Discovery of novel bacterial elongation condensing enzyme inhibitors by virtual screening

The elongation condensing enzymes in the bacterial fatty acid biosynthesis pathway represent desirable targets for the design of novel, broad-spectrum antimicrobial agents. A series of substituted benzoxazolinones was identified in this study as a novel class of elongation condensing enzyme (FabB and FabF) inhibitors using a two-step virtual screening approach. Structure activity relationships were developed around the benzoxazolinone scaffold showing that N-substituted benzoxazolinones were most active. The benzoxazolinone scaffold has high chemical tractability making this chemotype suitable for further development of bacterial fatty acid synthesis inhibitors.     

Identification of novel ASK1 inhibitors using virtual screening

Apoptosis signal-regulating kinase 1 (ASK1, also called MAP3K5) is a mitogen-activated protein kinase kinase kinase (MAP3K) that plays important roles in stress-induced cell death and inflammation, and is expected as a new therapeutic target for cancer, cardiovascular diseases, and neurodegenerative diseases. We identified novel ASK1 inhibitors by virtual screening from the public chemical library collected by Chemical Biology Research Initiative (CBRI) at the University of Tokyo.     

Discovery of the first SecA inhibitors using structure-based virtual screening

Bacterial protein secretion is a critical and complex process. The Sec machinery provides a major pathway for protein translocation across and integration into the cellular membrane in bacteria. Small molecule probes that perturb the functions of individual member proteins within the Sec machinery will be very important research tools as well as leads for future antimicrobial agent development. Herein we describe the discovery of inhibitors, through virtual screening, that specifically act on SecA ATPase, which is a critical member of the Sec system. These are the very first inhibitors reported for intrinsic SecA ATPase.     

Design and synthesis of novel cell wall inhibitors of Mycobacterium tuberculosis GlmM and GlmU

GlmM and GlmU are key enzymes in the biosynthesis of UDP-N-acetyl-d-glucosamine (UDP-GlcNAc), an essential precursor of peptidoglycan and the rhamnose–GlcNAc linker region in the mycobacterial cell wall. These enzymes are involved in the conversion of two important precursors of UDP-GlcNAc, glucosamine-6-phosphate (GlcN-6-P) and glucosamine-1-phosphate (GlcN-1-P). GlmM converts GlcN-6-P to GlcN-1-P, GlmU is a bifunctional enzyme, whereby GlmU converts GlcN-1-P to GlcNAc-1-P and then catalyzes the formation of UDP-GlcNAc from GlcNAc-1-P and uridine triphosphate. In the present study, methyl 2-amino-2-deoxyl-α-d-glucopyranoside 6-phosphate (1α), methyl 2-amino-2-deoxyl-β-d-glucopyranoside 6-phosphate (1β), two analogs of GlcN-6-P, were synthesized as GlmM inhibitors; 2-azido-2-deoxy-α-d-glucopyranosyl phosphate (2) and 2-amino-2,3-dideoxy-3-fluoro-α-d-glucopyranosyl phosphate (3), analogs of GlcN-1-P, were synthesized firstly as GlmU inhibitors. Compounds 1α, 1β, 2, and 3 as possible inhibitors of mycobacterial GlmM and GlmU are reported herein. Compound 3 showed promising inhibitory activities against GlmU, whereas 1α, 1β and 2 were inactive against GlmM and GlmU even at high concentrations.     

Design of inhibitors of the HIV-1 integrase core domain using virtual screening

Acquired immunodeficiency syndrome (AIDS) is a disease of the human immune system caused by the human immunodeficiencyvirus (HIV). The integrase (IN) enzyme of HIV interacts with several cellular and viral proteins during the integration process.Thus, it represents an appropriate target for antiretroviral drugs (ARVs). We performed virtual screening of database compoundsand designed analogues using Elvitegravir (EVG) as a standard compound. The 378 screened compounds were retrieved fromZINC, ChemSpider, PubChem, and ChemBank Chemical Databases based on chemical similarity and literature searches related tothe structure of EVG. The Physiochemical properties, Bioactivity, Toxicity and Absorption, Distribution, Metabolism and Excretionof Molecules (ADME) of these compounds were predicted and docking Experiments were conducted using Molegro VirtualDocker software. The docking and ADME suggested very significant results in regard to EVG. The MolDock and Rerank scoreswere used to analyze the results. The compounds ZINC26507991 (-84.22), Analogue 9 (-68.49), ZINC20731658 (-66.79),ZINC00210363 (-43.44) showed better binding orientation with IN receptor model with respect to EVG (182.52). The ZINC26507991has showed significant ADME result.     

