The bedrock of drug discovery and a key tool for understanding cellular function and drug mechanisms of action is the structure determination of chemical compounds, peptides, and proteins. The development of new structure characterization tools, particularly those that fill critical gaps in existing methods, presents important steps forward for structural biology and drug discovery. The emergence of microcrystal electron diffraction (MicroED) expands the application of cryo–electron microscopy to include samples ranging from small molecules and membrane proteins to even large protein complexes using crystals that are one-billionth the size of those required for X-ray crystallography. This review outlines the conception, achievements, and exciting future trajectories for MicroED, an important addition to the existing biophysical toolkit.
MurG (uridine diphosphate-N-acetylglucosamine/N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase) is an essential bacterial
glycosyltransferase that catalyzes the N-acetylglucosamine
(GlcNAc) transformation of lipid I to lipid II during peptidoglycan
biosynthesis. Park’s nucleotide has been a convenient biochemical
tool to study the function of MraY (phospho-MurNAc-(pentapeptide)
translocase) and MurG; however, no fluorescent probe has been developed
to differentiate individual processes in the biotransformation of
Park’s nucleotide to lipid II via lipid I. Herein, we report
a robust assay of MurG using either the membrane fraction of a M. smegmatis strain or a thermostable MraY and MurG
of Hydrogenivirga sp. as enzyme sources, along
with Park’s nucleotide or Park’s nucleotide-N
ε-C6-dansylthiourea and uridine
diphosphate (UDP)-GlcN-C6-FITC as acceptor and donor substrates.
Identification of both the MraY and MurG products can be performed
simultaneously by HPLC in dual UV mode. Conveniently, the generated
lipid II fluorescent analogue can also be quantitated via UV–Vis
spectrometry without the separation of the unreacted lipid I derivative.
The microplate-based assay reported here is amenable to high-throughput
MurG screening. A preliminary screening of a collection of small molecules
has demonstrated the robustness of the assays and resulted in rediscovery
of ristocetin A as a strong antimycobacterial MurG and MraY inhibitor.
Capuramycin displays narrow spectrum of antibacterial activity by targeting bacterial translocase I (MraY). In our program of development of new N-acetylglucosaminephosphotransferase1 (DPAGT1) inhibitor, we have identified that a capuramycin phenoxypiperidinylbenzylamide analogue (CPPB) inhibits DPAGT1 enzyme with an IC50 value of 200 nM. Despite a strong DPAGT1 inhibitory activity, CPPB does not show cytotoxicity against normal cells and a series of cancer cell lines. However, CPPB inhibits migrations of several solid cancers including pancreatic cancers that require high DPAGT1 expression in order for tumor progression. DPAGT1 inhibition by CPPB leads to a reduced expression level of Snail, but does not reduce E-cadherin expression level at the IC50 (DPAGT1) concentration. CPPB displays a strong synergistic effect with paclitaxel against growth-inhibitory action of a patient-derived pancreatic adenocarcinoma, PD002: paclitaxel (IC50 1.25 M) inhibits growth of PD002 at 0.0024-0.16 M in combination with 0.10-2.0 M of CPPB (IC50 35 M).
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