PMID- 15745743
OWN - NLM
STAT- MEDLINE
DCOM- 20051003
LR  - 20211203
IS  - 0003-2697 (Print)
IS  - 0003-2697 (Linking)
VI  - 338
IP  - 2
DP  - 2005 Mar 15
TI  - Development of a mechanism-based assay for tissue transglutaminase--results of a 
      high-throughput screen and discovery of inhibitors.
PG  - 237-44
AB  - Tissue transglutaminase (TGase) is a Ca(2+)-dependent enzyme that catalyzes 
      cross-linking of intracellular proteins through a mechanism that involves 
      isopeptide bond formation between Gln and Lys residues. In addition to its 
      transamidation activity, TGase can bind guanosine 5'-triphosphate (GTP) and does 
      so in a manner that is antagonized by calcium. Once bound, GTP undergoes 
      hydrolysis to form guanosine 5'-diphosphate and inorganic phosphate. TGase is 
      thought to play a pathogenic role in neurodegenerative diseases by promoting 
      aggregation of disease-specific proteins that accumulate in these disorders. 
      Thus, this enzyme represents a viable target for drug discovery. We now report 
      the development of a mechanism-based assay for TGase and the results of a screen 
      using this assay in which we tested 56,500 drug-like molecules for their ability 
      to inhibit TGase. In this assay, the Gln- and Lys-donating substrates are 
      N,N-dimethylated casein (NMC) and N-Boc-Lys-NH-CH(2)-CH(2)-NH-dansyl (KXD), 
      respectively. Through a combination of steady state kinetic experiments and 
      reaction progress curve simulations, we were able to calculate values for the 
      initial concentrations of NMC, KXD, and Ca(2+) that would produce a steady state 
      situation in which all thermodynamically significant forms of substrate-bound 
      TGase exist in equal concentration. Under these conditions, the assay is 
      sensitive to both competitive and mixed active-site inhibitors and to inhibitors 
      that bind to the GTP site. The assay was optimized for automated screening in 
      384-well format and was then used to test our compound library. From among these 
      compounds, 104 authentic hits that represent several mechanistic classes were 
      identified.
FAU - Case, April
AU  - Case A
AD  - Laboratory for Drug Discovery in Neurodegeneration, Harvard Center for 
      Neurodegeneration and Repair, Cambridge, MA 02139, USA.
FAU - Ni, Jake
AU  - Ni J
FAU - Yeh, Li-An
AU  - Yeh LA
FAU - Stein, Ross L
AU  - Stein RL
LA  - eng
PT  - Journal Article
PL  - United States
TA  - Anal Biochem
JT  - Analytical biochemistry
JID - 0370535
RN  - 0 (Caseins)
RN  - 0 (Dansyl Compounds)
RN  - 0 (Enzyme Inhibitors)
RN  - 0 (N,N-dimethylcasein)
RN  - 2418-95-3 (epsilon-tert-butyloxycarbonyl-lysine)
RN  - EC 2.3.2.13 (Protein Glutamine gamma Glutamyltransferase 2)
RN  - EC 2.3.2.13 (Transglutaminases)
RN  - EC 3.6.1.- (GTP-Binding Proteins)
RN  - K3Z4F929H6 (Lysine)
SB  - IM
MH  - Caseins/chemistry
MH  - Dansyl Compounds/chemistry
MH  - Drug Evaluation, Preclinical/*methods
MH  - Enzyme Inhibitors/*pharmacology
MH  - GTP-Binding Proteins/*antagonists & inhibitors
MH  - Lysine/analogs & derivatives/chemistry
MH  - Protein Glutamine gamma Glutamyltransferase 2
MH  - Transglutaminases/*antagonists & inhibitors
EDAT- 2005/03/05 09:00
MHDA- 2005/10/04 09:00
CRDT- 2005/03/05 09:00
PHST- 2004/09/10 00:00 [received]
PHST- 2005/03/05 09:00 [pubmed]
PHST- 2005/10/04 09:00 [medline]
PHST- 2005/03/05 09:00 [entrez]
AID - S0003-2697(04)00803-6 [pii]
AID - 10.1016/j.ab.2004.09.047 [doi]
PST - ppublish
SO  - Anal Biochem. 2005 Mar 15;338(2):237-44. doi: 10.1016/j.ab.2004.09.047.