A Specific Pharmacophore Model of Aurora B Kinase Inhibitors and Virtual Screening Studies Based on it

In this study, 3D‐pharmacophore models of Aurora B kinase inhibitors have been developed by using HipHop and HypoGen modules in Catalyst software package. The best pharmacophore model, Hypo1, which has the highest correlation coefficient (0.9911), consists of one hydrogen‐bond acceptor, one hydrogen‐bond donor, one hydrophobic aliphatic moiety and one ring aromatic feature. Hypo1 was validated by test set and cross‐validation methods. And the specificity of Hypo1 to Aurora B inhibitors was examined with the use of selective inhibitors against Aurora B and its paralogue Aurora A. The results clearly indicate that Hypo1 can differentiate selective inhibitors of Aurora B from those of Aurora A, and the ring aromatic feature likely plays some important roles for the specificity of Hypo1. Then Hypo1 was used as a 3D query to screen several databases including Specs, NCI, Maybridge and Chinese Nature Product Database (CNPD) for identifying new inhibitors of Aurora B. The hit compounds were subsequently subjected to filtering by Lipinski’s rule of five and docking studies to refine the retrieved hits, and some compounds selected from the top ranked hits have been suggested for further experimental assay studies.     

A Dual Non‐ATP Analogue Inhibitor of Aurora Kinases A and B,Derived from Resorcinol with a Mixed Mode of Inhibition

Aurora kinases are the most commonly targeted mitotic kinases in the intervention of cancer progression. Here, we report a resorcinol derivative, 5‐methyl‐4‐(2‐thiazolylazo) resorcinol (PTK66), a dual inhibitor of Aurora A and Aurora B kinases. PTK66 is a surface binding non‐ATP analogue inhibitor that shows a mixed pattern of inhibition against both of Aurora A and B kinases. The in vitro IC₅₀ is approximately 47 and 40 μm for Aurora A and Aurora B kinases, respectively. In cellular systems, PTK66 exhibits a substantially low cytotoxicity at micromolar concentrations but it can induce aneuploidy under similar dosages as a consequence of Aurora kinase inhibition. This result was corroborated by a drop in the histone H3 (S10) phosphorylation level detected via Western blot analysis using three different cell types. Altogether, our findings indicate that the ligand containing resorcinol backbone is one of the novel scaffolds targeting the Aurora family of kinases, which could be a target for antineoplastic drug development.     

Antifungal screening and in silico mechanistic studies of an in‐house azole library

Systemic Candida infections pose a serious public health problem with high morbidity and mortality. C. albicans is the major pathogen identified in candidiasis; however, non‐albicans Candida spp. with antifungal resistance are now more prevalent. Azoles are first‐choice antifungal drugs for candidiasis; however, they are ineffective for certain infections caused by the resistant strains. Azoles block ergosterol synthesis by inhibiting fungal CYP51, which leads to disruption of fungal membrane permeability. In this study, we screened for antifungal activity of an in‐house azole library of 65 compounds to identify hit matter followed by a molecular modeling study for their CYP51 inhibition mechanism. Antifungal susceptibility tests against standard Candida spp. including C. albicans revealed derivatives 12 and 13 as highly active. Furthermore, they showed potent antibiofilm activity as well as neglectable cytotoxicity in a mouse fibroblast assay. According to molecular docking studies, 12 and 13 have the necessary binding characteristics for effective inhibition of CYP51. Finally, molecular dynamics simulations of the C. albicans CYP51 (CACYP51) homology model's catalytic site complexed with 13 were stable demonstrating excellent binding.     