Discovery of novel HCV polymerase inhibitors using pharmacophore-based virtual screening

We report the use of pharmacophore-based virtual screening as an efficient tool for the discovery of novel HCV polymerase inhibitors. A three-dimensional pharmacophore model for the HCV-796 binding site, NNI site IV inhibitor, to the enzyme was built by means of the structure-based focusing module in Cerius2 program. Using these models as a query for virtual screening, we produced a successful example of using pharmacophore-based virtual screening to identify novel compounds with HCV replicon assay through inhibition of HCV polymerization. Among the hit compounds, compounds 1 and 2 showed 56% and 48% inhibition of NS5B polymerization activity at 20 μM, respectively. In addition, compound 1 also exhibited replicon activity with EC₅₀ value of 2.16 μM. Following up the initial hit, we obtained derivatives of compound 1 and evaluated polymerization inhibition activity and HCV replicon assay. These results provide information necessary for the development of more potent NS5B inhibitors.     

Identification of influenza PA-Nter endonuclease inhibitors using pharmacophore- and docking-based virtual screening

Searching for new antiviral agents, we focused our interest on the influenza PA-Nter endonuclease. Therefore, we developed a three-dimensional pharmacophore model which contains the binding features addressed to the metal-chelating active site. The obtained hypothesis has been fruitfully employed to select three “hit compounds” through an in silico screening campaign on our in-house database of small molecules. We studied the binding poses of these hit compounds using molecular docking, and subjected them to an enzymatic assay with recombinant PA-Nter endonuclease. Compound 20 proved the most active inhibitor of the endonucleolytic cleavage reaction, with an IC₅₀ value of 12?μM.     

An aminoquinazoline inhibitor of the essential bacterial cell wall synthetic enzyme GlmU has a unique non-protein-kinase-like binding mode

GlmU is a bifunctional enzyme with acetyltransferase and uridyltransferase activities, and is essential for the biosynthesis of the bacterial cell wall. Inhibition results in a loss of cell viability. GlmU is therefore considered a potential target for novel antibacterial agents. A HTS (high-throughput screen) identified a series of aminoquinazolines with submicromolar potency against the uridyltransferase reaction. Biochemical and biophysical characterization showed competition with UTP binding. We determined the crystal structure of a representative aminoquinazoline bound to the Haemophilus influenzae isoenzyme at a resolution of 2.0 ?. The inhibitor occupies part of the UTP site, skirts the outer perimeter of the GlcNAc1-P (N-acetylglucosamine-1-phosphate) pocket and anchors a hydrophobic moiety into a lipophilic pocket. Our SAR (structure-activity relationship) analysis shows that all of these interactions are essential for inhibitory activity in this series. The crystal structure suggests that the compound would block binding of UTP and lock GlmU in an apo-enzyme-like conformation, thus interfering with its enzymatic activity. Our lead generation effort provides ample scope for further optimization of these compounds for antibacterial drug discovery.     

Structure-based virtual screening and biological evaluation of potent and selective ADAM12 inhibitors

We describe a series of potent and selective inhibitors of ADAM12 that were discovered using computational screening of a focused virtual library. The initial structure-based virtual screening selected 64 compounds from a 3D database of 67,062 molecules. Being evaluated by a cell-based ADAM12 activity assay, compounds 5, 11, 14, 16 were further identified as the potent and selective inhibitors of ADAM12 with low nanomolar IC₅₀ values. The mechanism underlying the potency and selectivity of a representative compound, 5, was investigated through molecular docking studies.     

Discovery of LRRK2 inhibitors by using an ensemble of virtual screening methods

In this paper, we present the results of a ligand- and structure-based virtual screen targeting LRRK2, a kinase that has been implicated in Parkinson’s disease. For the ligand-based virtual screen, the structures of 12 competitor compounds were used as queries for a variety of 2D and 3D searches. The structure-based virtual screen relied on homology models of LRRK2, as no X-ray structure is currently available in the public domain. From the virtual screening, 662 compounds were purchased, of which 35 showed IC₅₀ values below 10 μM in wild-type and/or mutant LRRK2 (a hit rate of 5.3%). Of these 35 hits, four were deemed to have potential for medicinal chemistry follow-up.     

