Dataset Information

High-throughput screens for small-molecule inhibitors of Pseudomonas aeruginosa biofilm development.

ABSTRACT: Pseudomonas aeruginosa is both a model biofilm-forming organism and an opportunistic pathogen responsible for chronic lung infections in cystic fibrosis (CF) patients and infections in burn patients, among other maladies. Here we describe the development of an efficient high-throughput screen to identify small-molecule modulators of biofilm formation. This screen has been run with 66,095 compounds to identify those that prevent biofilm formation without affecting planktonic bacterial growth. The screen is a luminescence-based attachment assay that has been validated with several strains of P. aeruginosa and compared to a well-established but low-throughput crystal violet staining biofilm assay. P. aeruginosa strain PAO1 was selected for use in the screen both because it forms robust biofilms and because genetic information and tools are available for the organism. The attachment-inhibited mutant, strain PAO1 DeltafliC, was used as a screening-positive control. We have also developed and validated a complementary biofilm detachment assay that can be used as an alternative primary screen or secondary screen for the attachment screening-positive compounds. We have determined the potencies of 61 compounds against biofilm attachment and have identified 30 compounds that fall into different structural classes as biofilm attachment inhibitors with 50% effective concentrations of less than 20 microM. These small-molecule inhibitors could lead to the identification of their relevant biofilm targets or potential therapeutics for P. aeruginosa infections.

SUBMITTER: Junker LM

PROVIDER: S-EPMC2043262 | biostudies-literature | 2007 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

High-throughput screens for small-molecule inhibitors of Pseudomonas aeruginosa biofilm development.

Junker Lauren M LM Clardy Jon J

Antimicrobial agents and chemotherapy 20070730 10

Pseudomonas aeruginosa is both a model biofilm-forming organism and an opportunistic pathogen responsible for chronic lung infections in cystic fibrosis (CF) patients and infections in burn patients, among other maladies. Here we describe the development of an efficient high-throughput screen to identify small-molecule modulators of biofilm formation. This screen has been run with 66,095 compounds to identify those that prevent biofilm formation without affecting planktonic bacterial growth. The ...[more]

PMID: 17664319

Similar Datasets

Project description:Leishmania species are parasitic protozoa that have a tightly controlled cell cycle, regulated by cyclin-dependent kinases (CDKs). Cdc2-related kinase 3 (CRK3), an essential CDK in Leishmania and functional orthologue of human CDK1, can form an active protein kinase complex with Leishmania cyclins CYCA and CYC6. Here we describe the identification and synthesis of specific small molecule inhibitors of bacterially expressed Leishmania CRK3:CYC6 using a high throughput screening assay and iterative chemistry. We also describe the biological activity of the molecules against Leishmania parasites.In order to obtain an active Leishmania CRK3:CYC6 protein kinase complex, we developed a co-expression and co-purification system for Leishmania CRK3 and CYC6 proteins. This active enzyme was used in a high throughput screening (HTS) platform, utilising an IMAP fluorescence polarisation assay. We carried out two chemical library screens and identified specific inhibitors of CRK3:CYC6 that were inactive against the human cyclin-dependent kinase CDK2:CycA. Subsequently, the best inhibitors were tested against 11 other mammalian protein kinases. Twelve of the most potent hits had an azapurine core with structure activity relationship (SAR) analysis identifying the functional groups on the 2 and 9 positions as essential for CRK3:CYC6 inhibition and specificity against CDK2:CycA. Iterative chemistry allowed synthesis of a number of azapurine derivatives with one, compound 17, demonstrating anti-parasitic activity against both promastigote and amastigote forms of L. major. Following the second HTS, 11 compounds with a thiazole core (active towards CRK3:CYC6 and inactive against CDK2:CycA) were tested. Ten of these hits demonstrated anti-parasitic activity against promastigote L. major.The pharmacophores identified from the high throughput screens, and the derivatives synthesised, selectively target the parasite enzyme and represent compounds for future hit-to-lead synthesis programs to develop therapeutics against Leishmania species. Challenges remain in identifying specific CDK inhibitors with both target selectivity and potency against the parasite.

Dataset Information

High-throughput screens for small-molecule inhibitors of Pseudomonas aeruginosa biofilm development.

Publications

High-throughput screens for small-molecule inhibitors of Pseudomonas aeruginosa biofilm development.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets