High-throughput Screening Of Pentamidine Sensitized Small Molecules As Novel Antibacterials

The discovery of penicillin in the late 1920s opened up a new era of antibiotic chemotherapy,and many infectious diseases have been effectively controlled since then.The development and utilization of various antibiotics have developed rapidly.However,with the continuous development of bacterial resistance,the risk of death caused by drug-resistant infections is gradually increasing.Only a small number of new antibiotics are approved in the pass 20 years,and patients only have very limited treatment options with multi-drug resistant bacterial infection.Therefore,it is urgent to develop new antibacterial drugs or new treatment plan.Combination therapy of individual antibiotics increases the overall efficacy of antibiotic chemotherapy with minimized host toxicity,as well as suppresses the development of resistance.Bacteria are divided into two classes based on difference of their cell wall structure:gram-positive and gram-negative which has an additional outer membrane.The outer membrane of gram-negative bacteria is a permeability barrier to many small molecules,which results in diffential susceptibility of the two types of bacteria to a number of common antibiotics.Therefore,it is a valuable strategy to develop adjuvant that can sensitize gram-negative bacteira to gram-positive bacterial only antibiotics.Pentamidine,an aromatic diamidine drug,is not a traditional antibiotic.It has activity against a number of microorganisms including protozoa（Trypanosoma brucei,Leishmania spp.,and Babesia spp.）and fungi（Pneumocystis jirovecii）.Previously it is reported that,pentamidine displayed synergy with antibiotics typically restricted to gram-positive bacteria,offering effective drug combinations with activity against a wide range of gram-negative pathogens.In this study,we carried out high-thoughtput screening to identify additional small molecules that can be sensitized by pentamindine to inhibition the growth of E.coli.We have 1,584 small molecule compounds from the National Cancer Institute（NCI）Diversity Set VI compound library.The specific results are as follows:（1）In the screening of the NCI compound library,we obtained 6 small molecules that can be sensitized by pentamidine to produce antibacterial activity.These 6compounds are:NSC59984,NSC68982,NSC311727,NSC204262,NSC622608,NSC522131,NSC522131 and NSC204262 have better antibacterial effects.When there is 32μg/mL of pentamidine,the MIC values of NSC522131 and NSC204262 are reduced by>32 times.（2）Further research on the structural analogs of NSC522131,triclocarban and triclosan showed that,the synegistic effect of triclocarban is better than triclosan.Pentamidine combined with triclocarban and NSC522131 has various degrees of synergy for Escherichia coli,Klebsiella pneumoniae,and Acinetobacter baumannii,but for Pseudomonas aeruginosa.This bacterium is resistant to such synergy combination,which may be caused by another resistance mechanism other than lipopolysaccharide.（3）Toxicity is an important consideration in drug development.Here we tested the toxicity of pentamidine to Caenorhabditis elegans.LD_{5 0} was fitted by using Origin to give a value of 508.6μg/m L.When the concentration of pentamidine is lower than512μg/mL,the toxicity is low.The survival rate is about 90%after 12 hours of drug treatment.,and after 24 hours,and the survival rate is about 80%.When the dose of pentamidine is above 512μg/mL,it is much more toxic to C.elegans.The survival rate is about 60%after 12 hours of treatment,and the survival rate is only 7%after 24hours.