Discovery of d-amino acid oxidase inhibitors based on virtual screening against the lid-open enzyme conformation

d-Amino acid oxidase (DAAO) inhibitors are typically small polar compounds with often suboptimal pharmacokinetic properties. Features of the native binding site limit the operational freedom of further medicinal chemistry efforts. We therefore initiated a structure based virtual screening campaign based on the X-ray structures of DAAO complexes where larger ligands shifted the loop (lid opening) covering the native binding site. The virtual screening of our in-house collection followed by the in vitro test of the best ranked compounds led to the identification of a new scaffold with micromolar IC₅₀. Subsequent SAR explorations enabled us to identify submicromolar inhibitors. Docking studies supported by in vitro activity measurements suggest that compounds bind to the active site with a salt-bridge characteristic to DAAO inhibitor binding. In addition, displacement of and interaction with the loop covering the active site contributes significantly to the activity of the most potent compounds.     

Identification of novel BRAF kinase inhibitors with structure-based virtual screening

VRAF murine sarcoma viral oncogene homologue B1 (BRAF) kinase has proved to be a promising target for the development of therapeutics for the treatment of a variety of human cancers. Here, we report the first example of a successful application of the structure-based virtual screening to identify novel BRAF inhibitors. These inhibitors have desirable physicochemical properties as a drug candidate, and compound 1 revealed a submicromolar binding affinity (0.7 μM). Therefore, they may serve as promising lead compounds for further development by structure-activity relationship (SAR) studies to optimize the inhibitory activities. Structural features relevant to the stabilization of the newly identified inhibitors in the ATP-binding site of BRAF are discussed in detail.     

Identification of novel inhibitors of methionyl-tRNA synthetase (MetRS) by virtual screening

Multiple inhibitors of the antibacterial target, Staphylococcus aureus MetRS, were identified by virtual screening. The process consisted of building a Catalyst^® pharmacophore from a ligand-S. aureus MetRS structure and using this pharmacophore to screen a commercial database. The top hits from this search were then docked into the S. aureus MetRS structure and this information was used to select compounds for testing. This resulted in a high hit rate of compounds that are in distinct structural classes from the known MetRS ligands.     

Design checkpoint kinase 2 inhibitors by pharmacophore modeling and virtual screening techniques

Damage to DNA is caused by ionizing radiation, genotoxic chemicals or collapsed replication forks. When DNA is damaged or cells fail to respond, a mutation that is associated with breast or ovarian cancer may occur. Mammalian cells control and stabilize the genome using a cell cycle checkpoint to prevent damage to DNA or to repair damaged DNA. Checkpoint kinase 2 (Chk2) is one of the important kinases, which strongly affects DNA-damage and plays an important role in the response to the breakage of DNA double-strands and related lesions. Therefore, this study concerns Chk2. Its purpose is to find potential inhibitors using the pharmacophore hypotheses (PhModels) and virtual screening techniques. PhModels can identify inhibitors with high biological activities and virtual screening techniques are used to screen the database of the National Cancer Institute (NCI) to retrieve compounds that exhibit all of the pharmacophoric features of potential inhibitors with high interaction energy. Ten PhModels were generated using the HypoGen best algorithm. The established PhModel, Hypo01, was evaluated by performing a cost function analysis of its correlation coefficient (r), root mean square deviation (RMSD), cost difference, and configuration cost, with the values 0.955, 1.28, 192.51, and 16.07, respectively. The result of Fischer’s cross-validation test for the Hypo01 model yielded a 95% confidence level, and the correlation coefficient of the testing set (r_test) had a best value of 0.81. The potential inhibitors were then chosen from the NCI database by Hypo01 model screening and molecular docking using the cdocker docking program. Finally, the selected compounds exhibited the identified pharmacophoric features and had a high interaction energy between the ligand and the receptor. Eighty-three potential inhibitors for Chk2 are retrieved for further study.     

Structure-based virtual screening and biological evaluation of novel small-molecule BTK inhibitors

Bruton tyrosine kinase (BTK) is linked to multiple signalling pathways that regulate cellular survival, activation, and proliferation. A covalent BTK inhibitor has shown favourable outcomes for treating B cell malignant leukaemia. However, covalent inhibitors require a high reactive warhead that may contribute to unexpected toxicity, poor selectivity, or reduced effectiveness in solid tumours. Herein, we report the identification of a novel noncovalent BTK inhibitor. The binding interactions (i.e. interactions from known BTK inhibitors) for the BTK binding site were identified and incorporated into a structure-based virtual screening (SBVS). Top-rank compounds were selected and testing revealed a BTK inhibitor with >50% inhibition at 10 µM concentration. Examining analogues revealed further BTK inhibitors. When tested across solid tumour cell lines, one inhibitor showed favourable inhibitory activity, suggesting its potential for targeting BTK malignant tumours. This inhibitor could serve as a basis for developing an effective BTK inhibitor targeting solid cancers.     

