Activity-based protein profiling of host-virus interactions.

Virologists have benefited from large-scale profiling methods to discover new host-virus interactions and to learn about the mechanisms of pathogenesis. One such technique, referred to as activity-based protein profiling (ABPP), uses active site-directed probes to monitor the functional state of enzymes, taking into account post-translational interactions and modifications. ABPP gives insight into the catalytic activity of enzyme families that does not necessarily correlate with protein abundance. ABPP has been used to investigate several viruses and their interactions with their hosts. Differential enzymatic activity induced by viruses has been monitored using ABPP. In this review, we present recent advances and trends involving the use of ABPP methods in understanding host-virus interactions and in identifying novel targets for diagnostic and therapeutic applications.

Cellular consequences of copper complexes used to catalyze bioorthogonal click reactions.

Copper toxicity is a critical issue in the development of copper-based catalysts for copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions for applications in living systems. The effects and related toxicity of copper on mammalian cells are dependent on the ligand environment. Copper complexes can be highly toxic, can induce changes in cellular metabolism, and can be rapidly taken up by cells, all of which can affect their ability to function as catalysts for CuAAC in living systems. Herein, we have evaluated the effects of a number of copper complexes that are typically used to catalyze CuAAC reactions on four human cell lines by measuring mitochondrial activity based on the metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to study toxicity, inductively coupled plasma mass spectrometry to study cellular uptake, and coherent anti-Stokes Raman scattering (CARS) microscopy to study effects on lipid metabolism. We find that ligand environment around copper influences all three parameters. Interestingly, for the Cu(II)-bis-L-histidine complex (Cu(his)(2)), cellular uptake and metabolic changes are observed with no toxicity after 72 h at micromolar concentrations. Furthermore, we show that under conditions where other copper complexes kill human hepatoma cells, Cu(I)-L-histidine is an effective catalyst for CuAAC labeling of live cells following metabolic incorporation of an alkyne-labeled sugar (Ac(4)ManNAl) into glycosylated proteins expressed on the cell surface. This result suggests that Cu(his)(2) or derivatives thereof have potential for in vivo applications where toxicity as well as catalytic activity are critical factors for successful bioconjugation reactions.

Synthesis and bioorthogonal coupling chemistry of a novel cyclopentenone-containing unnatural tyrosine analogue.

Herein we report the synthesis of a novel amino acid with orthogonal functionality to the natural amino acid side chains. Tyrosine was O-alkylated with a cyclic 5-membered alpha,beta-unsaturated ketone ring (5). We have established that this amino acid analogue can undergo cycloaddition reactions in aqueous media with in situ generated nitrones. Nitrone formation occurred by micellar catalysis can undergo aqueous 1,3-dipolar cycloaddition reactions with the unnatural Tyr. We also performed a linear free energy analysis of the one pot bioconjugation reaction in water using cyclopentenone as a model for the Tyr analogue and seven different aryl nitrones. We found that the Hammett rho value was -0.94, suggesting that the reaction occurs in a concerted fashion with a slight positive charge buildup in the transition state. The Hammett rho value also suggests that the bioconjugation reaction is tolerant of different substituents and thus may be useful for introducing novel functionality into peptides and proteins containing the Tyr analogue 5. The aqueous 1,3-dipolar cycloaddition reactions, that use nitrones to trap the O-alkylated Tyr 5, establish a novel strategy for rapid, water compatible bioconjugation reactions.