Free-Wilson Method Research Articles

Abstract 2681: EWS-FLI1 as a molecular target: Small molecule inhibitors for a disordered protein

Abstract Ewing's sarcoma family of tumors are characterized by the EWS-FLI1 oncogenic fusion protein. Our previous studies show that RNA Helicase A (RHA) enhances EWS-FLI1 driven oncogenesis, and interruption of this protein-protein complex by small molecule inhibitors validate this interaction as a unique therapeutic target (NatMed 2009 Jul;15(7):750-6). EWS-FLI1 is a significantly hydrophobic disordered protein with unknown three-dimensional structure, which therefore precludes standard structure-based small molecule design. Given the challenges of drug design targeted to EWS-FLI1, we hypothesize that the optimization of a peptide displacement assay using small molecules can predict and direct the design of more potent analogs. Fluorescence polarization (FP) was originally used to measure RHA peptide displacement from EWS-FLI1, but is time consuming and reagent limiting. To identify new strategies for higher-throughput screening, a TECAN ULTRA 384 was used for assay detection in a 96-well plate format. However, this resulted in high background and low signal. A second approach, AlphaScreen (amplified luminescent proximity homogenous assay, PerkinElmer), uses a bead-based interaction system based on energy conversion and generation of chemiluminescence when acceptor and donor beads are brought together during binding. Results show a 6-fold dynamic range between bound and quenched beads, in comparison to just 2.5-fold for FP and less than two-fold for TECAN. Our small molecule, YK-4-279, is able to reduce the signal down to baseline, indicating that YK-4-279 dissociates the RHA peptide and EWS-FLI1. Key negative controls support specific binding between EWS-FLI1 and the RHA peptide. In addition, YK-4-279 was unable to quench the signal from a positive control using immunoglobulin, supporting the conclusion that YK-4-279 disrupts the specific protein-peptide binding rather than directly extinguishing the chemiluminescence. Data from the AlphaScreen will be used as input to a quantitative-structure activity relationship (QSAR) method. This QSAR method will predict biological activity from chemical structure and the knowledge gained from AlphaScreen will aid in the optimization and design of future analogs. In our initial work, we used the Free-Wilson QSAR method, converting the structure of the small molecules into a numerical format allowing for a mathematical comparison between structures, to populate a table with 32 small molecule analogs of YK-4-279. The AlphaScreen data may also be used with other QSAR methods, to which we may transition in later work. AlphaScreen technology provides a method to quantitatively compare binding of small molecules in a high-throughput screening system, yields a greater dynamic range with more specificity, and provides data that will help to predict the binding of other analogs with similar chemical structures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2681.

Abstract B181: EWS-FLI1 as a molecular target: Identification of inhibitors for a disordered protein

Abstract Ewing's sarcoma family of tumors (ESFT) are characterized by the EWS-FLI1 oncogenic fusion protein which results from a t(11;22) chromosomal translocation. Our previous studies show that RNA Helicase A (RHA) enhances EWS-FLI1 driven oncogenesis, and interruption of this protein-protein complex by small molecule inhibitors validate this interaction as a unique therapeutic target (NatMed 2009 Jul;15(7):750-6). EWS-FLI1 is a significantly hydrophobic disordered protein with unknown three-dimensional structure, which therefore precludes standard structure-based small molecule design. Fortunately, disordered proteins have a greater potential for binding with small molecule inhibitors due to structural flexibility. Given the challenges of drug design targeted to EWS-FLI1, we hypothesize that the optimization of a peptide displacement assay using small molecules can predict and direct the design of more potent analogs. Our methods originally used fluorescence polarization (FP) to measure RHA peptide displacement from EWS-FLI1. FP is a time consuming and reagent-limiting assay, so we evaluated new strategies using a 96-well plate format with the goal of high-throughput data collection. The first approach used a TECAN ULTRA 384 for assay detection and resulted in readings with high background noise and low signal. A second approach, AlphaScreen (amplified luminescent proximity homogenous assay, PerkinElmer), uses a bead-based interaction system based on energy conversion and generation of chemiluminescence when acceptor and donor beads are brought together during binding. The AlphaScreen assay uses approximately 3 ug of EWS-FLI1 protein per assay well and binds the 6-histadine tagged protein to the nickel-chelated donor beads while anti-FITC acceptor beads detect the flouresceintagged peptide. Our results show a 6-fold dynamic range between bound and quenched beads, in comparison to just 2.5-fold for FP and less than two-fold for the TECAN platform. Our small molecule YK-4-279 is able to reduce the signal back down to baseline, indicating that YK-4-279 dissociates the RHA peptide and EWS-FLI1. Key negative controls support specific binding between EWS-FLI1 and the RHA peptide. In addition, YK-4-279 was unable to quench the signal from a positive control using immunoglobulin, supporting the conclusion that YK-4-279 does indeed disrupt the specific protein-peptide binding rather than directly extinguishing the chemiluminescence. Data from the AlphaScreen will be used as input to a quantitative-structure activity relationship (QSAR) method. This QSAR method will predict biological activity from chemical structure and the knowledge gained from AlphaScreen will aid in the optimization and design of future analogs. In our initial work, we used the Free-Wilson QSAR method, converting the structure of the small molecules into a numerical format which allows for a mathematical comparison between structures, to populate a table with 32 small molecule analogs of YK-4-279. The AlphaScreen data may also be used with other QSAR methods, to which we may transition in later work. The AlphaScreen technology provides a method to quantitatively compare binding of small molecules in a high-throughput screening system, yields a greater dynamic range with more specificity, and provides data that will help to predict the binding of other analogs with similar chemical structures. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B181.

