Small-Molecule Inhibitors of the NusB-NusE Protein-Protein Interaction with Antibiotic Activity.

The NusB-NusE protein-protein interaction (PPI) is critical to the formation of stable antitermination complexes required for stable RNA transcription in all bacteria. This PPI is an emerging antibacterial drug target. Pharmacophore-based screening of the mini-Maybridge compound library (56 000 molecules) identified N,N'-[1,4-butanediylbis(oxy-4,1-phenylene)]bis(N-ethyl)urea 1 as a lead of interest. Competitive enzyme-linked immunosorbent assay screening validated 1 as a 20 µM potent inhibitor of NusB-NusE. Four focused compound libraries based on 1, comprising 34 compounds in total were designed, synthesized, and evaluated as NusB-NusE PPI inhibitors. Ten analogues displayed NusB-NusE PPI inhibition ≥50% at 25 µM concentration in vitro. In contrast to representative Gram-negative Escherichia coli and Gram-positive Bacillus subtilis species, these analogues showed up to 100% growth inhibition at 200 µM. 2-((Z)-4-(((Z)-4-(4-((E)-(Carbamimidoylimino)methyl)phenoxy)but-2-en-1-yl)oxy)benzylidene)hydrazine-1-carboximidamide 22 showed excellent activity against important pathogens. With minimum inhibitory concentration values of ≤3 µg/mL for Gram-positive Streptococcus pneumoniae and methicillin-resistant Staphylococcus aureus and ≤51 µg/mL for Gram-negative Pseudomonas aeruginosa and Acinetobacter baumannii, 22 is a potent lead for a novel antibacterial target. Epifluorescence studies in live bacteria were consistent with 22, inhibiting the NusB-NusE PPI as proposed.

Pyrimidine-Based Inhibitors of Dynamin I GTPase Activity: Competitive Inhibition at the Pleckstrin Homology Domain.

The large GTPase dynamin mediates membrane fission during clathrin-mediated endocytosis (CME). The aminopyrimidine compounds were reported to disrupt dynamin localization to the plasma membrane via the PH domain and implicate this mechanism in the inhibition of CME. We have used a computational approach of binding site identification, docking, and interaction energy calculations to design and synthesize a new library of aminopyrimidine analogues targeting site-2 of the pleckstrin homology (PH) domain. The optimized analogues showed low micromolar inhibition against both dynamin I (IC50 = 10.6 ± 1.3 to 1.6 ± 0.3 µM) and CME (IC50(CME) = 65.9 ± 7.7 to 3.7 ± 1.1 mM), which makes this series among the more potent inhibitors of dynamin and CME yet reported. In CME and growth inhibition cell-based assays, the data obtained was consistent with dynamin inhibition. CEREP ExpresS profiling identified off-target effects at the cholecystokinin, dopamine D2, histamine H1 and H2, melanocortin, melatonin, muscarinic M1 and M3, neurokinin, opioid KOP and serotonin receptors.

Synthesis and anti-mycobacterial activity of 4-(4-phenyl-1H-1,2,3-triazol-1-yl)salicylhydrazones: revitalizing an old drug.

The antitubercular drug; para-aminosalicylic acid (PAS) was used as the core scaffold for the design of a series of 1H-1,2,3-triazolylsalicylhydrazones upon coupling with triazole and arylhydrazone moietis to furnish a single molecular architecture. The obtained derivatives were screened against Mycobacterium tuberculosis H37Rv revealing good to high activity for the active compounds (MIC values of 0.39-1.5 µg/mL) compared to the marketed drugs isoniazid, rifampicin and ethambutol. Moreover, the most active analogue N-(1-(4-chlorobenzyl)-2-oxoindolin-3-ylidene)-2-hydroxy-4-(4-phenyl-1H-1,2,3-triazol-1-yl)-benzohydrazide (20) was found to be ten-fold more potent than PAS and equipotent to rifampicin (MIC 0.39 µg/mL), while exhibiting low cytotoxicity with a selectivity index of >128. In addition, this compound was shown to be active against persistent forms of mycobacteria comparable to standard drugs in nutrient starvation model. Accordingly, we introduce compound 20 as a valuable lead for further development. A 3D-QSAR study was also conducted to help in explaining the observed activity and to serve as a tool for further development.

Quinolone-1-(2H)-ones as hedgehog signalling pathway inhibitors.

