Design,synthesis and evaluation of d-amino acid-containing peptidomimetics targeting the polo-box domain of polo-like kinase 1

A series of d-amino acid-containing peptidomimetics were designed, synthesized as novel polo-like kinase 1 (Plk1) polo-box domain (PBD) inhibitors based on the reported peptide Plk1 PBD inhibitor. Their inhibitory activity to Plk1, Plk2, and Plk3 PBD were evaluated using our fluorescence polarization (FP) assay. Compound 18 bound to Plk1 PBD with IC₅₀ of 0.80 μM and showed nearly no inhibition to Plk2 PBD or Plk3 PBD at 100 μM. Compound 18 induced Hela cells to undergo apoptosis by increasing the ratio of the cells at the G2/M phase by decreasing the neosynthesized proteins in a dose-dependent manner from 50 to 150 μM. Compound 18 showed improved stability in rat plasma compared to l-peptide inhibitor LHSpTA. These novel d-amino acid modified selective Plk1 PBD inhibitors may provide new lead compounds for further optimization.     

Identification of novel peptidomimetics targeting the polo-box domain of polo-like kinase 1

A series of new peptidomimetics targeting the polo-box domain (PBD) of polo-like kinase 1 (Plk1) was identified based on the potent and selective pentapeptide Plk1 PBD inhibitor PLHSpT. Unnatural amino acid residues were introduced to the newly designed compound and the N-terminal substituent of the peptidomimetic was investigated. The optimized compound 9 inhibited the Plk1 PBD with IC₅₀ of 0.267 μM and showed almost no inhibition to Plk2 PBD or Plk3 PBD at 100 μM. Biolayer interferometry studies demonstrated that compound 9 showed potent binding affinity to Plk1 with a K_d value of 0.164 μM, while no K_d were detected against Plk2 and Plk3. Compound 9 showed improved stability in rat plasma compared to PLHSpT. Binding mode analysis was performed and in agreement with the observed experimental results. There are only two natural amino acids remained in the chemical structure of 9. This study may provide new information for further research on Plk1 PBD inhibitors.     

Design,synthesis and biological evaluation of phosphopeptides as Polo-like kinase 1 Polo-box domain inhibitors

Polo-like kinase 1 (Plk1) is an anti-cancer target due to its critical role in mitotic progression. A growing body of evidence has documented that Peptide-Plk1 inhibitors showed high Plk1 binding affinity. However, phosphopeptides-Plk1 inhibitors showed poor cell membranes permeability, which limits their clinical applications. In current study, nine candidate phosphopeptides consisting of non-natural amino acids were rationally designed and then successfully synthesized using an Fmoc-solid phase peptide synthesis (SPPS) strategy. Moreover, the binding affinities and selectivity were evaluated via fluorescence polarization (FP) assay. The results confirmed that the most promising phosphopeptide 6 bound to Plk1 PBD with the IC₅₀ of 38.99?nM, which was approximately 600-fold selectivity over Plk3 PBD (IC₅₀?=?25.44?μM) and nearly no binding to Plk2 PBD. Furthermore the intracellular activities and the cell membrane permeability of phosphopeptide 6 were evalutated. Phosphopeptide 6 demonstrated appropriate cell membrane permeability and arrested HeLa cells cycle in G2/M phase by regulating CyclinB1-CDK1. Further, phosphopeptide 6 showed typical apoptotic morphology and induced caspase-dependent apoptosis. In conclusion, we expect our discovery can provide new insights into the further optimization of Plk1 PBD inhibitors.     

