Two Distinct Mechanisms of Inhibition of LpxA Acyltransferase Essential for Lipopolysaccharide Biosynthesis

The
lipopolysaccharide biosynthesis pathway is considered an attractive
drug target against the rising threat of multi-drug-resistant Gram-negative
bacteria. Here, we report two novel small-molecule inhibitors (compounds 1 and 2) of the acyltransferase LpxA, the first
enzyme in the lipopolysaccharide biosynthesis pathway. We show genetically
that the antibacterial activities of the compounds against efflux-deficient Escherichia coli are mediated by LpxA inhibition. Consistently,
the compounds inhibited the LpxA enzymatic reaction in vitro. Intriguingly,
using biochemical, biophysical, and structural characterization, we
reveal two distinct mechanisms of LpxA inhibition; compound 1 is a substrate-competitive inhibitor targeting apo LpxA,
and compound 2 is an uncompetitive inhibitor targeting
the LpxA/product complex. Compound 2 exhibited more favorable
biological and physicochemical properties than compound 1 and was optimized using structural information to achieve improved
antibacterial activity against wild-type E. coli.
These results show that LpxA is a promising antibacterial target and
imply the advantages of targeting enzyme/product complexes in drug
discovery.

脂多糖生物合成通路（lipopolysaccharide biosynthesis pathway）被视为应对日益严峻的多重耐药革兰氏阴性菌威胁的极具吸引力的药物靶点。本研究报道了两类新型小分子抑制剂（化合物1与化合物2），可靶向脂多糖生物合成通路中的首个酶——酰基转移酶LpxA。我们通过遗传学实验证实，此类化合物针对外排缺陷型大肠杆菌（Escherichia coli）的抗菌活性，是通过抑制LpxA实现的。与之相符的是，体外实验表明此类化合物可抑制LpxA的酶促反应。有趣的是，通过生化、生物物理及结构表征手段，我们揭示了两种截然不同的LpxA抑制机制：化合物1为靶向空载LpxA（apo LpxA）的底物竞争性抑制剂，而化合物2则为靶向LpxA-产物复合物的反竞争性抑制剂。相较于化合物1，化合物2展现出更优异的生物学与理化性质，且我们通过结构信息对其进行了优化，使其针对野生型大肠杆菌的抗菌活性得到提升。上述结果证实LpxA是极具潜力的抗菌药物靶点，同时也提示在药物研发中靶向酶-产物复合物的优势。