A series of quinolone-2-(1H)-ones derived from the Ugi-Knoevenagel three- and four-component reaction were prepared exhibiting low micromolar cytotoxicity against a panel of eight human cancer cell lines known to possess the Hedgehog Signalling Pathway (HSP) components, as well as the seminoma TCAM-2 cell line. A focused SAR study was conducted and revealed core characteristics of the quinolone-2-(1H)-ones required for cytotoxicity. These requirements included a C3-tethered indole moiety, an indole C5-methyl moiety, an aliphatic tail or an ester, as well as an additional aromatic moiety. Further investigation in the SAG-activated Shh-LIGHT2 cell line with the most active analogues: 2-(3-cyano-2-oxo-4-phenylquinolin-1(2H)-yl)-2-(1-methyl-1H-indol-3-yl)-N-(pentan-2-yl)acetamide (5), 2-(3-cyano-2-oxo-4-phenylquinolin-1(2H)-yl)-2-(5-methyl-1H-indol-3-yl)-N-(pentan-2-yl)acetamide (23) and ethyl (2-(3-cyano-2-oxo-4-phenylquinolin-1(2H)-yl)-2-(5-methyl-1H-indol-3-yl)acetyl)glycinate (24) demonstrated a down regulation of the HSP via a reduction in Gli expression, and in the mRNA levels of Ptch1 and Gli2. Analogues 5, 23 and 24 returned in cell inhibition values of 11.6, 2.9 and 3.1 µM, respectively, making this new HSP-inhibitor pharmacophore amongst the most potent non-Smo targeted inhibitors thus far reported.

1,8-Naphthalimide derivatives: new leads against dynamin I GTPase activity.

Fragment-based in silico screening against dynamin I (dynI) GTPase activity identified the 1,8-naphthalimide framework as a potential scaffold for the design of new inhibitors targeting the GTP binding pocket of dynI. Structure-based design, synthesis and subsequent optimization resulted in the development of a library of 1,8-naphthalimide derivatives, called the Naphthaladyn™ series, with compounds 23 and 29 being the most active (IC50 of 19.1 ± 0.3 and 18.5 ± 1.7 µM respectively). Compound 29 showed effective inhibition of clathrin-mediated endocytosis (IC50(CME) 66 µM). The results introduce 29 as an optimised GTP-competitive lead Naphthaladyn™ compound for the further development of naphthalimide-based dynI GTPase inhibitors.

Further exploration of the heterocyclic diversity accessible from the allylation chemistry of indigo.

Diversity-directed synthesis based on the cascade allylation chemistry of indigo, with its embedded 2,2'-diindolic core, has resulted in rapid access to new examples of the hydroxy-8a,13-dihydroazepino[1,2-a:3,4-b']diindol-14(8H)-one skeleton in up to 51% yield. Additionally a derivative of the novel bridged heterocycle 7,8-dihydro-6H-6,8a-epoxyazepino[1,2-a:3,4-b']diindol-14(13H)-one was produced when the olefin of the allylic substrate was terminally disubstituted. Further optimisation also produced viable one-pot syntheses of derivatives of the spiro(indoline-2,9'-pyrido[1,2-a]indol)-3-one (65%) and pyrido[1,2,3-s,t]indolo[1,2-a]azepino[3,4-b]indol-17-one (72%) heterocyclic systems. Ring-closing metathesis of the N,O-diallylic spiro structure and subsequent Claisen rearrangement gave rise to the new (1R,8aS,17aS)-rel-1,2-dihydro-1-vinyl-8H,17H,9H-benz[2',3']pyrrolizino[1',7a':2,3]pyrido[1,2-a]indole-8,17-(2H,9H)-dione heterocyclic system.

Phenothiazine-derived antipsychotic drugs inhibit dynamin and clathrin-mediated endocytosis.