Selective blockade of cancer cell proliferation and anchorage-independent growth by Plk1 activity–dependent suicidal inhibition of its polo-box domain

Polo-like kinase 1 (Plk1) plays a critical role in proper M-phase progression and cell proliferation. Plk1 is overexpressed in a broad spectrum of human cancers and is considered an attractive anticancer drug target. Although a large number of inhibitors targeting the catalytic domain of Plk1 have been developed, these inhibitors commonly exhibit a substantial level of cross-reactivity with other structurally related kinases, thus narrowing their applicable dose for patient treatment. Plk1 contains a C-terminal polo-box domain (PBD) that is essentially required for interacting with its binding targets. However, largely due to the lack of both specific and membrane-permeable inhibitors, whether PBD serves as an alternative target for the development of anticancer therapeutics has not been rigorously examined. Here, we used an intracellularly expressed 29-mer-long PBIP1-derived peptide (i.e., PBIPtide), which can be converted into a “suicidal” PBD inhibitor via Plk1-dependent self-priming and binding. Using this highly specific and potent system, we showed that Plk1 PBD inhibition alone is sufficient for inducing mitotic arrest and apoptotic cell death in cancer cells but not in normal cells, and that cancer cell–selective killing can occur regardless of the presence or absence of oncogenic RAS mutation. Intriguingly, PBD inhibition also effectively prevented anchorage-independent growth of malignant cancer cells. Thus, targeting PBD represents an appealing strategy for anti-Plk1 inhibitor development. Additionally, PBD inhibition–induced cancer cell–selective killing may not simply stem from activated RAS alone but, rather, from multiple altered biochemical and physiological mechanisms, which may have collectively contributed to Plk1 addiction in cancer cells.     

New phenolic cinnamic acid derivatives as selective COX-2 inhibitors. Design,synthesis, biological activity and structure-activity relationships

Selective inhibition of cyclooxygenase (COX)-2 enzyme is an important achievement when looking for potent anti-inflammatory agents, with fewer gastrointestinal side effects. In this work, a new series of cinnamic acid derivatives, namely hexylamides, have been designed, synthesized and evaluated in human blood for their inhibitory activity of COX-1 and COX-2 enzymes. From this, new structure-activity relationships were built, showing that phenolic hydroxyl groups are essential for both COX-1 and COX-2 inhibition. Furthermore, the presence of bulky hydrophobic di-tert-butyl groups in the phenyl ring strongly contributes for selective COX-2 inhibition. In addition, a correlation with the theoretical log P has been carried out, showing that lipophilicity is particularly important for COX-2 inhibition. Further, a plasma protein binding (PPB) prediction has been performed revealing that PPB seems to have no influence in the activity of the studied compounds. From the whole study, effective selective inhibitors of COX-2 were found, namely compound 9 (IC₅₀ = 3.0 ± 0.3 μM), 10 (IC₅₀ = 2.4 ± 0.6 μM) and 23 (IC₅₀ = 1.09 ± 0.09 μM). Those can be considered starting point hit compounds for further optimization as potential non-steroidal anti-inflammatory drugs.     

A high-throughput assay based on fluorescence polarization for inhibitors of the polo-box domain of polo-like kinase 1

The serine/threonine kinase polo-like kinase 1 (Plk1) is critically involved in multiple mitotic processes and has been established as an adverse prognostic marker for tumor patients. Plk1 localizes to its substrates and its intracellular anchoring sites via its polo-box domain (PBD), which is unique to the family of polo-like kinases. Therefore, inhibition of the Plk1 PBD has been suggested as an approach to the inhibition of Plk1 that circumvents specificity problems associated with the inhibition of the conserved adenosine triphosphate (ATP) binding pocket. Here we report on the development of a high-throughput assay based on fluorescence polarization that allows the discovery of small-molecule inhibitors of the Plk1 PBD. The assay is based on binding of the Plk1 PBD to a phosphothreonine-containing peptide comprising its optimal binding motif with a K_d of 26 ± 2 nM. It is stable with regard to dimethyl sulfoxide (DMSO) and time, and it has a Z′ value of 0.73 ± 0.06 in a 384-well format.     

