| Tetracycline antibiotics are widely used in veterinary clinic and agricultural production because of their broad antibacterial spectrum,low price,fewer side effects and promoting animal growth.In recent years,due to the abuse of tetracyclines,the drug resistance rate has increased year by year,especially to Escherichia coli,and the drug resistance rate has reached 80%.More seriously,the research and development of new antibacterial drugs for gram-negative bacteria such as Escherichia coli is lagging behind,and some tetracycline antibiotics may be on the verge of elimination in the long run.Therefore,it is imperative to develop new treatment strategies.Antibiotic adjuvant is one of the feasible strategies to solve the current tetracycline antibiotic resistance crisis.The new use of old drugs,that is,to discover new tetracycline antibiotic adjuvants from drugs that have been approved for marketing,can save a lot of economic costs and time costs of new drug research and development.Guanidine compounds have a unique structure,can be protonated and positively charged in physiological environment,and can form hydrogen bonds or electrostatic reactions with many bacterial targets,so they have the potential to become antibiotic adjuvants.In this study,new tetracycline antibiotic adjuvants were discovered from guanidine-containing drugs that have been approved for marketing,and their mechanism of action,structureactivity relationship,safety and effectiveness were preliminarily explored.The first is the discovery of guanethidine,a new adjuvant of tetracycline antibiotics.In this study,the second part of Chinese Pharmacopoeia(2010 and 2015editions)was used as the library.According to the chemical structure,from 2,603 drugs,29 drugs containing guanidine or substituted guanidine were retrieved.Screening was carried out by measuring the graded inhibition concentration index of drug combination application.It was found that guanethidine,an antihypertensive drug,has the potential to act as a synergist for tetracycline antibiotics.It can reverse the high resistance of multidrug-resistant bacteria to tetracycline antibiotics,reduce the minimum inhibitory concentration of multidrug-resistant bacteria E.coil C3 by up to 128 times,and restore its sensitivity.Guanethidine can also delay the development of bacterial drug resistance,reduce the frequency of bacterial drug resistance,and help tetracycline completely kill bacteria within 8 to 12 hours.Among the bacteria collected in this study,guanethidine can increase the antibacterial activity of tetracycline against Escherichia coli carrying tetracycline efflux pump resistance gene tet A.In addition,guanethidine significantly increased the antibacterial activity of rifampicin against E.coil C3.The study on the synergistic mechanism of guanethidine showed that guanethidine could significantly affect the outer membrane permeability of multidrug-resistant strain E.coil C3,also destroy the proton dynamics of its energy supply system,and affect the intracellular ATP content and ROS level related to proton dynamics.Guanethidine had no significant effect on nonspecific efflux pump,However,it can affect the activity of tetracycline-specific efflux pump Tet A,and destroy its conformation by destroying the proton power of its energy supply system or combining with the acidic amino acid residues in the active pocket of efflux pump protein Tet A by hydrogen bonding,thus increasing the accumulation of tetracycline antibiotics in bacterial cells and increasing its antibacterial activity.In addition,it has not been found that guanethidine affects protein synthesis and genetic material.The study on the structure-activity relationship of guanidine showed that guanidine was the necessary group for the synergistic action of guanidine,and heptamethyleneimine was the synergistic group.Among them,acidic groups are completely unfavorable to the synergistic effect of guanidine compounds and have no synergistic activity.Basic groups,all synergistic,but decreased in activity.In addition,guanethidine and tetracycline do not work as a whole,but perform their functions separately.We also found that extracellular p H changes and magnesium ions can affect the synergistic effect of guanethidine and tetracycline.The study of safety and effectiveness showed that the combination of guanethidine and tetracycline did not cause serious hemolysis and cytotoxicity in vitro,nor did it affect the blood routine,blood biochemical indexes,organ coefficient and other indexes of healthy mice,and the safety of the combination of guanethidine and tetracycline was within the controllable range.Moreover,guanethidine combined with tetracycline can effectively reduce the intracellular E.coil C3 load of mammalian cells,significantly improve the survival rate of animals,and reduce the bacterial load in organs,which has certain therapeutic potential.To sum up,this study discovered the antihypertensive drug guanethidine,the synergistic mechanism and structure-activity relationship of guanethidine were preliminarily explored,and its safety and effectiveness in vivo and in vitro were evaluated,which proved that guanethidine has the potential to become a new adjuvant of tetracycline antibiotics. |
