in carcinogenesis and tumor progression, constitutes a new approach for combating cancer. We carried out a structure–activity study of new compounds derived from a well‐known PHM substrate analogue, the olefinic compound 4‐phenyl‐3‐butenoic acid (PBA). We designed, synthesized, and tested various PBA derivatives both in vitro and in silico. We show that it is possible to increase PBA affinity for PHM by appropriate functionalization of its aromatic nucleus. Compound 2 d , for example, bears a meta‐benzyloxy substituent, and exhibits better inhibition features (K_i=3.9 μM , k_inact/K_i=427 M ^?1 s^?1) than the parent PBA (K_i=19 μM , k_inact/K_i=82 M ^?1 s^?1). Docking calculations also suggest two different binding modes for PBA derivatives; these results will aid in the development of further PHM inhibitors with improved features.     

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.     

Synthesis and Evaluation of 1‐(1‐(Benzo[b]thiophen‐2‐yl)cyclohexyl)piperidine (BTCP) Analogues as Inhibitors of Trypanothione Reductase

Thirty two analogues of phencyclidine were synthesised and tested as inhibitors of trypanothione reductase (TryR), a potential drug target in trypanosome and leishmania parasites. The lead compound BTCP ( 1 , 1‐(1‐benzo[b]thiophen‐2‐yl‐cyclohexyl) piperidine) was found to be a competitive inhibitor of the enzyme (K_i=1 μM ) and biologically active against bloodstream T. brucei (EC₅₀=10 μM ), but with poor selectivity against mammalian MRC5 cells (EC₅₀=29 μM ). Analogues with improved enzymatic and biological activity were obtained. The structure–activity relationships of this novel series are discussed.     

Design,Synthesis, in vitro MAO‐B Inhibitory Evaluation,and Computational Studies of Some 6‐Nitrobenzothiazole‐Derived Semicarbazones

Monoamine oxidase B (MAO‐B) is an important drug target for the treatment of neurological disorders. A series of 6‐nitrobenzothiazole‐derived semicarbazones were designed, synthesized, and evaluated as inhibitors of the rat brain MAO‐B isoenzyme. Most of the compounds were found to be potent inhibitors of MAO‐B, with IC₅₀ values in the nanomolar to micromolar range. Molecular docking studies were performed with AutoDock 4.2 to deduce the affinity and binding mode of these inhibitors toward the MAO‐B active site. The free energies of binding (ΔG) and inhibition constants (K_i) of the docked compounds were calculated by the Lamarckian genetic algorithm (LGA) of AutoDock 4.2. Good correlations between the calculated and experimental results were obtained. 1‐[(4‐Chlorophenyl)(phenyl)methylene]‐4‐(6‐nitrobenzothiazol‐2‐yl)semicarbazide emerged as the lead MAO‐B inhibitor, with top ranking in both the experimental MAO‐B assay (IC₅₀: 0.004±0.001 μM ) and in computational docking studies (K_i: 1.08 μM ). Binding mode analysis of potent inhibitors suggests that these compounds are well accommodated by the MAO‐B active site through stable hydrophobic and hydrogen bonding interactions. Interestingly, the 6‐nitrobenzothiazole moiety is stabilized in the substrate cavity with the aryl or diaryl residues extending up into the entrance cavity of the active site. According to our results, docking experiments could be an interesting approach for predicting the activity and binding interactions of this class of semicarbazones against MAO‐B. Thus, a binding site model consisting of three essential pharmacophoric features is proposed, and this can be used for the design of future MAO‐B inhibitors.     

Molecular Recognition of Two 2,4‐syn‐Functionalized (S)‐Glutamate Analogues by the Kainate Receptor GluK3 Ligand Binding Domain

The kainate receptors are the least studied subfamily of ionotropic glutamate receptors. These receptors are thought to have a neuromodulatory role and have been associated with a variety of disorders in the central nervous system. This makes kainate receptors interesting potential drug targets. Today, structures of the ligand binding domain (LBD) of the kainate receptor GluK3 are only known in complex with the endogenous agonist glutamate, the natural product kainate, and two synthetic agonists. Herein we report structures of GluK3 LBD in complex with two 2,4‐syn‐functionalized (S)‐glutamate analogues to investigate their structural potential as chemical scaffolds. Similar binding affinities at GluK3 were determined for the 2‐(methylcarbamoyl)ethyl analogue (K_i=4.0 μM ) and the 2‐(methoxycarbonyl)ethyl analogue (K_i=1.7 μM ), in agreement with the similar positioning of the compounds within the binding pocket. As the binding affinity is similar to that of glutamate, this type of Cγ substituent could be used as a scaffold for introduction of even larger substituents reaching into unexplored binding site regions to achieve subtype selectivity.     