Several inhibitors of the Plk1 Polo-Box Domain turn out to be non-specific protein alkylators

For almost a decade, there has been much interest in the development of chemical inhibitors of Polo-like kinase 1 (Plk1) protein interactions. Plk1 is a master regulator of the cell division cycle that controls numerous substrates. It is a promising target for cancer drug development. Inhibitors of the kinase domain of Plk1 had some success in clinical trials. However, they are not perfectly selective. In principle, Plk1 can also be inhibited by interfering with its protein interaction domain, the Polo-Box Domain (PBD). Selective chemical inhibitors of the PBD would constitute tools to probe for PBD-dependent functions of Plk1 and could be advantageous in cancer therapy. The discovery of Poloxin and thymoquinone as PBD inhibitors indicated that small, cell-permeable chemical inhibitors could be identified. Other efforts followed, including ours, reporting additional molecules capable of blocking the PBD. It is now clear that, unfortunately, most of these compounds are non-specific protein alkylators (defined here as groups covalently added via a carbon) that have little or no potential for the development of real Plk1 PBD-specific drugs. This situation should be minded by biologists potentially interested in using these compounds to study Plk1. Further efforts are needed to develop selective, cell-permeable PBD inhibitors.     

Novel pyridazinone derivatives as butyrylcholinesterase inhibitors

In the current study, forty-four new [3-(2/3/4-methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl carbamate derivatives were synthesized and evaluated for their ability to inhibit electric eel acetylcholinesterase (EeAChE) and equine butyrylcholinesterase (eqBuChE) enzymes. According to the inhibitory activity results, [3-(2-methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl heptylcarbamate (16c, eqBuChE, IC₅₀ = 12.8 μM; EeAChE, no inhibition at 100 μM) was the most potent eqBuChE inhibitor among the synthesized compounds and was found to be a moderate inhibitor compared to donepezil (eqBuChE, IC₅₀ = 3.25 μM; EeAChE, IC₅₀ = 0.11 μM). Kinetic and molecular docking studies indicated that compounds 16c and 14c (hexylcarbamate derivative, eqBuChE, IC₅₀ = 35 μM; EeAChE, no inhibition at 100 μM) were mixed-type inhibitors which accommodated within the catalytic active site (CAS) and peripheral anionic site (PAS) of hBuChE through stable hydrogen bonding and π-π stacking. Furthermore, it was determined that [3-(2-methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl (4-methylphenyl)carbamate 7c (eqBuChE, IC₅₀ = 34.5 μM; EeAChE, 38.9% inhibition at 100 μM) was the most active derivative against EeAChE and a competitive inhibitor binding to the CAS of hBuChE. As a result, 6-(2-methoxyphenyl)pyridazin-3(2H)-one scaffold is important for the inhibitory activity and compounds 7c, 14c and 16c might be considered as promising lead candidates for the design and development of selective BuChE inhibitors for Alzheimer’s disease treatment.     

Design,synthesis and biological evaluation of novel 4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine derivatives as potential BRD4 inhibitors

Recently, diverse kinase inhibitors were reported having interaction with BRD4. It provided a strategy for developing a new structural framework for the next-generation BRD4-selective inhibitors. Starting from PLK1 kinase inhibitor BI-2536, we designed 18 compounds by modifying dihydropteridine core. Compound 23 showed potent BRD4 inhibitory activities with IC₅₀ of 79 nM and no inhibitory activities for PLK1. Cell antiproliferation assay was performed and potent inhibitory activity against MV4;11 with IC₅₀ of 1.53 μM. Cell apoptosis and western blotting indicated compound 23 induced apoptosis by down-regulating c-Myc. These novel selective BRD4 inhibitors provided new lead compounds for further drug development.     

Novel non-peptide β-secretase inhibitors derived from structure-based virtual screening and bioassay

This Letter describes an efficient approach by integrating virtual screening with bioassay technology for finding small organic inhibitors targeting β-secretase (BACE-1). Fifteen hits with inhibitory potencies ranging from 2.8 to 118 μM (IC₅₀) against β-secretase were successfully identified. Compound 12 with IC₅₀ of 2.8 μM is the most potent hit against BACE-1. Docking simulation from gold 3.0 suggests putative binding mode of 12 in BACE-1 and potential key pharmacophore groups for further designing of non-peptide compounds as more powerful inhibitors against BACE-1.     