Chiral distinction in square planar Pt and Pd complexes of 2,2′-bipyridine derivatives

A computational study of square planar organometallic complexes formed by the ligand 2,2′-bipyridine and all its possible difluoro derivatives in analogous position of the aromatic rings (n,n′-difluoro-2,2′-bipyridine, where n = n′ = 3, 4, 5, and 6) and two M–X2 (M = Pd and Pt and X = F, Cl, Br, and I) fragments has been carried out amounting to a total of 80 complexes. Relationships have been found between the chiral distinction energy and the different chemical moieties present. Using the statistical Free-Wilson method, the relative energies between the diastereomeric complexes have been correlated with the position of the substituent, the counter anion, and the central metal cation.

The Oriented Development of Antituberculotics (Part II): Halogenated 3‐(4‐Alkylphenyl)‐1,3‐benzoxazine‐2,4‐(3H)‐diones

Based on our previous studies, 21 new halogenated 3-(4-alkylphenyl)-1,3-benzoxazine-2,4-(3H)-diones were synthesized by the reaction of salicylanilides and methyl-chloroformate. All compounds were screened in vitro against three different strains of mycobacterium, and Free-Wilson method was used to establish structure-activity relationships. 6-Bromo-3-(4-butylphenyl)-1,3-benzoxazine-2,4-(3H)-dione 3b proved to be the most active compound of the series.

The antimycobacterial derivatives against potential pathogenic strains: 2-Hydroxy-3-(4-phenylpiperazin-1-yl)-propylphenylcarbamates

According to our previous study 29 derivatives of 2-hydroxy-3-(4-phenylpiperazin-1-yl)-propylphenylcarbamates were tested for in vitro antimycobacterial activity against potential pathogenic strains Mycobacterium kansasii and Mycobacterium avium. The variations in group of compounds were by the substitution on phenyl rings. The Free-Wilson method was used to evaluate structure-antimycobacterial activity relationships. The advantage of compounds under study is in the activity against M. kansasii.

The Oriented Development of Antituberculotics: Salicylanilides

On the basis of our previous results 22 salicylanilides were synthesized. The compounds were tested for in vitro antimycobacterial activity against Mycobacterium tuberculosis, Mycobacterium kansasii, and Mycobacterium avium. The Free-Wilson method was used to evaluate structure-antimycobacterial activity relationships. 4-Chloro-N-(4-propylphenyl)salicylamide and 5-chloro-N-(4-propylphenyl)salicylamide were selected for preclinical studies.

Antimycobacterial activity of piperidinylpropyl esters of alkoxy-substituted phenylcarbamic acids.

A series of 17 hydrochlorides of piperidinylpropyl esters of alkoxy-substituted phenylcarbamic acids with the alkoxy group in position 2, 3 or 4 on the phenyl ring, and basic substituents attached to the moiety in position 3, were evaluated for in vitro antimycobacterial activity against the strains of Mycobacterium tuberculosis, M. kansasii and M. avium. To describe the structure-antimycobacterial activity relationships (QSAR), an approach based on the Free-Wilson method was employed to express the differences between individual moieties (including propyl and ethyl). The change of ethyl to propyl moiety increases the activity to M. tuberculosis but decreases the antimycobacterial activity to all potentially pathogenic strains under study.

Antimycobacterial and antifungal isosters of salicylamides.