Chlorpromazine is a phenothiazine-derived antipsychotic drug (APD) that inhibits clathrin-mediated endocytosis (CME) in cells by an unknown mechanism. We examined whether its action and that of other APDs might be mediated by the GTPase activity of dynamin. Eight of eight phenothiazine-derived APDs inhibited dynamin I (dynI) in the 2-12 µm range, the most potent being trifluoperazine (IC50 2.6 ± 0.7 µm). They also inhibited dynamin II (dynII) at similar concentrations. Typical and atypical APDs not based on the phenothiazine scaffold were 8- to 10-fold less potent (haloperidol and clozapine) or were inactive (droperidol, olanzapine and risperidone). Kinetic analysis showed that phenothiazine-derived APDs were lipid competitive, while haloperidol was uncompetitive with lipid. Accordingly, phenothiazine-derived APDs inhibited dynI GTPase activity stimulated by lipids but not by various SH3 domains. All dynamin-active APDs also inhibited transferrin (Tfn) CME in cells at related potencies. Structure-activity relationships (SAR) revealed dynamin inhibition to be conferred by a substituent group containing a terminal tertiary amino group at the N2 position. Chlorpromazine was previously proposed to target AP-2 recruitment in the formation of clathrin-coated vesicles (CCV). However, neither chlorpromazine nor thioridazine affected AP-2 interaction with amphiphysin or clathrin. Super-resolution microscopy revealed that chlorpromazine blocks neither clathrin recruitment by AP-2, nor AP-2 recruitment, showing that CME inhibition occurs downstream of CCV formation. Overall, potent dynamin inhibition is a shared characteristic of phenothiazine-derived APDs, but not other typical or atypical APDs, and the data indicate that dynamin is their likely in-cell target in endocytosis.

Development of quinone analogues as dynamin GTPase inhibitors.

Virtual screening of the ChemDiversity and ChemBridge compound databases against dynamin I (dynI) GTPase activity identified 2,5-bis-(benzylamino)-1,4-benzoquinone 1 as a 273 ± 106 µM inhibitor. In silico lead optimization and focused library-led synthesis resulted in the development of four discrete benzoquinone/naphthoquinone based compound libraries comprising 54 compounds in total. Sixteen analogues were more potent than lead 1, with 2,5-bis-(4-hydroxyanilino)-1,4-benzoquinone (45) and 2,5-bis(4-carboxyanilino)-1,4-benzoquinone (49) the most active with IC50 values of 11.1 ± 3.6 and 10.6 ± 1.6 µM respectively. Molecular modelling suggested a number of hydrogen bonding and hydrophobic interactions were involved in stabilization of 49 within the dynI GTP binding site. Six of the most active inhibitors were evaluated for potential inhibition of clathrin-mediated endocytosis (CME). Quinone 45 was the most effective CME inhibitor with an IC50(CME) of 36 ± 16 µM.

In silico docking, molecular dynamics and binding energy insights into the bolinaquinone-clathrin terminal domain binding site.

Clathrin-mediated endocytosis (CME) is a process that regulates selective internalization of important cellular cargo using clathrin-coated vesicles. Perturbation of this process has been linked to many diseases including cancer and neurodegenerative conditions. Chemical proteomics identified the marine metabolite, 2-hydroxy-5-methoxy-3-(((1S,4aS,8aS)-1,4a,5-trimethyl-1,2,3,4,4a,7,8,8a-octahydronaphthalen-2-yl)methyl)cyclohexa- 2,5-diene-1,4-dione (bolinaquinone) as a clathrin inhibitor. While being an attractive medicinal chemistry target, the lack of data about bolinaquinone's mode of binding to the clathrin enzyme represents a major limitation for its structural optimization. We have used a molecular modeling approach to rationalize the observed activity of bolinaquinone and to predict its mode of binding with the clathrin terminal domain (CTD). The applied protocol started by global rigid-protein docking followed by flexible docking, molecular dynamics and linear interaction energy calculations. The results revealed the potential of bolinaquinone to interact with various pockets within the CTD, including the clathrin-box binding site. The results also highlight the importance of electrostatic contacts over van der Waals interactions for proper binding between bolinaquinone and its possible binding sites. This study provides a novel model that has the potential to allow rapid elaboration of bolinaquinone analogues as a new class of clathrin inhibitors.

Novel non-cyclooxygenase inhibitory derivatives of naproxen for colorectal cancer chemoprevention.

A structure-based medicinal chemistry strategy was applied to design new naproxen derivatives that show growth inhibitory activity against human colon tumor cells through a cyclooxygenase (COX)-independent mechanism. In vitro testing of the synthesized compounds against the human HT-29 colon tumor cell line revealed enhanced growth inhibitory activity compared to the parent naproxen with 3a showing IC50 of 11.4 µM (two orders of magnitude more potent than naproxen). Selectivity of 3a was investigated against a panel of three tumor and one normal colon cell lines and showed up to six times less toxicity against normal colonocytes. Compound 3a was shown to induce dose-dependent apoptosis of HT116 colon tumor cells as evidenced by measuring the activity of caspases-3 and 7. None of the synthesized compounds showed activity against COX-1 or COX-2 isozymes, confirming a COX-independent mechanism of action. Compound 3k was found to have no ulcerogenic effect in rats as indicated by electron microscope scanning of the stomach after oral administration. A pharmacophore model was developed for elucidating structure-activity relationships and subsequent chemical optimization for this series of compounds as colorectal cancer chemopreventive drugs.

3,5-disubstituted thiadiazine-2-thiones: new cell-cycle inhibitors.

Two series, a and b, of 3-cyclopentyl or (3-cyclohexyl)-5-substituted-3,4,5,6-tetrahydro-2H-1,3,5-thiadiazine-2-thiones (THTT) 2a-9a and 3b, 4b, 6b-9b, were synthesized to develop new cell cycle inhibitors. Variable and promising in vitro antiproliferative activities were shown with the synthesized THTT derivatives. Compound 5a with a 5-cyclopentyl group on position-3 and a glutamine residue on position-5 of the THTT moiety showed maximum activity (IC(50) = 8.98 µM). Compound 5a possessed notable cell cycle disrupting and apoptotic activities with enhanced selectivity against cancer cells, suggesting the potential for the development of new selective cell cycle inhibitors. There is no evident relationship between the cytotoxic activity of the tested compounds and their lipophilicity. In addition, a pharmacophore based study was performed to explain the biological activity on structural bases. A successful model was generated with a good correlation with the observed activity.

Schiff bases of indoline-2,3-dione: potential novel inhibitors of Mycobacterium tuberculosis (Mtb) DNA gyrase.

In the present study a series of Schiff bases of indoline-2,3-dione were synthesized and investigated for their Mtb gyrase inhibitory activity. Promising inhibitory activity was demonstrated with some of these derivatives, which exhibited IC(50) values ranging from 50-157 mM. The orientation and the ligand-receptor interactions of such molecules within the Mtb DNA gyrase A subunit active site were investigated applying a multi-step docking protocol using Molecular Operating Environment (MOE) and Autodock4 docking software. The results revealed the importance of the isatin moiety and the connecting side chain for strong interactions with the enzyme active site. Among the tested compounds the terminal aromatic ring benzofuran showed the best activity. Promising new leads for developing a novel class of Mtb gyrase inhibitors were obtained from Schiff bases of indoline-2,3-dione.

Quantitative structure-activity relationship (QSAR) studies on a series of 1,3,4-thiadiazole-2-thione derivatives as tumor-associated carbonic anhydrase IX inhibitors.

A linear quantitative structure-activity relationship (QSAR) study that encodes various aspects of physicochemical, topological and electronic descriptors has been developed for a series of 1,3,4-thiadiazole-2-thione derivatives (1a-r and 2a-c). The carbonic anhydrase IX inhibitory activity of the candidates under study (1a-r and 2a-c) were correlated to the selected parameters using stepwise linear regression analyses to achieve the best QSAR model. Promising results were obtained with the employed tetra-parametric model indicating that the information approach used in the present investigation is quite useful for modeling carbonic anhydrase IX inhibitors.

Design, synthesis, and docking studies of new 1,3,4-thiadiazole-2-thione derivatives with carbonic anhydrase inhibitory activity.

A new series of 1,3,4-thiadiazole-2-thione derivatives have been prepared and assayed for the inhibition of three physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic human isozymes I and II, and the transmembrane, tumor-associated hCA IX. Against hCA I the investigated thiones, showed inhibition constants in the range of 2.55-222 microM, against hCA II in the range of 2.0-433 microM, and against hCA IX in the range of 1.25-148 microM. Compound 5c, 4-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl)-1-(5-nitro-2-oxoindolin-3-ylidene)semicarbazide showed interesting inhibition of the tumor-associated hCA IX with K(I) value of 1.25 microM, being the first non-sulfonamide type inhibitor of such activity. This result is rather important taking into consideration the known antitumor activity of thiones. In addition, docking of the tested compounds into CA II active site was performed in order to predict the affinity and orientation of these compounds at the isozyme active site. The results showed similar orientation of the target compounds at CA II active site compared with reported sulfonamide type CAIs with the thione group acting as a zinc-binding moiety.