ABSTRACT
A library of queuine analogues targeting the modification of tRNA isoacceptors for Asp, Asn, His and Tyr catalysed by queuine tRNA ribosyltransferase (QTRT, also known as TGT) was evaluated in the treatment of a chronic multiple sclerosis model: murine experimental autoimmune encephalomyelitis. Several active 7-deazaguanines emerged, together with a structure-activity relationship involving the necessity for a flexible alkyl chain of fixed length.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental , Animals , Mice , Encephalomyelitis, Autoimmune, Experimental/drug therapy , RNA, Transfer , Structure-Activity Relationship , Pentosyltransferases/metabolismABSTRACT
Base-modification can occur throughout a transfer RNA molecule; however, elaboration is particularly prevalent at position 34 of the anticodon loop (the wobble position), where it functions to influence protein translation. Previously, we demonstrated that the queuosine modification at position 34 can be substituted with an artificial analogue via the queuine tRNA ribosyltransferase enzyme to induce disease recovery in an animal model of multiple sclerosis. Here, we demonstrate that the human enzyme can recognize a very broad range of artificial 7-deazaguanine derivatives for transfer RNA incorporation. By contrast, the enzyme displays strict specificity for transfer RNA species decoding the dual synonymous NAU/C codons, determined using a novel enzyme-RNA capture-release method. Our data highlight the broad scope and therapeutic potential of exploiting the queuosine incorporation pathway to intentionally engineer chemical diversity into the transfer RNA anticodon.
Subject(s)
Pentosyltransferases/metabolism , RNA, Transfer/metabolism , Guanine/analogs & derivatives , Guanine/metabolism , Humans , RNA/metabolism , RNA, Transfer/chemistry , Substrate SpecificityABSTRACT
Queuine is a modified pyrrolopyrimidine nucleobase derived exclusively from bacteria. It post-transcriptionally replaces guanine 34 in transfer RNA isoacceptors for Asp, Asn, His and Tyr, in almost all eukaryotic organisms, through the activity of the ancient tRNA guanine transglycosylase (TGT) enzyme. tRNA hypomodification with queuine is a characteristic of rapidly-proliferating, non-differentiated cells. Autoimmune diseases, including multiple sclerosis, are characterised by the rapid expansion of T cells directed to self-antigens. Here, we demonstrate the potential medicinal relevance of targeting the modification of tRNA in the treatment of a chronic multiple sclerosis modelmurine experimental autoimmune encephalomyelitis. Administration of a de novo designed eukaryotic TGT substrate (NPPDAG) led to an unprecedented complete reversal of clinical symptoms and a dramatic reduction of markers associated with immune hyperactivation and neuronal damage after five daily doses. TGT is essential for the therapeutic effect, since animals deficient in TGT activity were refractory to therapy. The data suggest that exploitation of the eukaryotic TGT enzyme is a promising approach for the treatment of multiple sclerosis.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/therapy , Genetic Therapy/methods , Multiple Sclerosis/therapy , Pyrimidinones/pharmacology , Pyrroles/pharmacology , RNA, Transfer/pharmacology , Animals , Brain/pathology , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/pathology , Mice, Inbred C57BL , Multiple Sclerosis/genetics , Pentosyltransferases/genetics , Pentosyltransferases/metabolism , Pyrimidinones/chemistry , Pyrroles/chemistry , RNA, Transfer/chemistry , Thioguanine/chemistryABSTRACT
The TAM subfamily of Receptor Tyrosine Kinases (RTKs) contains three human proteins of therapeutical interest, Axl, Mer, and Tyro3. Our goal was to design a type II inhibitor specific for this family, i.e. able to interact with the allosteric pocket and with the hinge region of the kinase. We report the synthesis of several series of purine analogues of BMS-777607. The structural diversity of the designed inhibitors was expected to modify the interactions formed in the binding site and consequently to modulate their selectivity profiles. The most potent inhibitor 6g exhibits Kds of 39, 42, 65 and 200 nM against Axl, Mer, Met and Tyro3 respectively. Analysis of the affinity of 6g for active and inactive forms of Abl1, an RTK protein that does not belong to the TAM subfamily, together with the binding modes of 6g predicted by docking studies, indicates that 6g displays some selectivity for the TAM family and may act as a type II inhibitor.
Subject(s)
Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Animals , Cells, Cultured , Chlorocebus aethiops , Dose-Response Relationship, Drug , Humans , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemistry , Receptor Protein-Tyrosine Kinases/metabolism , Structure-Activity Relationship , Vero CellsABSTRACT
An efficient microwave-assisted palladium/copper comediated C-8 direct alkenylation of purines with styryl bromides has been developed. The method is regioselective, functional group tolerant, rapid, and compatible with other related azoles. Combined with subsequent nucleophilic substitution, it provides an easy access to new 6,8,9-trisubstituted purines.
