Outer membrane: a key obstacle for new antimicrobial agents

Abstract

INTRODUCTION: Antimicrobial resistance is one of the world’s major challenges in both microbiology and public health since infections caused by multidrug-resistant bacteria are reaching alarming levels. The world is currently facing a global antibiotics crisis and some new strategies need to be explored to tackle these resistant infections.

The outer membrane is a differential structure of Gram-negative bacteria that works as a highly effective selective permeability barrier. Thus, the permeation through the Gram- negative cell envelope is a challenge for drug compounds to reach their targets.

One path to open new antimicrobial perspectives is the chemical modification of old antimicrobial compounds that may result in optimized drugs with improved antimicrobial properties. In this context, the activity exploration of the derivatives of Microcin J25 and Trimethoprim was carried out.

HYPOTHESIS: The main hypotheses of this thesis are that chemical modifications of current antibiotics may significantly contribute to overcoming the problems arising from the increase and spread of antibiotic resistance in bacteria. Moreover, the combination of these modified compounds with old-rescued antibiotics may contribute to the solution bases of the problem caused by resistance.

Objectives: The main objective of this thesis is to determine the antimicrobial properties of chemically modified drug compounds. The secondary objectives of this thesis are divided in two parts:

Microcin J25: Study of the antimicrobial activity of the modified Microcin J25 as well as determination of its toxicity.

Trimethoprim: Study of the antimicrobial activity and cytotoxicity of the Trimethoprim derivatives, exploration of the effect of the new derivatives on the Dihydrofolate Reductase (DHFR) enzyme and start a computational approach to decipher the intimal mechanisms of action.

METHODOLOGY: The antimicrobial activity of the derived compounds was explored against planktonic bacteria (studying the Minimum Inhibitory Concentration and the FIC index) and against sessile bacteria (exploring the Minimum Biofilm Eradication Concentration and the Biofilm Prevention Concentration). The growth curves of several microorganisms in contact with these compounds and in combination with colistin were also studied. The cytotoxicity of all the compounds was tested. An enzymatic assay with

E. coli DHFR as well as a docking modelling were carried out to investigate the mechanism of action of TMP derivatives.

RESULTS: With respect to Microcin MccJ25, it has been detected that the chemical modification of the compound resulted in a new peptide without antimicrobial activity. It acted synergistically with sublethal concentrations of colistin.

When referring to Trimethoprim (TMP), some of the new derivatives showed antibacterial similar to that of TMP. Moreover, almost all the new TMP-like compounds acted synergistically with SMX. P. aeruginosa PAO1 was fully resistant to TMP and all its derivatives as well as to the combination of TMP-SMX. The combination of TMP, TMP- like molecules and SMX with colistin enhanced their antimicrobial efficacy against E. coli, P. aeruginosa and S. marcescens. Compounds 1a and 1b, like TMP, strongly inhibited the activity of the E. coli DHFR. Additionally, it was detected that the heterocyclic ring of the compound 1a fills the pocket occupied by the nicotinamide ring of NADPH.

CONCLUSIONS: The present PhD thesis has led to some relevant conclusions. With respect to Microcin MccJ25, the chemical modification of the compound avoided its detection by the membrane receptor FhuA. Moreover, it did not affect the interaction with the target and, as the polymyxin facilitated the microcin entrance across the membrane, once inside the cell, the new compound retained its ability to inhibit the growth of bacteria.

When referring to Trimethoprim (TMP), the derivatives showed interesting antimicrobial activities acting synergistically with SMX. The combination of TMP, TMP- like molecules and SMX with colistin enhances their antimicrobial efficacy by permeabilizing the cells. Compounds 1a and 1b, like TMP, strongly inhibited the activity of the E. coli DHFR and it was suggested that both molecules interact with the analogues during inhibition.

The search of new antimicrobial compounds is one of the main pathways to overtake bacterial resistance to antibiotics. All putative compounds should be tested in conditions in which outer membrane role as permeability barrier is inactivated. Their assay together with sublethal concentrations of colistin is proposed as one of the methods of election.

INTRODUCCIÓN: La resistencia a los antimicrobianos es uno de los principales desafíos del mundo tanto en microbiología como en salud pública, ya que las infecciones causadas por bacterias multirresistentes están alcanzando niveles alarmantes.

Una opción para abrir nuevas perspectivas antimicrobianas es la modificación química de compuestos antimicrobianos ya existentes que pueden resultar medicamentos optimizados con propiedades antimicrobianas mejoradas. En este contexto, en esta tesis se llevó a cabo la exploración de la actividad de los derivados de Microcina J25 y Trimethoprim.

HIPÓTESIS: Las principales hipótesis de esta tesis son que las modificaciones químicas de los antibióticos actuales y la combinación de estos compuestos modificados con antibióticos antiguos rescatados pueden contribuir significativamente a superar los problemas que surgen del aumento y la propagación de la resistencia a los antibióticos en las bacterias.

Objetivos: El principal objetivo de esta tesis es determinar las propiedades antimicrobianas de compuestos químicamente modificados.

Metodología: Se exploró la actividad antimicrobiana de los compuestos derivados frente bacterias planctónicas y frente bacterias sésiles. También se estudiaron las curvas de crecimiento de varios microorganismos en contacto con estos compuestos y en combinación con colistina. Se ensayó la citotoxicidad de todos los compuestos. Se llevó a cabo un ensayo enzimático con E. coli DHFR así como un modelo de docking.

RESULTADOS Y CONCLUSIONES: Con respecto a la Microcina MccJ25, se detectó que la modificación química del compuesto dio como resultado un nuevo péptido sin actividad antimicrobiana ya que se evitó su detección por el receptor de membrana FhuA. Esta actuó de forma sinérgica con concentraciones subletales de colistina debido a que la polimixina facilitó la entrada de la microcina a través de la membrana y, una vez dentro de la célula, el nuevo compuesto conservó su capacidad para inhibir el crecimiento de las bacterias.

En cuanto a Trimethoprim (TMP), algunos de los nuevos derivados mostraron un efecto antibacteriano similar al de TMP. Además, casi todos los nuevos compuestos actuaron sinérgicamente con SMX. P. aeruginosa PAO1 fue totalmente resistente a TMP y todos sus derivados, así como a la combinación de TMP-SMX. La combinación de TMP, análogos de TMP y SMX con colistina mejoró su eficacia antimicrobiana contra E. coli, P. aeruginosa y S. marcescens ya que ésta permeabilizó las células. Los compuestos 1a y 1b, como TMP, inhibieron la actividad de la DHFR de E. coli. Además, se observó que el anillo heterocíclico del compuesto 1a llena el bolsillo ocupado por el anillo de nicotinamida de NADPH y se sugirió que ambas moléculas interactúan con los análogos durante la inhibición.