A set of 40 derivatives of 3-hydroxypicolinic acid and 2-sulfanylbenzoic acid, isosteric to salicylanilides was synthesized. The compounds were evaluated for in vitro activity against Mycobacterium tuberculosis, Mycobacterium kansasii and Mycobacterium avium, Candida albicans, Candida tropicalis, Candida krusei, Candida glabrata, Trichosporon beigelii, Aspergillus fumigatus, Absidia corymbifera, Trichophyton mentagrophytes and Microsporum gypseum. Structure-activity relationships of antimycobacterial activity and antifungal activity against T. mentagrophytes and M. gypseum were analyzed by the Free-Wilson method. An increase in antimycobacterial activity was observed only for the sulfanylbenzoic acid derivatives, especially those with the benzyl moiety. The antifungal activity was not significant.

Synthesis and QSAR Studies of New Conjugated Benzothiazole Derivatives.

AbstractFor Abstract see ChemInform Abstract in Full Text.

In vitro antifungal activity of 3-phenyl-2H-benzoxazine-2,4(3H)-diones.

A series of 81 3-phenyl-2H-benzoxazine-2,4(3H)-diones with substitution at C(6) on the benzoxazine ring and on the phenyl moiety was synthesized; the compounds were evaluated for antifungal activity against five strains of potentially pathogenic fungi (Absidia corymbifera, Aspergillus fumigatus, Candida albicans, Microsporum gypseum and Trichophyton mentagrophytes). Structure-activity relationships against T. mentagrophytes and M. gypseum were determined using the Free-Wilson method, which was further combined with the approach of Hansch. In vitro antifungal activity becomes higher with increasing electron-accepting ability of the substituents on the phenyl ring, and with increasing lipophilicity.

Synthesis and QSAR Studies of New Conjugated Benzothiazole Derivatives

A series of new push-pull 2-(ω-phenylalkenyl)benzothiazolium compounds with methyl groups in different positions of the conjugated alkenyl bridge were synthesized by alkylation and condensation. The antimicrobial testing has been carried out and the effect of the alkenyl substituent was investigated by the QSAR Free-Wilson method.

Joint QSAR Analysis Using the Free-Wilson Approach and Quantum Chemical Parameters

A new quantitative structure-activity relationship (QSAR) technique combining the Free-Wilson method and constructed quantum chemical parameters was used to simulate the aqueous solubility (S w), 1-octanol/water partition coefficient (K ow) of 14 new synthesized benzanilide derivatives and their 96 h acute toxicity (EC50) to Daphnia magna. The mode of action of the 14 selected compounds to Daphnia magna was shown to be a complex process involving a physical partition stage and a biochemical reaction stage. The results also indicated that the joint (QSAR) analysis was much effective than the original Free-Wilson method and Hansch method not only in predicting properties/toxicity, but also in investigating the mode of action of chemicals.

ChemInform Abstract: New Groups of Antimycobacterial Agents: 6-Chloro-3-phenyl-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones and 6-Chloro-3-phenyl-2H-1,3-benzoxazine-2,4(3H)-dithiones.

Abstract A series of 6-chloro-3-phenyl-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones 3 and a series of 6-chloro-3-phenyl-2H-1,3-benzoxazine-2,4(3H)-dithiones 4 were synthesized by melting 6-chloro-3-phenyl-2H-1,3-benzoxazine-2,4(3H)-dione and its derivatives substituted on the phenyl ring 2 with tetraphosphorus decasulfide. Compounds 2c–e, 3 and 4 exhibited in vitro activity against Mycobacterium tuberculosis, M. kansasii (two strains) and M. avium better than or comparable to that of isoniazid. Replacement of the oxo group by a thioxo group at position 4 led to improvement in activity against M. tuberculosis and M. kansasii. The Free-Wilson method and procedure developed by the authors were used to analyse the structure–activity and structure–antimycobacterial profile relationships, respectively.

New groups of antimycobacterial agents: 6-chloro-3-phenyl-4-thioxo-2 H-1,3-benzoxazine-2(3 H)-ones and 6-chloro-3-phenyl-2 H-1,3-benzoxazine-2,4(3 H)-dithiones

A series of 6-chloro-3-phenyl-4-thioxo-2 H-1,3-benzoxazine-2(3 H)-ones 3 and a series of 6-chloro-3-phenyl-2 H-1,3-benzoxazine-2,4(3 H)-dithiones 4 were synthesized by melting 6-chloro-3-phenyl-2 H-1,3-benzoxazine-2,4(3 H)-dione and its derivatives substituted on the phenyl ring 2 with tetraphosphorus decasulfide. Compounds 2c–e, 3 and 4 exhibited in vitro activity against Mycobacterium tuberculosis, M. kansasii (two strains) and M. avium better than or comparable to that of isoniazid. Replacement of the oxo group by a thioxo group at position 4 led to improvement in activity against M. tuberculosis and M. kansasii. The Free-Wilson method and procedure developed by the authors were used to analyse the structure–activity and structure–antimycobacterial profile relationships, respectively.

