ABSTRACT
Thioamides are found in a few natural products and two known protein assemblies: the Escherichia coli ribosome and methyl-coenzyme M reductase (MCR) from methane-metabolizing archaea. Compared to an amide, thioamides alter the physical and chemical properties of peptide backbones, including the conformation dynamics, proteolytic stability, hydrogen-bonding capabilities, and possibly reactivity of a protein when installed. Recently, there has been significant progress in elucidating enzymatic post-translational thioamide installation, with most work leveraging the archaeal MCR-modifying enzymes. This chapter describes the protocols used for the in vitro enzymatic thioamidation of MCR-derived peptides, including polypeptide overexpression, purification, reaction reconstitution, and mass spectrometry-based product analysis. In addition, we highlight the protocols used for the biochemical, kinetics, and binding studies using recombinant enzymes obtained heterologously from E. coli. We anticipate that these methods will serve to guide future studies on peptide post-translational thioamidation, as well as other peptide backbone modifications using a parallel workflow.
Subject(s)
Escherichia coli , Thioamides , Archaea , Escherichia coli/genetics , Kinetics , PeptidesABSTRACT
Six anti-AIDS drugs were assessed for in vivo genotoxicity and cytotoxicity at human clinical doses with the mouse bone marrow micronucleus assay. These included four dideoxynucleosides (azidothymidine, dideoxycytidine, dideoxyadenosine, and dideoxyinosine), an anthracycline antibiotic (doxorubicin), and a chelating agent (D-penicillamine). Cytological analysis of the mouse bone marrow cells revealed: (i) The dideoxynucleosides and D-penicillamine failed to induce significant number of micronuclei, and except for one of the five doses of dideoxyinosine, none of the dideoxynucleosides were cytotoxic at the doses tested. (ii) Doxorubicin induced micronuclei in a dose-dependent manner which was statistically significant at 4-times the clinical dose but was not cytotoxic at any of the doses tested.
Subject(s)
Acquired Immunodeficiency Syndrome/drug therapy , Antiviral Agents/toxicity , Dideoxynucleosides/toxicity , Mutagens/toxicity , Animals , Antiviral Agents/therapeutic use , Bone Marrow/drug effects , Bone Marrow Cells , Dideoxynucleosides/therapeutic use , Doxorubicin/toxicity , Female , Male , Mice , Micronucleus Tests , Penicillamine/toxicityABSTRACT
Selegiline hydrochloride (1-deprenyl) and bromocriptine mesylate (2-bromo-alpha-ergocryptine) are two drugs that have shown considerable promise in the treatment of Parkinson's disease. The in vivo mouse bone marrow micronucleus assay was used to examine their clastogenic and cytotoxic potential in human clinical dose range. Our results indicate that both drugs failed to induce significant number of micronuclei and were not cytotoxic at any of the doses tested, in vivo in mouse bone marrow cells, at doses as high as 16-times the clinical dose used in humans.
