Initial screening of compound libraries

Screen two libraries of 10,000 chemically diverse drug-like compounds each (DIVERSet1 and DIVERSet2) for inhibition of prion protein aggregation using molecular SIFT screening assay

Not specifiedNot specified

Note: Compounds supplied in DMSO solution on 96-well microtiter plates

View evidence from paper

“we tested a library of 10,000 chemically diverse drug-like compounds in regard to inhibition of prion protein aggregation in a molecular SIFT screening assay”

2

Identification of initial lead structure

Based on SIFT screening results combined with testing of primary hits in cellular anti-prion assay, identify N-benzylidene-benzohydrazide (NBB) derivatives as new lead structure with anti-prion activity

Not specifiedNot specified

Note: NBB compounds also inhibit Poly-Q and α-syn aggregation both in vitro and in vivo, but contain Schiff's base structure prone to rapid metabolism in vivo

View evidence from paper

“Based on this data in combination with testing of primary hits in a cellular anti-prion assay, we identified N -benzylidene-benzohydrazide (NBB) derivatives as a new lead structure with anti-prion activity”

3

Secondary screening of additional compounds

Screen 10,000 additional compounds and perform parallel analysis of all 20,000 compounds in microtiter plate based high-throughput anti-prion cell culture assay followed by structure-activity and cluster analysis

Not specifiedNot specified

Note: Analysis identified cluster of highly active compounds belonging to 3,5-diphenyl-pyrazole (DPP) derivatives chemical class

View evidence from paper

“we continued by screening of 10,000 additional compounds and performed a parallel analysis of all these 20,000 compounds in a microtiter plate based high-throughput anti-prion cell culture assay followed by structure–activity and cluster analysis”

4

Pilot in vivo experiment with DPP compound

Conduct pilot experiment with one DPP compound from initial screening library in prion-infected mice to assess effect on survival

Not specifiedNot specified

Note: Pilot experiment showed significant effect on survival in prion-infected mice

View evidence from paper

“A pilot experiment with one DPP compound from the initial screening library showed a significant effect on survival in prion-infected mice”

5

Synthesis of focused DPP compound library

Synthesize focused library of approximately 150 DPP-related compounds for further testing in vitro, in cell culture, and in vivo

Not specifiedNot specified

Note: Details regarding synthesis and quality control provided in supplementary 'Compound Synthesis' section. Materials and solvents purchased from Sigma-Aldrich and Alfa Aesar

View evidence from paper

“we synthesized a focused library of ~150 DPP-related compounds (Suppl. 'Compound Synthesis') for further testing in vitro, in cell culture, and in vivo”

6

Selection of compounds for in vivo testing

Based on results from SIFT and cell culture anti-prion assays, select 38 compounds for in vivo testing in prion-infected mice

Not specifiedNot specified

Note: Selection criteria based on performance in SIFT and cell culture assays

View evidence from paper

“Based on results from the SIFT and cell culture anti-prion assays, 38 compounds were selected for in vivo testing in prion-infected mice”

7

In vivo testing of selected compounds

Test 38 selected DPP-related compounds in prion-infected mice to assess therapeutic effects on disease progression and survival

Not specifiedNot specified

Note: anle138b showed highest anti-prion activity and efficacy in in vitro and in vivo models for synucleinopathies

View evidence from paper

“In these experiments, the compound anle138b [3-(1,3-benzodioxol-5-yl)-5-(3-bromophenyl)-1 H -pyrazole] showed the highest anti-prion activity”

Anle138b: a novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and Parkinson’s disease

In Vivo Prion Mouse Study

Equipment2

Materials7

Software1

Protocol Steps

Initial screening of compound libraries

Identification of initial lead structure

Secondary screening of additional compounds

Pilot in vivo experiment with DPP compound

Synthesis of focused DPP compound library

Selection of compounds for in vivo testing

In vivo testing of selected compounds