QSAR Analysis of Substituted 2-Phenylhydrazonoacetamides Acting as Inhibitors of 15-Lipoxygenase

An evaluation of quantitative structure-activity relationships (QSAR) for 28 N1-phenyl-2-phenylhydrazonoacetamides, that are inhibitors of soybean 15-lipoxygenase was carried out with different statistical methods. Initially the variation of structure was characterized by the Free-Wilson method and analyzed by multiple linear regression (MLR) and partial least squares analysis (PLS). Both methods revealed an increase in activity, if the N1-phenyl substituent of the parent molecule is meta-substituted with groups, that show a positive resonance effect at the annular structure. To include physicochemical aspects a combined Free-Wilson-Hansch approach was used. Because of high intercorrelations among some physicochemical parameters a principal component-analysis (PCA) was performed to extract information from those intercorrelated variables in a few principal components (PC's). The resulting equations indicate that besides an electron donating group at the central amidrazone moiety electronic effects at the arylhydrazone substituent play an important role for the biological activity.

A quantitation of stereoselectivity by the Free-Wilson method

The stereoselectivity of muscarinic receptors was quantified by the Free-Wilson method. The analysis was performed with the affinity data, published by Gualtieri for certain muscarinic ligands (1,3-oxathiolanes) in three different tissues. The 2 and 3 positions in the 1,3-oxathiolane ring were important to differentiate between the receptors in those tissues. The results also showed that the three chiral centers in the 1,3-oxathiolanes displayed different stereoselective discrimination. The results of the Free-Wilson analysis were compared with those of a previous study on the same compounds obtained by epimeric eudismic analysis. The agreements and differences between the results obtained by these two methods are discussed in depth. Chirality 11:139–143, 1999. © 1999 Wiley-Liss, Inc.

Combination of the Topliss Approach with the Free-Wilson Analysis in the Study of Antimycobacterial Activityof 4-Alkylthiobenzanilides

On the basis of a preliminary study of the antimycobacterial activity of thiobenzanilides, a group of 4'-isopropyl- and 4'-butylthiobenzanilides have been synthesized and tested against Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium avium and Mycobacterium fortuitum. The effect of the substituents on minimum inhibitory concentrations was calculated by the Free-Wilson method. The separated values were analyzed by the Topliss approach. The substitution of the thiobenzanilide in position 4' by the isopropyl group or butyl group increased the antimycobacterial activity less than the cyclohexyl group. The substitution in position 4 decreased the activity by the steric effect.

A New Pharmacophore of Potential Antimycobacterial Activity: The Disulfide Group in the Ring of Alicyclic Compounds

A series of thirty 2,3-dithiabicyclo[3.3.0]octa-4,8-diene derivatives was examined for antimycobacterial activity against Mycobacterium tuberculosis, M. kansasii, M. avium, and M. fortuitum. The structure-activity relationships were analysed by Free-Wilson method. The authors consider the disulfide moiety to be the pharmacophore of antimycobacterial activity. To verify the idea, several other monocyclic sulfides, 3,4-dihydro-1,2-dithiolane derivatives, were also evaluated.

New Groups of Potential Antituberculotics: 5-Alkylthio-1-aryltetrazoles

A series of 5-alkylthio-1-aryltetrazoles 1-14 was prepared by alkylation of the corresponding 1-aryltetrazole-5-thiols with alkyl bromides in the cyclohexane-aqueous potassium hydroxide system. The new compounds were evaluated for their activity against Mycobacterium tuberculosis, M. kansasii, M. avium and M. fortuitum. The effects of aryl and alkyl fragments on minimum inhibitory concentrations (MIC) against M. tuberculosis and M. kansasii were analyzed by Free-Wilson method. On basis of calculated fragment contributions, 5-butylthio-1-(3,4-dimethylphenyl)tetrazole (15) was predicted to be the most antimycobacterially active derivative in the present series studied and its activity was verified experimentally. MIC values of 30 μmol l-1 and 61 μmol l-1 were obtained for its activity against M. tuberculosis and M. kansasii, respectively.

Synergism ofQSAR models: The Free-Wilson method in a baricentric coordinate system

Synergism ofQSAR models: The Free-Wilson method in a baricentric coordinate system