Finding the Achilles Heel in Gram Negative Bacteria. Role of Medicinal Chemistry

Transcription

1 Finding the Achilles Heel in Gram Negative Bacteria. Role of Medicinal Chemistry Gabriele Costantino

2 Bacterial Resistance: A Global Threat 2

3 Bacterial Resistance: A Global Threat

4 Resistance: An Inextricable Process The present strategy of discovering new antibiotics and waiting for new resistance to evolve is untenable in the long term. Vertical Transmission Horizontal Transmission

5 Vertical Transmission Resistance: An Inextricable Process

6 Horizontal Transmission Resistance: An Inextricable Process

7 Resistance: An Inextricable Process Mechanisms of Resistance Enzymatic Inactivation Decreased Permeability Efflux Alteration of target Overproduction of target(s)

8 Resistance: An Inextricable Process Mechanisms of Resistance

9 Resistance: An Inextricable Process Mechanisms of Resistance Directing new and more sophisticated drugs against known bacterial targets is more and more perceived as a hopeless race Alternative mechanisms and/or targets and/or host-based targets must be identified

10 Resistance: An Inextricable Process Today s outline : Social evolution in micro-organisms and a Trojan horse approach to medical intervention strategies Exploit Plasmids in Antibacterial Therapy Siderophores Interfering with protons and ph.

11 Social evolution in micro-organisms and a Trojan horse approach to medical intervention strategies Bacteria are social organisms. They show social behaviours and they cooperate

12 Social evolution in micro-organisms and a Trojan horse approach to medical intervention strategies The most common way for bacteria to cooperate with one another is the release of exoproducts which facilitate bacterial growth o Siderophores o Beta-lactamases o Proteases o. A second layer of complexity is that the release of exoproducts is regulated in a cell-density dependent manner, via diffusible signal molecules by a process termed Quorum Sensing (QS)

13 Bridging Bacterial Biochemistry with Sociobiology Taxation is an economically costly process for an individual Production of exoproducts is a metabolically costly process for an individual Taxes produce public goods which benefits the whole community Exoproducts are public (chemical) goods which benefits the whole (bacterial) community A tax-evader (cheater) does not pay the cost of taxation but benefits of the public goods A non-cooperative individual (cheater) does not pay the metabolic cost of exoproduct synthesis but benefits of the public goods In the absence of a punishment mecanism, the tax-evader is richer than the tax-paying In the absence of a punishment mecanisms, the cheater has a greater fitness than cooperants, and the cheater genes are spread through the population

14 Bridging Bacterial Biochemistry with Sociobiology Cheater Cooperant Exoproducts

15 Bridging Bacterial Biochemistry with Sociobiology A society where tax-evaders are predominant will decrease its social wealth, since public goods are consumed and not produced. vs Bacterial population containing cheaters that do not synthesize exoproducts will show lower virulence, will be less productive and relatively poor in transmission and in initiating new colonies. The ability of cheater strains to invade cooperative populations is a potential Achilles heel of bacterial virulence (and also resistance)

16 Bridging Bacterial Biochemistry with Sociobiology 1. Introduction of invasive cheats can lead to direct reduction in bacterial virulence and to direct decrease of bacterial population size. The infection will thus result more susceptible to other intervention strategies. 2. The cheaters can be used as Trojan horses to introduce alleles such as sensitivity to antibiotics into population that was previously antibiotics resistant 3. The cheaters can be used for the introduction of lethal toxin under the control of an inducible promoter which, when activated, will eliminate both the cooperators and the cheaters

17 Bridging Bacterial Biochemistry with Sociobiology Introduction of invasive cheats can lead to direct reduction in bacterial virulence and to direct decrease of bacterial population size

18 Bridging Bacterial Biochemistry with Sociobiology The cheaters can be used as Trojan horses to introduce alleles such as sensitivity to antibiotics into population that was previously antibiotics resistant Plasmids and Phage-like approach

19 Exposing Plasmids as the Achille s heel of Drug Resistant Bacteria Many multi-drug resistant bacterial pathogens harbor large plasmids that encode proteins conferring resistance to antibiotics. While the acquisition of these plasmids often enables bacteria to survive in the presence of antibiotics, it is possible that plasmids also represent a vulnerability that can be exploited in tailored antibacterial therapy. o Inhibition of plasmid conjugation o Inhibition of plasmid replication o Exploitation of plasmid-encoded toxinantitoxin systems.