Chemoenzymatic Synthesis of GDP‐L‐Fucose Derivatives as Potent and Selective α‐1,3‐Fucosyltransferase Inhibitors

Fucosyltransferases (FucTs) usually catalyze the final step of glycosylation and are critical to many biological processes. High levels of specific FucT activities are often associated with various cancers. Here we report the development of a chemoenzymatic method for synthesizing a library of guanosine diphosphate β‐L ‐fucose (GDP‐Fuc) derivatives, followed by in situ screening for inhibitory activity against bacterial and human α‐1,3‐FucTs. Several compounds incorporating appropriate hydrophobic moieties were identified from the initial screening. These were individually synthesized, purified and characterized in detail for their inhibition kinetics. Compound 5 had a K_i of 29 nM for human FucT‐VI, and is 269 and 11 times more selective than for Helicobacter pylori FucT (K_i=7.8 μM) and for human FucT‐V (K_i=0.31 μM).     

Structure‐Based Design of Microsomal Prostaglandin E2 Synthase‐1 (mPGES‐1) Inhibitors using a Virtual Fragment Growing Optimization Scheme

A small library of 2,3‐dihydroxybenzamide‐ and N‐(2,3‐dihydroxyphenyl)‐4‐sulfonamide‐based microsomal prostaglandin E₂ synthase‐1 (mPGES‐1) inhibitors was identified following a step‐by‐step optimization of small aromatic fragments selected to interact in focused regions in the active site of mPGES‐1. During the virtual optimization process, the 2,3‐dihydroxybenzamide moiety was first selected as a backbone of the proposed new chemical entities; the identified compounds were then synthesized and biologically evaluated, identifying derivatives with very promising inhibitory activities in the micromolar range. Subsequent structure‐guided replacement of the 2,3‐dihydroxybenzamide by the N‐(2,3‐dihydroxyphenyl)sulfonamide moiety led to the identification of N‐(2,3‐dihydroxyphenyl)‐4‐biphenylsulfonamide ( 6 ), the most potent small molecule of the series (IC₅₀=0.53±0.04 μm ). The simple synthetic procedure and the possibility of enhancing the potency of this class of inhibitors through additional structural modifications pave the way for further development of new molecules with mPGES‐1‐inhibitory activity, with potential application as anti‐inflammatory and anticancer agents.     

5‐(Piperidin‐4‐yl)‐3‐Hydroxypyrazole: A Novel Scaffold for Probing the Orthosteric γ‐Aminobutyric Acid Type A Receptor Binding Site

A series of bioisosteric N₁‐ and N₂‐substituted 5‐(piperidin‐4‐yl)‐3‐hydroxypyrazole analogues of the partial GABA_AR agonists 4‐PIOL and 4‐PHP have been designed, synthesized, and characterized pharmacologically. The unsubstituted 3‐hydroxypyrazole analogue of 4‐PIOL ( 2 a ; IC₅₀～300 μM ) is a weak antagonist at the α₁β₂γ₂ GABA_AR, whereas substituting the N₁‐ or N₂‐position with alkyl or aryl substituents resulted in antagonists with binding affinities in the high nanomolar to low micromolar range at native rat GABA_ARs. Docking studies using a α₁β₂γ₂ GABA_AR homology model along with the obtained SAR indicate that the N₁‐substituted analogues of 4‐PIOL and 4‐PHP, 2 a – k , and previously reported 3‐substituted 4‐PHP analogues share a common binding mode to the orthosteric binding site in the receptor. Interestingly, the core scaffold of the N₂‐substituted analogues of 4‐PIOL and 4‐PHP, 3 b – k , are suggested to flip 180° thereby adapting to the binding pocket and addressing a cavity situated above the core scaffold.     