Computer‐aided drug design of small molecule inhibitors of the ERCC1‐XPF protein–protein interaction

The heterodimer of DNA excision repair protein ERCC‐1 and DNA repair endonuclease XPF (ERCC1‐XPF) is a 5′–3′ structure‐specific endonuclease essential for the nucleotide excision repair (NER) pathway, and it is also involved in other DNA repair pathways. In cancer cells, ERCC1‐XPF plays a central role in repairing DNA damage induced by chemotherapeutics including platinum‐based and cross‐linking agents; thus, its inhibition is a promising strategy to enhance the effect of these therapies. In this study, we rationally modified the structure of F06, a small molecule inhibitor of the ERCC1‐XPF interaction (Molecular Pharmacology, 84 , 2013 and 12), to improve its binding to the target. We followed a multi‐step computational approach to investigate potential modification sites of F06, rationally design and rank a library of analogues, and identify candidates for chemical synthesis and in vitro testing. Our top compound, B5 , showed an improved half‐maximum inhibitory concentration (IC₅₀) value of 0.49 µM for the inhibition of ERCC1‐XPF endonuclease activit, and lays the foundation for further testing and optimization. Also, the computational approach reported here can be used to develop DNA repair inhibitors targeting the ERCC1‐XPF complex.     

Structure‐based pharmacophore models to probe anticancer activity of inhibitors of protein kinase B‐beta (PKB β)

Protein kinase B ‐ beta (PKBβ/Akt2) is a non‐receptor kinase that has attracted a great deal of attention as a promising cancer therapy drug target. In mammalian cells, hyperactivation of Akt2 exclusively facilitates the survival of solid tumors by interfering with cell cycle progression. This definite function of Akt2 in tumor survival/maintenance provides the basis for the development of its antagonists with the aim of desensitizing cell proliferation. In order to find novel and potent Akt2 inhibitors, structure ‐ based pharmacophore models have been developed and validated by the test set prediction. The final pharmacophore model was used for hits identification using public chemical databases. The hits were further prioritized using drug ‐ like filters which revealed 14 potential hit compounds having novel chemical scaffolds. Our results elucidate the importance of three hydrogen bond acceptors (A), one hydrogen bond donor (D), one hydrophobic group (H), and one positive ionic charge (P) toward inhibition of the Ak2. One of our selected hits showed 68% cell apoptosis at 8 μg/ml concentration. We proposed various chemical scaffolds including benzamide, carboxamide, and methyl benzimidazole targeting Akt2 and thus may act as potential leads for the further development of new anticancer agents.     

In vitro and in vivo pharmacokinetic characterizations of AMG 900, an orally bioavailable small molecule inhibitor of aurora kinases

1. AMG 900 is a small molecule being developed as an orally administered, highly potent, and selective pan-aurora kinase inhibitor. The aim of the investigations was to characterize in vitro and in vivo pharmacokinetic (PK) properties of AMG 900 in preclinical species.

2. AMG 900 was rapidly metabolized in liver microsomes and highly bound to plasma proteins in the species tested. It was a weak Pgp substrate with good passive permeability.

3. AMG 900 exhibited a low-to-moderate clearance and a small volume of distribution. Its terminal elimination half-life ranged from 0.6 to 2.4?h. AMG 900 was well-absorbed in fasted animals with an oral bioavailability of 31% to 107%. Food intake had an effect on rate (rats) or extent (dogs) of AMG 900 oral absorption.

4. The clearance and volume of distribution at steady state in humans were predicted to be 27.3?mL/h/kg and 93.9?mL/kg, respectively.

5. AMG 900 exhibited acceptable PK properties in preclinical species and was predicted to have low clearance in humans. AMG 900 is currently in Phase I clinical testing as a treatment for solid tumours. Preliminary human PK results appear to be consistent with the predictions.