20 Exposing Plasmids as the Achille s heel of Drug Resistant Bacteria

21 Exposing Plasmids as the Achille s heel of Drug Resistant Bacteria Plasmids are transferred between bacteria through conjugation. Inhibition of the relaxase enzyme (blue oval) has been proposed as an antibacterial strategy Plasmids replicate to maintain themselves in the bacterial population. Strategies have been devised and small molecules have been identified that inhibit plasmid replication, thus resulting in the elimination of the plasmid from the bacterial population and resensitizing the bacteria to antibiotics. One of the most common plasmid maintenance systems is the toxin-antitoxin (TA) post-segregational killing mechanism. In this mechanism, if a plasmid-free daughter cell arises, the labile antitoxin is degraded and the toxin induces cell death Williams, JJ. et al. Curr Opin Chem Biol August ; 12(4):

22 Exposing Plasmids as the Achille s heel of Drug Resistant Bacteria The relaxase domain (shown as the blue circle) domain of TrwC binds the orit (1) of the T-strand. Tyrosine-19 (shown as an orange circle) of relaxase catalyzes a cleavage at the nic site and forms a covalent phosphotyrosyl intermediate with the 5 terminus of orit(2). Rolling circle replication using the uncleaved strand as a template displaces the T-strand(3) and Y26 cleaves at the T-strand nic site (4). The T-strand is re-ligated and transferred to the recipient cell (5). Williams, JJ. et al. Curr Opin Chem Biol August ; 12(4):

23 Exposing Plasmids as the Achille s heel of Drug Resistant Bacteria

24 Exposing Plasmids as the Achille s heel of Drug Resistant Bacteria Antiplasmid antibiotics induce plasmid elimination from the bacterial cell population, thus resensitizing bacteria to antibiotics.

25 Exposing Plasmids as the Achille s heel of Drug Resistant Bacteria Chromosomally-encoded TA systems are possibly used by the cell to respond to stress. Stress causes toxin activation and reversible inhibition of cell growth; if the point of no return is reached, cell death occurs Plasmid-encoded TA systems function as a post-segregational killing system, resulting in the elimination of plasmidfree bacterial cells.

26 Exposing Plasmids as the Achille s heel of Drug Resistant Bacteria Toxin-Antitoxin (TA) has no human ortholog and the gene product could serve as an ideal target via two mechanisms:

27 Exposing Plasmids as the Achille s heel of Drug Resistant Bacteria Medicinal Chemistry Approaches toward TA activators Extracellular death factors Inhibitors of the PemI PemK/MoxX MoxT interactions Disruptors of e - z

28 Siderophores: Trojan Horses for Antibacterials Extracellular free iron is scarce in vertebrates, yet essential for microbial growth. A mechanism displayed by microbial pathogens to cope with iron scarcity involves the production of siderophores. These low molecular weight molecules bear an affinity to iron that exceeds by several orders of magnitude that of transferrin, the main protein in blood for iron transport. Under iron starvation, siderophores are excreted, scavenge ferric ions and the complex is shuttled inside the cell.

29 Siderophores: Trojan Horses for Antibacterials Under aerobic conditions and physiological ph, Fe 3+ exists as hydroxide polymers, with a solubility products of M The Fe 3+ free concentration in serum and tissue is about M, but the concentration required for bacterial growth is about 10-6 M As a matter of fact, in infected hosts Fe 3+ witholding is an important defense mechanism mainly constituted by iron-binding proteins transferrin and lactoferrin Bacteria have evolved high affinity uptake pathways formed by ferric ion specific carriers (siderophores) and their cognate membrane receptors. Siderophores are excreted and sequester Fe3+ with very high affinity. Then the complex binds the cognate receptor and internalized. Ferric ion is thus released by esteratic activity and reduced to ferreous ion.

