Department of Molecular and Cell Biology, Brown University, Providence, Rhode Island, United States of America.
Department of Chemistry, Brown University, Providence, Rhode Island, United States of America.
PLoS Comput Biol. 2022 Jun 27;18(6):e1009944. doi: 10.1371/journal.pcbi.1009944. eCollection 2022 Jun.
The rate of modern drug discovery using experimental screening methods still lags behind the rate at which pathogens mutate, underscoring the need for fast and accurate predictive simulations of protein evolution. Multidrug-resistant bacteria evade our defenses by expressing a series of proteins, the most famous of which is the 29-kilodalton enzyme, TEM β-lactamase. Considering these challenges, we applied a covalent docking heuristic to measure the effects of all possible alanine 237 substitutions in TEM due to this codon's importance for catalysis and effects on the binding affinities of commercially-available β-lactam compounds. In addition to the usual mutations that reduce substrate binding due to steric hindrance, we identified two distinctive specificity-shifting TEM mutations, Ala237Arg and Ala237Lys, and their respective modes of action. Notably, we discovered and verified through minimum inhibitory concentration assays that, while these mutations and their bulkier side chains lead to steric clashes that curtail ampicillin binding, these same groups foster salt bridges with the negatively-charged side-chain of the cephalosporin cefixime, widely used in the clinic to treat multi-resistant bacterial infections. To measure the stability of these unexpected interactions, we used molecular dynamics simulations and found the binding modes to be stable despite the application of biasing forces. Finally, we found that both TEM mutants also bind strongly to other drugs containing negatively-charged R-groups, such as carumonam and ceftibuten. As with cefixime, this increased binding affinity stems from a salt bridge between the compounds' negative moieties and the positively-charged side chain of the arginine or lysine, suggesting a shared mechanism. In addition to reaffirming the power of using simulations as molecular microscopes, our results can guide the rational design of next-generation β-lactam antibiotics and bring the community closer to retaking the lead against the recurrent threat of multidrug-resistant pathogens.
使用实验筛选方法进行现代药物发现的速度仍然落后于病原体变异的速度，这突显出需要快速准确地预测蛋白质进化。多药耐药菌通过表达一系列蛋白质来逃避我们的防御，其中最著名的是 29 千道尔顿的酶 TEM β-内酰胺酶。考虑到这些挑战，我们应用了一种共价对接启发式方法来衡量由于该密码子对催化的重要性以及对市售β-内酰胺化合物结合亲和力的影响，TEM 中所有可能的丙氨酸 237 取代的影响。除了由于空间位阻而降低底物结合的常见突变外，我们还确定了两种独特的特异性转移 TEM 突变，Ala237Arg 和 Ala237Lys，及其各自的作用模式。值得注意的是，我们通过最低抑菌浓度测定发现并验证了，尽管这些突变及其更大的侧链导致阻碍氨苄西林结合的空间位阻，但这些相同的基团与头孢菌素头孢克肟的负电荷侧链形成盐桥，头孢克肟广泛用于临床治疗多耐药菌感染。为了测量这些意外相互作用的稳定性，我们使用分子动力学模拟发现结合模式很稳定，尽管施加了偏置力。最后，我们发现两种 TEM 突变体也与其他含有负 R 基团的药物（如卡芦莫南和头孢布烯）强烈结合。与头孢克肟一样，这种结合亲和力的增加源于化合物的负部分与精氨酸或赖氨酸的正侧链之间的盐桥，表明存在共同的机制。除了再次证实使用模拟作为分子显微镜的力量外，我们的结果还可以指导下一代β-内酰胺抗生素的合理设计，并使社区更接近重新夺回对抗多药耐药病原体反复威胁的主导地位。