Novel chromanone-dithiocarbamate hybrids as multifunctional AChE inhibitors with β-amyloid anti-aggregation properties for the treatment of Alzheimer’s disease

By connecting chromanone with dithiocarbamate moieties through flexible linkers, a series of hybrids as novel multifunctional AChE inhibitors have been designed and synthesized. Most of these compounds displayed strong and excellently selective inhibition to eeAChE as well as potent inhibition to self- and AChE-induced Aβ aggregation. Among them, compound 6c showed the best activity to inhibit eeAChE (IC₅₀ = 0.10 μM) and AChE-induced Aβ aggregation (33.02% at 100 μM), and could effectively inhibit self-induced Aβ aggregation (38.25% at 25 μM). Kinetic analysis and docking study indicated that compound 6c could target both the CAS and PAS, suggesting that it was a dual binding site inhibitor for AChE. Besides, it exhibited good ability to penetrate the BBB and low neurotoxicity in SH-SY5Y cells. More importantly, compound 6c was well tolerated in mice (2500 mg/kg, po) and could attenuate the memory impairment in a scopolamine-induced mouse model. Overall, these results highlight 6c as a promising multifunctional agent for treating AD and also demonstrate that the dithiocarbamate is a valid scaffold for design of multifunctional AChE inhibitors.     

Development of high-throughput assays based on fluorescence polarization for inhibitors of the polo-box domains of polo-like kinases 2 and 3

The serine/threonine kinases Plk1, Plk2, and Plk3 harbor a protein–protein interaction domain dubbed polo-box domain (PBD). Recently, the inhibition of the PBD of the cancer target Plk1 has been successfully explored as an alternative to the inhibition of the kinase by ATP-competitive ligands. However, because the PBDs of Plk1, Plk2, and Plk3 have very similar optimal binding motifs, absolute specificity for the PBD of Plk1 over the PBDs of Plk2 and Plk3 may also represent a big challenge for a small molecule. To aid in the activity profiling of Plk PBD inhibitors, and to identify selective small molecules that will reveal the cellular consequences of inhibiting the PBDs of Plk2 and Plk3, we have developed high-throughput assays based on fluorescence polarization against the PBDs of Plk2 and Plk3. The assays are stable with regard to time and 10% dimethyl sulfoxide and have Z′ values 0.7, making them well-suited for high-throughput screening. Moreover, our data provide insights into the binding preferences of the PBDs of Plk2 and Plk3.     

Molecular design and engineering of phosphopeptide ligands to target lung cancer polo-like kinase

Polo-like kinase (Plk) plays a central role in centrosome cycle and is closely associated with the oncogenesis of lung cancer. The protein consists of a catalytic kinase domain (KD) and a regulatory polo-box domain (PBD); either direct inhibition of the KD’s catalytic activity or indirect disruption of the PBD–substrate interaction can be used to potentially suppress the pathological activation of lung cancer Plk. Here, we reported a successful molecular design and engineering of phosphopeptide ligands to target Plk PBD domain by integrating in silico modeling and in vitro assay. In the procedure, a helical peptide segment hps was derived from dimerization interface of the complex crystal structure of domain dimer using bioinformatics approach, which was then used as sequence template to generate potent phosphopeptide binders of Plk PBD domain in terms of a systematic residue mutation profile. Fluorescence anisotropy assays were conducted to substantiate the findings and conclusions obtaining from the molecular engineering. Consequently, three helical phosphopeptides, including the native hps and its two mutants hps-m ₁ and hps-m ₂, were successfully designed that can independently rebind to Plk PBD domain with a moderate or high affinity (K _d = 127, 26, and 5 μM, respectively). These peptide ligands can be considered as potent self-competitors to disrupt PBD dimerization in lung cancer metastasis. Structural and energetic analysis revealed that hydrophobic forces and van der Waals contacts confer strong stability for domain–peptide complex system, while hydrogen bonds and electrostatic interactions contribute specificity and selectivity to the complex recognition.     

