Whole organism high-content screening by label-free, image-based Bayesian classification for parasitic diseases.

Sole reliance on one drug, Praziquantel, for treatment and control of schistosomiasis raises concerns about development of widespread resistance, prompting renewed interest in the discovery of new anthelmintics. To discover new leads we designed an automated label-free, high content-based, high throughput screen (HTS) to assess drug-induced effects on in vitro cultured larvae (schistosomula) using bright-field imaging. Automatic image analysis and Bayesian prediction models define morphological damage, hit/non-hit prediction and larval phenotype characterization. Motility was also assessed from time-lapse images. In screening a 10,041 compound library the HTS correctly detected 99.8% of the hits scored visually. A proportion of these larval hits were also active in an adult worm ex-vivo screen and are the subject of ongoing studies. The method allows, for the first time, screening of large compound collections against schistosomes and the methods are adaptable to other whole organism and cell-based screening by morphology and motility phenotyping.

cis-6-oxo-hexahydro-2-oxa-1,4-diazapentalene and cis-6-oxo-hexahydropyrrolo[3,2-c]pyrazole based scaffolds: design rationale, synthesis and cysteinyl proteinase inhibition.

The 5,5-bicycles cis-6-oxo-hexahydro-2-oxa-1,4-diazapentalene 3 and cis-6-oxo-hexahydropyrrolo[3,2-c]pyrazole 4 were designed as rotationally restricted templates towards the preparation of inhibitors of CAC1 cysteinyl proteinases. The design strategy was exemplified through the solution and solid phase preparation of potent inhibitors of human cathepsin K and may potentially be applied to inhibitors of other CAC1 proteinases.

Synthesis and evaluation of cis-hexahydropyrrolo[3,2-b]pyrrol-3-one peptidomimetic inhibitors of CAC1 cysteinyl proteinases.

A stereoselective synthesis of functionalised cis-hexahydropyrrolo[3,2-b]pyrrol-3-ones has been developed through Fmoc and Cbz-protected intermediates 5 and 6. Building blocks 5 and 6 were prepared via the intramolecular cyclisation of anti-epoxide 17. The intramolecular reaction occurred exclusively through the anti-epoxide to provide the 5,5-cis-fused bicycle, whereas the syn-epoxide, which theoretically would provide the 5,5-trans-fused bicycle, remained unchanged. These experimental observations are consistent with a key design element that we have introduced within this novel bicyclic ketone scaffold. Our bicyclic design strategy provides chiral stability to the bridgehead stereocentre that is situated alpha to the ketone because the cis-fused geometry is both thermodynamically and kinetically stable. Building blocks 5 and 6 have been utilised in both solid phase and solution phase syntheses of peptidomimetics 22, 36-40, which exhibit potent in vitro inhibition against a range of CAC1 cysteinyl proteinases. Compound 22, a potent and selective inhibitor of human cathepsin K exhibited good primary DMPK properties along with promising activity in an in vitro cell-based human osteoclast assay of bone resorption.

Bicyclic peptidomimetic tetrahydrofuro[3,2-b]pyrrol-3-one and hexahydrofuro[3,2-b]pyridine-3-one based scaffolds: synthesis and cysteinyl proteinase inhibition.

A stereoselective synthesis of (3aS,6aR)-tetrahydrofuro[3,2-b]pyrrol-3-ones and (3aS,7aR)-hexahydrofuro[3,2-b]pyridine-3-ones has been developed through Fmoc protected scaffolds 12 and 13. A key design element within these novel bicyclic scaffolds, in particular the 5,5-fused system, was the inherent stability of the cis-fused geometry in comparison to that of the corresponding trans-fused. Since the bridgehead stereocentre situated beta to the ketone was of a fixed and stable configuration, the fact that cis ring fusion is both kinetically and thermodynamically stable with respect to trans ring fusion provides chiral stability to the bridgehead stereocentre that is situated alpha to the ketone. To exemplify this principle, building blocks 12 and 13 were designed, prepared and utilised in a solid phase combinatorial synthesis of peptidomimetic inhibitors 10, 45a-e, 11 and 46. Both series were chirally stable with 5,5-series 10 and 45a-e exhibiting potent in vitro activity against a range of CAC1 cysteinyl proteinases. Compound 10, a potent and selective inhibitor of cathepsin K, possessed good primary DMPK properties along with promising activity in an in vitro cell-based human osteoclast assay of bone resorption.

Functionalised 2,3-dimethyl-3-aminotetrahydrofuran-4-one and N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)acylamide based scaffolds: synthesis and cysteinyl proteinase inhibition.

A stereoselective synthesis of functionalised (2R,3R)-2,3-dimethyl-3-amidotetrahydrofuran-4-one, its (2S,3R)-epimer and (3aR,6aR)-N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)acylamide cysteinyl proteinase inhibitors has been developed using Fmoc-protected scaffolds 6-8 in a solid-phase combinatorial strategy. Within these scaffolds, the introduction of an alkyl substituent alpha to the ketone affords chiral stability to an otherwise configurationally labile molecule. Preparation of scaffolds 6-8 required stereoselective syntheses of suitably protected alpha-diazomethylketone intermediates 9-11, derived from appropriately protected alpha-methylthreonines (2R,3R)-12, (2R,3S)-13 and a protected analogue of (1R,2R)-1-amino-2-hydroxycyclopentanecarboxylic acid 14. Application of standard methods for the preparation of amino acid alpha-diazomethylketones, through treatment of the mixed anhydride or pre-formed acyl fluorides of intermediates 12-14 with diazomethane, proved troublesome giving complex mixtures. However, the desired alpha-diazomethylketones were isolated and following a lithium chloride/acetic acid promoted insertion reaction provided scaffolds 6-8. Elaboration of 6-8 on the solid phase gave alpha,beta-dimethyl monocyclic ketone based inhibitors 38a-f, 39a,b,d,e,f and bicyclic inhibitors 40a-e that exhibited low micromolar activity against a variety of cysteinyl proteinases.