     

A bioinformatics search for selective histamine h4 receptor antagonists through structure-based virtual screening strategies

The prevalence of allergic disease is increasing dramatically in the developed world. Studies of allergic diseases have clearly demonstrated that histamine plays an important role in the pathogenesis of the early-phase allergic response. Histamine effects are mediated by H1, H2, H3, and H4 receptors. The presence of the histamine H4 receptors on leukocytes and mast cells suggests that the new histamine receptor H4 plays an important role in the modulation of the immune system. Thus, histamine H4 receptor is an attractive target for anti-allergic therapy. In our present study, we have generated a histamine H4 receptor model using I-TASSER based on human B2-adrenergic G-protein-coupled receptor. Structurally similar compounds of the three known antagonists JNJ777120, thioperamide, and Vuf6002 were retrieved from PubChem, and database was prepared. Virtual screening of those databases was performed, and six compounds with high docking score were identified. Also the binding mode revealed that all the six compounds had interaction with Asp94 of the receptor. Our results serve as a starting point in the development of novel lead compounds in anti-allergic therapy.     

Fragment‐ and negative image‐based screening of phosphodiesterase 10A inhibitors

A novel virtual screening methodology called fragment‐ and negative image‐based (F‐NiB) screening is introduced and tested experimentally using phosphodiesterase 10A (PDE10A) as a case study. Potent PDE10A‐specific small‐molecule inhibitors are actively sought after for their antipsychotic and neuroprotective effects. The F‐NiB combines features from both fragment‐based drug discovery and negative image‐based (NIB) screening methodologies to facilitate rational drug discovery. The selected structural parts of protein‐bound ligand(s) are seamlessly combined with the negative image of the target's ligand‐binding cavity. This cavity‐ and fragment‐based hybrid model, namely its shape and electrostatics, is used directly in the rigid docking of ab initio generated ligand 3D conformers. In total, 14 compounds were acquired using the F‐NiB methodology, 3D quantitative structure–activity relationship modeling, and pharmacophore modeling. Three of the small molecules inhibited PDE10A at ~27 to ~67 μM range in a radiometric assay. In a larger context, the study shows that the F‐NiB provides a flexible way to incorporate small‐molecule fragments into the drug discovery.     

Proposing novel MDM2 inhibitors: Combined physics‐driven high‐throughput virtual screening and in vitro studies

The mouse double minute 2 (MDM2) protein acts as a negative regulator of the p53 tumor suppressor. It directly binds to the N terminus of p53 and promotes p53 ubiquitination and degradation. Since the most common p53‐suppressing mechanisms involve the MDM2, proposing novel inhibitors has been the focus of many in silico and also experimental studies. Thus, here we screened around 500,000 small organic molecules from Enamine database at the binding pocket of this oncogenic target. The screening was achieved systematically with starting from the high‐throughput virtual screening method followed by more sophisticated docking approaches. The initial high number of screened molecules was reduced to 100 hits which then were studied extensively for their therapeutic activity and pharmacokinetic properties using binary QSAR models. The structural and dynamical profiles of the selected molecules at the binding pocket of the target were studied thoroughly by all‐atom molecular dynamics simulations. The free energy of the binding of the hit molecules was estimated by the MM/GBSA method. Based on docking simulations, binary QSAR model results, and free energy calculations, 11 compounds ( E1 – E11 ) were selected for in vitro studies. HUVEC vascular endothelium, colon cancer, and breast cancer cell lines were used for testing the binding affinities of the identified hits and for further cellular effects on human cancer cell. Based on in vitro studies, six compounds ( E1 , E2 , E5 , E6 , E9 , and E11 ) in breast cancer cell lines and six compounds ( E1 , E2 , E5 , E6 , E8 , and E10 ) in colon cancer cell lines were found as active. Our results showed that these compounds inhibit proliferation and lead to apoptosis.     

Searching for Dual Inhibitors of the MDM2‐p53 and MDMX‐p53 Protein–Protein Interaction by a Scaffold‐Hopping Approach

Two libraries of substituted benzimidazoles were designed using a ‘scaffold‐hopping’ approach based on reported MDM2‐p53 inhibitors. Substituents were chosen following library enumeration and docking into an MDM2 X‐ray structure. Benzimidazole libraries were prepared using an efficient solution‐phase approach and screened for inhibition of the MDM2‐p53 and MDMX‐p53 protein–protein interactions. Key examples showed inhibitory activity against both targets.     

Hyperforin induces apoptosis through extrinsic/intrinsic pathways and inhibits EGFR/ERK/NF‐κB‐mediated anti‐apoptotic potential in glioblastoma

Glioblastoma is the most common primary brain tumor with poor survival rate and without effective treatment strategy. Notably, amplification and active mutation of epidermal growth factor receptor (EGFR) occur frequently in glioblastoma patient that may be a potential treatment target. Several studies indicated that various type of herbal compounds not only regulate anti‐depressant effect but also shown capacity to suppress glioblastoma growth via inducing apoptosis and inhibiting oncogene signaling transduction. Hyperforin, an herb compound derived from St. John's wort was used to treat depressive disorder by inhibiting neuronal reuptake of several neurotransmitters. Although hyperforin can reduce matrix metallopeptidases‐2 (MMPs) and ‐9‐mediated metastasis of glioblastoma, the detail mechanism of hyperforin on glioblastoma is remaining unclear. Here, we suggested that hyperforin may induce extrinsic/intrinsic apoptosis and suppress anti‐apoptotic related proteins expression of glioblastoma. We also indicated that hyperforin‐mediated anti‐apoptotic potential of glioblastoma was correlated to inactivation of EGFR/extracellular signal‐regulated kinases (ERK)/nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) signaling.     

A small molecule UPR modulator for diabetes identified by high throughput screening

Unfolded protein response (UPR) is a stress response that is specific to the endoplasmic reticulum (ER). UPR is activated upon accumulation of unfolded (or misfolded) proteins in the ER's lumen to restore protein folding capacity by increasing the synthesis of chaperones. In addition, UPR also enhances degradation of unfolded proteins and reduces global protein synthesis to alleviate additional accumulation of unfolded proteins in the ER. Herein, we describe a cell-based ultra-high throughput screening (uHTS) campaign that identifies a small molecule that can modulate UPR and ER stress in cellular and in vivo disease models. Using asialoglycoprotein receptor 1 (ASGR) fused with Cypridina luciferase (CLuc) as reporter assay for folding capacity, we have screened a million small molecule library and identified APC655 as a potent activator of protein folding, that appears to act by promoting chaperone expression. Furthermore, APC655 improved pancreatic β cell viability and insulin secretion under ER stress conditions induced by thapsigargin or cytokines. APC655 was also effective in preserving β cell function and decreasing lipid accumulation in the liver of the leptin-deficient (ob/ob) mouse model. These results demonstrate a successful uHTS campaign that identified a modulator of UPR, which can provide a novel candidate for potential therapeutic development for a host of metabolic diseases.     

Lipoxin A4 ameliorates renal ischaemia–reperfusion‐induced acute lung injury in rats

Lipoxin A4 (LA4), a bioactive product of arachidonic acid, has been shown to exert strong anti‐inflammatory activity. By contrast, the anti‐inflammatory action of LA4 in a renal ischaemia–reperfusion (RIR)‐mediated acute lung inflammation (ALI) model and the potential pathogenesis of the condition is still unclear. The aim of the current research was to investigate the effect of LA4 on RIR‐induced ALI. The rat ALI model was induced by RIR. LA4 was injected via the tail vein immediately after RIR. The results indicate that LA4 markedly inhibits inflammatory cells infiltration, attenuates myeloperoxidase activity, and reduces the concentration of inflammatory mediators and Toll‐like receptor 4 (TLR4) in RIR‐induced ALI. Furthermore, LA4 suppressed nuclear factor kappa B (NF‐κB) p65 and mitogen‐activated protein kinase (MAPK) activation. The protective effect of LA4 in RIR‐stimulated ALI was reversed by BOC‐2 (an antagonist of the LA4 receptor). These results indicate that LA4 exerts powerful anti‐inflammatory functions in RIR‐induced ALI by attenuating TLR4 expression via MAPK and NF‐κB signalling. Accordingly, LA4 might be an underlying treatment drug for RIR‐induced ALI.