Design,synthesis and biological evaluation of pyrimidine-based derivatives as VEGFR-2 tyrosine kinase inhibitors

Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a crucial role in tumor angiogenesis, and inhibition of the VEGFR-2 signaling pathway has already become an attractive approach for cancer therapy. In this study, a novel pyrimidine-based derivative 7j was designed as lead compound, and three series of potent VEGFR-2 inhibitors were synthesized and biologically evaluated against A549 and HepG2 cell lines. Compounds 7d, 9s and 13n exhibited superior inhibitory activities against A549 cell with IC₅₀ ranged from 9.19 to 13.17 μM and HepG2 cell with IC₅₀ ranged from 11.94 to 18.21 μM compared to those of Pazopanib (IC₅₀ = 21.18 and 36.66 μM). In addition, molecular docking study was performed to investigate the binding capacity and binding mode between target compounds and VEGFR-2.     

A new entry into the portfolio of α-glucosidase inhibitors as potent therapeutics for type 2 diabetes: Design,bioevaluation and one-pot multi-component synthesis of diamine-bridged coumarinyl oxadiazole conjugates

Diabetes mellitus (DM), a chronic multifarious metabolic disorder resulting from impaired glucose homeostasis has become one of the most challenging diseases with severe life threat to public health. The inhibition of α-glucosidase, a key carbohydrate hydrolyzing enzyme, could serve as one of the effective methodology in both preventing and treating diabetes through controlling the postprandial glucose levels and suppressing postprandial hyperglycemia. In this context, three series of diamine-bridged bis-coumarinyl oxadiazole conjugates were designed and synthesized by one-pot multi-component methodology. The synthesized conjugates (4a–j, 5a–j, 6a–j) were evaluated as potential inhibitors of glucosidases. Compound 6f containing 4,4′-oxydianiline linker was identified as the lead and selective inhibitor of α-glucosidase enzyme with an IC₅₀ value of 0.07 ± 0.001 μM (acarbose: IC₅₀ = 38.2 ± 0.12 μM). This inhibition efficacy was ∼545-fold higher compared to the standard drug. Compound 6f was also emerged as the lead molecule against intestinal maltase-glucoamylase with good inhibition strength (IC₅₀ = 0.04 ± 0.02 μM) compared to acarbose (IC₅₀ = 0.06 ± 0.01 μM). Against β-glucosidase enzyme, compound 6 g was noted as the lead inhibitor with IC₅₀ value of 0.08 ± 0.002 μM. Michaelis–Menten kinetic experiments were performed to explore the mechanism of inhibition. Molecular docking studies of the synthesized library of hybrid structures against glucosidase enzyme were performed to describe ligand-protein interactions at molecular level that provided an insight into the biological properties of the analyzed compounds. The results suggested that the inhibitors could be stabilized in the active site through the formation of multiple interactions with catalytic residues in a cooperative fashion. In addition, strong binding interactions of the compounds with the amino acid residues were effective for the successful identification of α-glucosidase inhibitors.     

Histidine N(τ)-cyclized macrocycles as a new genre of polo-like kinase 1 polo-box domain-binding inhibitors

Transition toward peptide mimetics of reduced size is an important objective of peptide macrocyclization. We have previously shown that PLH¹SpT (2a) (where H¹ indicates the presence of a –(CH₂)₈Ph group at the N(π) position and pT indicates phosphothreonine) is an extremely high affinity ligand of the polo-like kinase 1 (Plk1) polo-box domain (PBD). Herein we report that C-terminal macrocyclization of 2a employing N(π),N(τ)-bis-alkylated His residues as ring junctions can be achieved in a very direct fashion. The resulting macrocycles are highly potent in biochemical assays and maintain good target selectivity for the Plk1 PBD versus the PBDs of Plk2 and Plk3. Importantly, as exemplified by 5d, our current approach permits deletion of the N-terminal “Pro-Leu” motif to yield tripeptide ligands with decreased molecular weight, which retain high affinity and show improved target selectivity. These findings could fundamentally impact the future development of peptide macrocycles in general and Plk1 PBD-binding peptide mimetics in particular.     

Synthesis and exploration of 2-morpholino-4-phenylthiazol-5-yl acrylamide derivatives for their effects against carbonic anhydrase I,II, IX and XII isoforms as a non-sulfonamide class of inhibitors

Novel series of 2-morpholino-4-phenylthiazol-5-yl acrylamide derivatives (8a–s) have been synthesized and explored as a non-sulfonamide class of carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The newly synthesized molecules were evaluated for their CA inhibitory potency against four isoforms: the cytosolic isozyme hCA I, II as well as trans-membrane tumor associated isoform hCA IX and hCA XII taking acetazolamide (AAZ) as standard drug. The results revealed that most of the compounds showed good activity against hCA II, IX, and XII whereas none of them were active against hCA I (K_i >100 μM). It is observed that the physiologically most important cytosolic isoform hCA II was inhibited by these molecules in the range of K_i 9.3–77.7 μM. It is also found the both the transmembrane isoforms hCA IX and XII were also inhibited with K_is ranging between 54.7–96.7 μM and 4.6–8.8 μM, respectively. The binding modes of the active compounds within the catalytic pockets of hCA II, IX and XII were evaluated by docking studies. This new non-sulfonamide class of selective inhibitors of hCA II, IX and XII over the hCA I isoform may be used for further understanding the physiological roles of some of these isoforms in various pathologies.     

Discovery,molecular dynamic simulation and biological evaluation of structurally diverse cholinesterase inhibitors with new scaffold through shape-based pharmacophore virtual screening

Designing small molecule inhibitors targeting cholinesterases (ChEs) is considered as an efficient strategy for the treatment of Alzheimer′s disease (AD). In the present study, based on a shaped-based pharmacophore (SBP) model that we reported previously, virtual screening was performed on four commercial compound databases, from which eight small molecules containing new structurally scaffolds were retained and evaluated. In general, six of these potential hits were identified to be selective ChEs inhibitors. Three compounds exhibited IC₅₀ values and K_i values in micromolar range on acetylcholinesterase (AChE), the most active compound 4 showed IC₅₀ value of 6.31 ± 2.68 μM and K_i value of 4.76 μM. Other three compounds displayed IC₅₀ values and K_i values in micromolar range on butyrylcholinesterase (BChE) with high target selectivity, the most active compound 1 showed IC₅₀ value of 3.87 ± 2.48 μM and K_i value of 1.52 μM. Multiple biological evaluations were performed to determine their cytotoxicity, cyto-protective effects, antioxidant effect as well as druglike properties. These compounds provide new cores for the further design and optimization, with the aim to discover new ChEs inhibitors for the treatment of AD.     

Novel aromatic sulfonyl naphthalene-based boronates as 20S proteasome inhibitors

A novel series of non-peptide proteasome inhibitors (PIs) that act on chymotrypsin-like (ChT-L) of the proteasome were developed. These PIs bearing 4-aromatic sulfonyl naphthalene-based scaffold and Leu-boronic moiety as covalent bonding group displayed far better activity than PI-8182 for inhibiting ChT-L in preliminary biological activity test. The results showed that 2a (IC₅₀?=?6.942?μM, MCF-7) and 2c (IC₅₀?=?6.905?μM, MCF-7) displayed higher anti-proliferative activities than Bortezomib (IC₅₀?=?18.37?μM, MCF-7) under our experimental conditions. Furthermore, in the microsomal stability assay, 2a demonstrated excellent metabolic stability profiles with 56% remaining after 40?min, as compared to Bortezomib of which approximately 30% was remaining. The compounds 2a, 2c emerged as promising lead compounds for the development of novel non-peptide boronate PIs.