High throughput testing of drug library substances and monoclonal antibodies for capacity to reduce formation of cystatin C dimers to identify candidates for treatment of hereditary cystatin C amyloid angiopathy.
OBJECTIVE
To establish a high-throughput system for testing the ability of drugs or monoclonal antibodies to reduce the in vitro formation of cystatin C dimers to identify substances potentially useful for treatment of patients with hereditary cystatin C amyloid angiopathy (HCCAA).


METHODS
Various combinations of incubation temperature, time period, guanidinium chloride concentration and concentration of cystatin C monomers were tested in low-volume formats to induce dimer formation of recombinant cystatin C. The extent of dimerization was analysed by gel filtration chromatography and agarose gel electrophoresis.


RESULTS
A high-throughput system based upon agarose gel electrophoresis was developed and used to test 1040 drugs in a clinical drug library for their capacity to reduce cystatin C dimer formation in vitro. Seventeen substances reducing dimer formation by more than 30% were identified. A similar system for testing the capacity of monoclonal antibodies against cystatin C to reduce the in vitro formation of cystatin C dimers was also developed and used to test a panel of 12 monoclonal antibodies. Seven antibodies reducing dimer formation by more than 30% were identified and the two most potent, Cyst28 and HCC3, reduced dimerization by 75 and 60%, respectively.


CONCLUSIONS
We constructed a simple high-throughput system for testing the capacity of drugs and monoclonal antibodies to reduce the in vitro formation of cystatin C dimers and several candidates for treatment of HCCAA could be identified.
Department of Clinical Chemistry, Lund University Hospital, Lund, Sweden.
Gustav
G
Östner
Veronica
V
Lindström
Alexander B
AB
Postnikov
Tatiana I
TI
Solovyeva
Össur I
ÖI
Emilsson
Anders
A
Grubb
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D016657:Cerebral Amyloid Angiopathy / Q000151:congenital / Q000188:drug therapy / Q000378:metabolism* / Q000503:physiopathology; D002536:Cerebral Arteries / Q000187:drug effects / Q000378:metabolism* / Q000503:physiopathology; D002850:Chromatography, Gel; D055316:Cystatin C / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019281:Dimerization; D055808:Drug Discovery / Q000379:methods*; D004587:Electrophoresis, Agar Gel; D019791:Guanidine / Q000009:adverse effects; D057166:High-Throughput Screening Assays; D006801:Humans; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054852:Small Molecule Libraries / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D012996:Solutions
D000911:Antibodies, Monoclonal; D055316:Cystatin C; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D012996:Solutions; D019791:Guanidine