Lead generation of heat shock protein 90 inhibitors by a combination of fragment-based approach, virtual screening, and structure-based drug design

Heat shock protein 90 (Hsp90) is a molecular chaperone which regulates maturation and stabilization of its substrate proteins, known as client proteins. Many client proteins of Hsp90 are involved in tumor progression and survival and therefore Hsp90 can be a good target for developing anticancer drugs. With the aim of efficiently identifying a new class of orally available inhibitors of the ATP binding site of this protein, we conducted fragment screening and virtual screening in parallel against Hsp90. This approach quickly identified 2-aminotriazine and 2-aminopyrimidine derivatives as specific ligands to Hsp90 with high ligand efficiency. In silico evaluation of the 3D X-ray Hsp90 complex structures of the identified hits allowed us to promptly design CH5015765, which showed high affinity for Hsp90 and antitumor activity in human cancer xenograft mouse models.     

Identification of novel PARP-1 inhibitors by structure-based virtual screening

Poly(ADP-ribose)polymerase-1 (PARP-1) is an abundant and ubiquitous chromatin-bound nuclear protein. PARP-1, a DNA repair enzyme, has been in the limelight as a chemotherapeutic target. In this study, we demonstrated the successful use of structure-based virtual screening to identify inhibitors of PARP-1 from Otava databases comprised of nearly 260,000 compounds. Five novel inhibitors belonging to thienopyrimidinone, isoquinolinoquinazolinone, pyrroloquinazolinone, and cyclopentenothienopyrimidinone scaffolds revealed inhibitory potencies with IC₅₀ values ranged from 9.57 μM to 0.72 μM. Structural features relevant to the activity of these novel compounds within the active site of PARP-1 are discussed in detail and will guide future SAR investigation on these scaffolds.     

Development of selective and reversible pyrazoline based MAO-B inhibitors: virtual screening, synthesis and biological evaluation

In an effort to develop selective MAO (monoamine oxidase) B inhibitors, structure based virtual screening was initiated on an in-house library. Top 10 HITS were synthesized and evaluated for MAO (A and B) inhibitory activity, both against human and rat enzymes. All the compounds were found selective, reversible and active in nM range (100 times more potent than selegeline) towards MAO-B. Outstanding co-relation between predicted and experimental K_i values were observed.     

Identification of small-molecule urea derivatives as novel NAMPT inhibitors via pharmacophore-based virtual screening

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the condensation of nicotinamide (NAM) with 5-phosphoribosyl-1-prophosphate (PRPP) to yield nicotinamide mononucleotide (NMN), a rate limiting enzyme in a mammalian salvage pathway of nicotinamide adenine dinucleotide (NAD⁺) synthesis. Recently, intracellular NAD⁺ has received substantial attention due to the recent discovery that several enzymes including poly(ADP-ribose) polymerases (PARPs), mono(ADP-ribose) transferases (ARTs), and sirtuins (SIRTs), use NAD⁺ as a substrate, suggesting that intracellular NAD⁺ level may regulate cytokine production, metabolism, and aging through these enzymes. NAMPT is found to be upregulated in various types of cancer, and given its importance in the NAD⁺ salvage pathway, NAMPT is considered as an attractive target for the development of new cancer therapies. In this study, the reported NAMPT inhibitors bearing amide, cyanoguanidine, and urea scaffolds were used to generate pharmacophore models and pharmacophore-based virtual screening studies were performed against ZINC database. Following the filtering steps, ten hits were identified and evaluated for their in vitro NAMPT inhibitory effects. Compounds GF4 (NAMPT IC₅₀ = 2.15 ± 0.22 μM) and GF8 (NAMPT IC₅₀ = 7.31 ± 1.59 μM) were identified as new urea-typed inhibitors of NAMPT which also displayed cytotoxic activities against human HepG2 hepatocellular carcinoma cell line with IC₅₀ values of 15.20 ± 1.28 and 24.28 ± 6.74 μM, respectively.