Elucidating biological and pathological functions of protein lysine acetyltransferases (KATs) greatly depends on the knowledge of the dynamic and spatial localization of their enzymatic targets in the cellular proteome. Here we report the design and application of chemical probes for facile labeling and detection of substrates of the three major human KAT enzymes. In this approach, we attempted to create engineered KATs in junction with synthetic Ac-CoA surrogates to effectively label KAT substrates even in the presence of competitive nascent cofactor acetyl-CoA. The functionalized and transferable acyl moiety of the Ac-CoA analogs further allowed the labeled substrates to be probed with alkynyl or azido-tagged fluorescent reporters by the copper-catalyzed azide–alkyne cycloaddition. The synthetic co-factors, in combination with either native or rationally engineered KAT enzymes, provide a versatile chemical biology strategy to label and profile cellular targets of KATs at the proteomic level.
Histone acetyltransferases are important enzymes that regulate various cellular functions, such as epigenetic control of DNA transcription. Development of HAT inhibitors with high selectivity and potency will provide powerful mechanistic tools for the elucidation of the biological functions of HATs and may also have pharmacological value for potential new therapies. In this work, analogs of the known HAT inhibitor anacardic acid were synthesized and evaluated for inhibition of HAT activity. Biochemical assays revealed novel anacardic acid analogs that inhibited the human recombinant enzyme Tip60 selectively compared to PCAF and p300. Enzyme kinetics studies demonstrated that inhibition of Tip60 by one such novel anacardic acid derive, 20, was essentially competitive with Ac-CoA and noncompetitive with the histone substrate. In addition, these HAT inhibitors effectively inhibited acetyltransferase activity of nuclear extracts on the histone H3 and H4 at micromolar concentrations.
A series of ferrous complexes (L)FeCl2 (3a−f) and cobaltous complexes (L)CoCl2 (4a−f)
were prepared by the reaction of 2-(carboxylato)-6-iminopyridines 2a−f (2-COOEt-6-(2,6-R2C6H3NCCH3)C5H3N: 2a, R = CH3; 2b, R = Et; 2c, R = i-Pr; 2d, R = F; 2e, R = Cl; 2f,
R = Br) with FeCl2 or CoCl2. The obtained complexes were characterized by elemental
analysis and IR spectroscopy, and the solid-state structures of 2b,c, 3b,c,e,f, and 4c,f were
determined by X-ray diffraction analysis. X-ray crystallographic analyses of complexes 3c,e,f
and 4c,f reveal a four-coordinated distorted-tetrahedral geometry, except for complex 3b,
in which the tridentate ligand is coordinated through a weak bonding between iron and the
carbonyl oxygen of the ester group. The complexes were studied for ethylene oligomerization
and polymerization in the presence of methylaluminoxane (MAO) under various reaction
conditions. It was found that the ferrous complexes exhibit higher activities for ethylene
polymerization than for oligomerization, while the cobaltous complexes show higher activities
for ethylene oligomerization. In addition, the ferrous catalytic systems predominantly produce
linear oligomers and polyethylene with unsaturated end groups.
Purpose
To develop a family of 700 nm zwitterionic pentamethine indocyanine near-infrared fluorophores that would permit dual-channel image-guided surgery.
Procedures
Three complementary synthetic schemes were used to produce novel zwitterionic chemical structures. Physicochemical, optical, biodistribution, and clearance properties were compared to Cy5.5, a conventional pentamethine indocyanine now used for biomedical imaging.
Results
ZW700-1a, ZW700-1b, and ZW700-1c were synthesized, purified, and analyzed extensively in vitro and in vivo. All molecules had extinction coefficients ≥ 199,000 M−1cm−1, emission ≥ 660 nm, and stability ≥ 99% after 24 h in warm serum. In mice, rats, and pigs, ≥ 80% of the injected dose was completely eliminated from the body via renal clearance within 4 h. Either alone or conjugated to a tumor targeting ligand, ZW700-1a permitted dual-channel, high SBR, and simultaneous imaging with 800 nm NIR fluorophores using the FLARE® imaging system.
Conclusions
Novel 700 nm zwitterionic NIR fluorophores enable dual-NIR image-guided surgery.
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