ABSTRACT
Thalidomide and the immunomodulatory drug, lenalidomide, are therapeutically active in hematological malignancies. The ubiquitously expressed E3 ligase protein cereblon (CRBN) has been identified as the primary teratogenic target of thalidomide. Our studies demonstrate that thalidomide, lenalidomide and another immunomodulatory drug, pomalidomide, bound endogenous CRBN and recombinant CRBN-DNA damage binding protein-1 (DDB1) complexes. CRBN mediated antiproliferative activities of lenalidomide and pomalidomide in myeloma cells, as well as lenalidomide- and pomalidomide-induced cytokine production in T cells. Lenalidomide and pomalidomide inhibited autoubiquitination of CRBN in HEK293T cells expressing thalidomide-binding competent wild-type CRBN, but not thalidomide-binding defective CRBN(YW/AA). Overexpression of CRBN wild-type protein, but not CRBN(YW/AA) mutant protein, in KMS12 myeloma cells, amplified pomalidomide-mediated reductions in c-myc and IRF4 expression and increases in p21(WAF-1) expression. Long-term selection for lenalidomide resistance in H929 myeloma cell lines was accompanied by a reduction in CRBN, while in DF15R myeloma cells resistant to both pomalidomide and lenalidomide, CRBN protein was undetectable. Our biophysical, biochemical and gene silencing studies show that CRBN is a proximate, therapeutically important molecular target of lenalidomide and pomalidomide.
Subject(s)
Adjuvants, Immunologic/pharmacology , Antineoplastic Agents/pharmacology , Peptide Hydrolases/drug effects , Thalidomide/analogs & derivatives , Adaptor Proteins, Signal Transducing , HEK293 Cells , Humans , Lenalidomide , Thalidomide/pharmacology , Ubiquitin-Protein Ligases , UbiquitinationABSTRACT
We have examined analogs of the previously reported 7-deaza-2'-deoxypurine nucleoside triphosphate series of human telomerase inhibitors. Two new telomerase-inhibiting nucleotides are reported: 6-methoxy-7-deaza-2'-deoxyguanosine 5'-triphosphate (OMDG-TP) and 6-thio-7-deaza-2'-deoxyguanosine 5'-triphosphate (TDG-TP). In particular, TDG-TP is a very potent inhibitor of human telomerase with an IC(50) of 60 nM. TDG-TP can substitute for dGTP as a substrate for telomerase, but only at relatively high concentrations. Under conditions in which TDG-TP is the only available guanosine substrate, telomerase becomes nonprocessive, synthesizing short products that appear to contain only one to three TDG residues. Similarly, the less potent telomerase inhibitor OMDG-TP gives rise to short telomerase products, but less efficiently than TDG-TP. We show here that TDG-TP, and to a lesser extent OMDG-TP, can serve as substrates for both templated (Klenow exo) and nontemplated (terminal transferase) DNA polymerases. For either polymerase, the products arising from TDG-TP are relatively short, and give rise to bands of unusual mobility under PAGE conditions. These anomalous bands revert, under treatment with DTT, to normal mobility bands, indicating that these products may contain thiol-labile disulfide linkages involving the incorporated TDG residues. This observation of potential TDG-crosslinks may have bearing on the mechanism of telomerase inhibition by this nucleotide analog.
Subject(s)
Deoxyguanine Nucleotides/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Telomerase/antagonists & inhibitors , DNA Polymerase I/antagonists & inhibitors , Deoxyguanine Nucleotides/pharmacology , Humans , In Situ Nick-End Labeling , Structure-Activity RelationshipABSTRACT
We have developed a novel strategy for the preparation of tetrahedral transition state analogs for aspartic acid and metallo-proteases based upon the sulfonimidamide functional group. Our best alpha-des-amino dipeptide analog binds at least 100-fold tighter than the corresponding ground state structure (i.e., amide). A previously unpublished five-membered cyclic sulfonimidamide was also synthesized.
Subject(s)
Aspartic Acid/analogs & derivatives , Metalloendopeptidases/chemistry , Sulfonamides/chemistry , Carboxypeptidases/antagonists & inhibitors , Carboxypeptidases A , HIV Protease Inhibitors/pharmacology , Inhibitory Concentration 50 , Models, Chemical , Pepsin A/antagonists & inhibitorsSubject(s)
Anthraquinones/chemical synthesis , DNA/chemistry , Enzyme Inhibitors/chemical synthesis , Telomerase/antagonists & inhibitors , Anthraquinones/chemistry , Anthraquinones/pharmacology , Base Sequence , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Molecular Structure , Nucleic Acid Conformation , Nucleic Acid Denaturation , Oligodeoxyribonucleotides/chemistryABSTRACT
We have developed a general strategy for assaying proteases that does not require the use of fluorogenic, chromogenic, or radiolabeled peptide substrates. The endo- or exoproteolytic hydrolysis of simple peptides can be followed spectrophotometrically by coupling the proteolytic event via enzyme-catalyzed reactions to a chromogenic redox dye. The couple can be used directly to follow the action of carboxy or amino peptidases on peptide substrates or can be coupled by use of carboxy or amino peptidases to follow the action of endoproteases on peptide substrates that are blocked at the amino or carboxy terminus, respectively. Liberated amino acids are detected by use of amino acid oxidase, oxygen, horseradish peroxidase, and the redox dye 2,2'-azino-bis-(3-ethyl-benzthiazoline-6-sulfonic acid (epsilon 414nm = 36,000 M-1 cm-1).
