 Tony from Roma 3, thank you. Now, can you hear me? No, I can't hear you. Okay? No, not yet. It's me, can you hear me? Better? Okay. So, good morning. First of all, I would like to thank the organizers and in particular Vittorio for inviting me to give this talk at this nice meeting. And as you know, also from previous talks, the number of pathogenic bacteria with antibiotic resistance is increasing over years. But in the meanwhile, the research about new antibiotics is slowing down and there are no new antibiotics which have entered into clinical trials in the last years. So, this is a very big problem and we need to find new strategies to overcome this problem. Antivirulent drugs can be used alone or in combination with antibiotics and they target differently from antibiotics are not essential functions. So, antivirulent drugs do not kill bacteria. They inhibit the ability of bacteria to cause the infection. So, they prevent or inhibit the establishment of an infection and in this way, they reduce the capability of pathogens to cause damage to the host. Since they do not target essential functions, they are very specific for pathogens. So, they are supposed to... They should have less adverse effects on the host microbiota and since they don't kill bacteria, they should impose a weaker selective pressure for drug resistance. And this is not trivial because actually you can find mutants resistant to antivirulent drugs and the mechanism would be the same as for antibiotic resistance such as modification of the target, modification of the molecule of the drug, efflux pumps, surface, bacterial surface modifications and so on. Yeah, I'm going to explain this in the next slide. So, in this cartoon, the emergence of resistance to antibiotics is compared to the emergence of resistance to antivirulent drugs. And okay, in the case of antibiotics, if you administer an antibiotic to an infecting population and the mutant resistant is present, all the susceptible cells will die. The resistant mutant will... Sorry, the resistant mutant will survive and will generate a new population that will be totally resistant to the antibiotic. In the case of antivirulent drugs, we should take in mind that the majority of virulent factors are secreted virulent factors. They are proteases, hemolysines, toxins, all public goods, all secreted factors that are shared by the population. And all the population takes advantage of these public goods. So, and these public goods are needed to disrupt the host tissues and get nutrition for the bacterial growth of the entire population. And they are also important for resistance to the immune system. If you give an antivirulent drug, to an infecting population, what will happen is that the susceptible cells will not die, they will just stop to produce the virulent factors. And if a mutant resistant to antivirulent drug is present, this will be the only cell continuing to produce virulent factors. But this amount will be not sufficient to sustain the growth of the entire population. So, what will happen is that the entire population will be attenuated for the production of virulent factors and the immune system will have better chance to clear along the infection. And this is not just a theory. This concept has been proved in several papers. And one of these, my favorite one is from Martin Schuster, who is over there and Mel Bay, which proved this concept. He's interested in the discovery of new antivirulent drugs. And we use as a model organism, pseudomonas aeruginosa. We have learned about these microorganisms in the previous lectures. And we know that it is an important nosocomial pathogen, is one of the most-breed nosocomial pathogens in developed countries. It causes infections in intensive care unit infections, catheter-associated infections, wound infections. And it is very important in cystic fibrosis because more or less 80% of the patients affected by this genetic disease die as a consequence of the chronic lung infection caused by pseudomonas aeruginosa in their lungs. And the severity of the infection is aggravated by the high resistance of these microorganisms to antibiotics. It is resistant, intrinsically resistant to the majority of the antibiotics used in clinics. And it also acquires very easily new antibiotic resistance by a horizontal gene transfer. And so, in pseudomonas aeruginosa, virulence is multifactorial. This bug produces an array of different virulence factors, most of which, the majority are secreted virulence factors, so they are public goods. And these are, for instance, Renu Lipids, hydrogen cyanide, piosynine, that is a secondary metabolite with cytotoxic effects, protease, cydrophores, and it also produces biofilm. And all these virulence factors, the production of all these virulence factors is positively controlled by quorum sensing. And we already know, thanks to Pete and other speakers, what quorum sensing is. If we inactivate quorum sensing, all the expression of all these virulence factors will be shut down. And indeed, there are many papers published every year about quorum sensing inhibition. On aerugin, in the last four years, 100 papers per year have been published, and I use this query, quorum sensing inhibitor, but half of these papers, more or less, are about the inhibition of pseudomonas aeruginosa quorum sensing. However, very few papers show activity in any more models, and as far as I know, there are no clinical trials so far. And one reason could be that when you find a new active molecule and you want to bring this molecule to the clinics, you need a lot of research, a lot of money, and a lot of time to develop a drug. It takes from 10 to 12 years of research, starting from the identification of the active principle, then you have to characterize the mechanism of action, make the experiments in vitro and in animal models, make formulations, make all the toxicity and pharmacokinetic studies, and this takes a lot of time and money also. So a lot of people working in the