30 Siderophores: Trojan Horses for Antibacterials

31 Siderophores: Trojan Horses for Antibacterials

32 Siderophores: Trojan Horses for Antibacterials The Trojan Horse Strategy

33 Siderophores: Trojan Horses for Antibacterials

34 Siderophores: Trojan Horses for Antibacterials

35 Siderophores: Trojan Horses for Antibacterials

36 Siderophores: Trojan Horses for Antibacterials

37 Siderophores: Trojan Horses for Antibacterials

38 Siderophores: Trojan Horses for Antibacterials Natural sideromycins of known structure. The iron-binding siderophore unit is black, the antibiotic warhead is shown in red, and if a linker is present it is shown in blue

39 Interfering with ph homeostasis. The energy source in aerobic bacteria is derived from synthesis of ATP driven by proton movement down an electrochemical gradient for protons ATP synthesis depends on the flux of protons across the F 1 -F 0 ATP synthase energized by the ph and membrane-potential gradient

40 Interfering with ph homeostasis. As the hydrogen ions accumulate on one side of a membrane, the concentration of hydrogen ions creates an electrochemical gradient or potential difference (voltage) across the membrane. The energized state of the membrane as a result of this charge separation is called proton motive force (pmf) The proton motive force (pmf) equation predicts that there will be a reciprocal relationship between the ph gradient and the potential difference across the relevant bacterial membrane pmf (mv) = Δµ H+ = RT/nF ln[h + out]/[h + in] +Δψ= 61 ΔpH + Δψ Exposure to an acid ph causes their periplasmic ph to fall, increasing the inward proton gradient and decreasing the membrane potential or even causing development of a positive potential that could hinder proton influx

41 Interfering with ph homeostasis. Gram negative neutralophiles are unable to buffer the periplasmic ph. When exposed to very acidic media, such as in the stomach, the proton influx is blocked and the metabolism is slown down. Thus, Nnutralophiles can survive in an acidic environment by maintaining a cytoplasmic ph=5, but they cannot grow or colonize the acidic tissue. Examples are Salmonella typhimurium, Vibrio cholera, and pathogenic strains of Escherichia coli and Yersinia enterocolitica. They can pass through the stomach but not colonize it. A particular case is given by Helicobacter pylori, which is also a neutralophile but can colonize and grow in the stomach, being the cause of severe conditions, such as ulcerative lesions and gastric cancer.

42 Interfering with ph homeostasis

43 Interfering with ph homeostasis Twelve isoforms so far isolated. Carbonic anydrase is the most efficient enzyme known, with a k cat = 10 6 s -1

44 Periplasmic Buffering in Helicobacter Pylori Periplasmic buffering by Helicobacter pylori. Urea crosses the outer membrane (OM) and then the inner membrane (IM) through the acid-activated urea channel (UreI) at an external ph of <6.0. Cytoplasmic urease forms 2NH 3 + H 2 CO 3, and the latter is converted to CO 2 by cytoplasmic β-carbonic anhydrase ( β- CA). These gases cross the IM, and the CO 2 is converted to HCO 3 by the membranebound α-carbonic anhydrase (α-ca), thereby maintaining periplasmic ph at ~6.1, which is the effective pka ( log10 Ka, in which Ka is the acid dissociation constant) of the CO 2 /HCO 3 couple. Exiting NH 3 neutralizes the H + that is produced by carbonic anhydrase, as well as the entering H +, and can also exit the OM to alkalize the medium. Krulwic et al. Nat. Rev. Microb. VOLUME 9 MAY 2011,330

45 Carbonic Anhydrase is a Druggable Target for Antibacterials Capasso C, Supuran CT. Curr Med Chem. 2015;22(18):