A Virtual Screening Study of the 18 kDa Translocator Protein using Pharmacophore Models Combined with 3D‐QSAR Studies

In the present study, we considered various pharmacophore hypotheses for TSPO ligands and an optimal one was selected on the basis of 3D‐QSAR studies. This hypothesis was used in a ligand‐based virtual screening study on the Maybridge database with the aim of identifying new TSPO ligands. Binding assays revealed that all selected compounds displayed TSPO affinity at 10 μM , and among them two compounds exhibited sub‐micromolar K_i values. These results validated our applied methodologies, and the two compounds with sub‐micromolar affinity could be used as interesting leads for the development of new active TSPO ligands.     

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.     

GluN2B‐Selective N‐Methyl‐d‐aspartate (NMDA) Receptor Antagonists Derived from 3‐Benzazepines: Synthesis and Pharmacological Evaluation of Benzo[7]annulen‐7‐amines

Given their high neuroprotective potential, ligands that block GluN2B‐containing N‐methyl‐D ‐aspartate (NMDA) receptors by interacting with the ifenprodil binding site located on the GluN2B subunit are of great interest for the treatment of various neuronal disorders. In this study, a novel class of GluN2B‐selective NMDA receptor antagonists with the benzo[7]annulene scaffold was prepared and pharmacologically evaluated. The key intermediate, N‐(2‐methoxy‐5‐oxo‐6,7,8,9‐tetrahydro‐5H‐benzo[7]annulen‐7‐yl)acetamide ( 11 ), was obtained by cyclization of 3‐acetamido‐5‐(3‐methoxyphenyl)pentanoic acid ( 10 b ). The final reaction steps comprise hydrolysis of the amide, reduction of the ketone, and reductive alkylation, leading to cis‐ and trans‐configured 7‐(ω‐phenylalkylamino)benzo[7]annulen‐5‐ols. High GluN2B affinity was observed with cis‐configured γ‐amino alcohols substituted with a 3‐phenylpropyl moiety at the amino group. Removal of the benzylic hydroxy moiety led to the most potent GluN2B antagonists of this series: 2‐methoxy‐N‐(3‐phenylpropyl)‐6,7,8,9‐tetrahydro‐5H‐benzo[7]annulen‐7‐amine ( 20 a , K_i=10 nM ) and 2‐methoxy‐N‐methyl‐N‐(3‐phenylpropyl)‐6,7,8,9‐tetrahydro‐5H‐benzo[7]annulen‐7‐amine ( 23 a , K_i=7.9 nM ). The selectivity over related receptors (phencyclidine binding site of the NMDA receptor, σ₁ and σ₂ receptors) was recorded. In a functional assay measuring the cytoprotective activity of the benzo[7]annulenamines, all tested compounds showed potent NMDA receptor antagonistic activity. Cytotoxicity induced via GluN2A subunit‐containing NMDA receptors was not inhibited by the new ligands.     

Experimental Characterization of the Binding Affinities between Proapoptotic BH3 Peptides and Antiapoptotic Bcl‐2 Proteins

The Bcl‐2 family proteins are key regulators of the intrinsic apoptotic pathway and are among the validated targets for developing anticancer drugs. Protein–protein interactions between the pro‐ and antiapoptotic members of this family determine mitochondrial outer‐membrane permeabilization. Elucidating such protein–protein interactions in a quantitative way is helpful for network pharmacology studies on the Bcl‐2 family, which, in turn, will provide valuable guidance for developing new anticancer therapies. In this study, the binding affinities of the BH3 peptides derived from eight proapoptotic BH3‐only proteins (i.e., Bid, Bim, Puma, Noxa, Bad, Bmf, Bik, Hrk) against five well‐studied antiapoptotic proteins (i.e., Bcl‐x_L, Bcl‐2, Mcl‐1, Bcl‐w, Bfl‐1) in the Bcl‐2 family have been measured. Three different types of binding assay (i.e., surface plasmon resonance, fluorescence polarization, and homogeneous time‐resolved fluorescence) were employed for cross‐validation. The results confirmed that each proapoptotic BH3 peptide exhibited a distinct binding profile against the five antiapoptotic proteins. The binding data obtained herein serve as a fresh update or correction to existing knowledge. It is expected that such binding data will be helpful for building more accurate mathematical network models for depicting the complex protein–protein interactions within the Bcl‐2 family.