drug discovery field is using this drug repurposing approach to, as a shortcut, to deliver, fastly, a new drug, I would say an old drug, to a new use, to a new application. And this can be done by different approaches, searching for secondary activity in drugs already approved for use in humans for the treatments of whatever illnesses. And it can be done by different approaches if new insights are discovered about the molecular mechanisms underlying pathology, then rationally you can decide to locate an old drug to a new application. Other drugs are relocated repurposed by serendipity approaches. For the most famous example is a sildenium field. This molecule was developed in the 80s as for the treatment of heart disease, angina pectoris, and they went to clinical trials, and but the people involved in the trials did not find any effect on angina pectoris on the reason why the drug was developed. However, they experienced an interesting side effect. And this side effect was so interesting that at the end, the drug was used for its secondary effect. And okay, you can imagine what the effect was. The approach that we chose was the screening-based repurposing approach. In this approach, you can make a screening of a commercial library of FDA-approved drugs already used for the treatment of whatever illnesses and you can find for a secondary activity of interest. In this case, quantum sensing inhibition. And the drug can be the heat compound could be either directly tested in clinical trials if you are lucky. Otherwise, you can start an optimization program to develop the drug for a new application. And okay, so we used this approach to search for quantum sensing inhibitors using commercial libraries of FDA-approved compounds and on-pure-pure-built screening systems. So this is an important point in the searching for anti-virulent drugs because in the case of, if you want to search for new antibiotics, it's very simple because you just go for growth inhibition so you just need a spectrophotometer. But if you go for a virulent inhibitor, you need a special screening system, a target-oriented screening system. So first of all, the target. Pseudomonasaurogenosis has four different quantum sensing systems. Each one relying on a different signal molecule, two homosexual lactons. We have heard about it yesterday by Pete Greenberg, Trioxocit-12-Homocerylacton and C4-Homocerylacton. I will call them Trioxocit-12 and C4 for brevity. And Alkyl-Kinonon, PQS. And this is the most recently discovered, is a Hydrocyphenolthiazole. And more or less these quantum sensing systems, they work all in the same way. So there is an enzyme or a group of enzymes producing a signal that is secreted and when the signal is produced in a way that is proportional to the density of the bacterial population. So when the signal reaches a threshold concentration corresponding to a specific cell density, these will bind to an intracellular receptor and the complex between the signal and the receptor will activate a number of genes. And in pseudomonasero genomes, hundreds of genes are upregulated by the quantum sensing systems. Each of these quantum sensing systems regulates many, many genes including virulence genes. And all these systems are also organized in a hierarchical fashion so that the last system, relying on trioxy-C12, a signal molecule, positively controls the expression of the other quantum sensing systems. And this is the reason why we at first focused on the last system as our main target for the identification of anti-virulence drugs by drug repurposing approach. Then we had to construct a specific system for to make the screening. And in the screening system, one problem is sometimes that if you use strains different from the target organism, the drug will penetrate into the strain but the pseudomonasero genosa is quite impermeable to many substances so we decided to make the screening system directly in pseudomonasero genosa. And we constructed a mutant unable to produce the signal molecule, trioxy-C12, and carrying a transcriptional fusion between a promoter dependent on a laser that is the receptor of the signal and the lax genes for the bioluminescence production. So this strain can make a very low level, basal level of light in the absence of the signal but if you provide the signal by culturing this cell together with a wall type, the wall type will produce the signal which will activate the receptor and the activated receptor will trigger light emission. And you can make this culture in very tiny micro walls, in micro walls, and in each micro wall you can test a different molecule and any molecule having an inhibitory effect on any step of the consensing process will give a reduction of light emission with respect to the untreated control. That you can, in this way we can hit even the synthesis. So we can hit any step of the process from the synthesis to the reception. So expression or activity of the synthase, expression or activity of the receptor or even the import-export mechanisms of the signal molecules. Whatever the mechanism of action if something is affecting quantum sensing, you can find it. So in this way we screen a library of about 1,000 compounds already approved for use in humans. So they are, what I forget to tell you before is the advantage of course is that these molecules are already, have been already optimized for use, for be used as good drugs. So they have good pharmacological properties, they are supposed to be not toxic for humans. So we screened this library and the criteria were to take, to select the compounds with more than 50% inhibition of light emission and no effect on growth or at least less than 20% growth reduction because this is a very key point in anti-virulence drugs. They shouldn't affect growth. So we selected among, sorry? If these are already known to be... Yeah, indeed several hits were antibiotics so we didn't select them. Even if these are antibiotics towards, to which Pseudomonas aeruginosa is resistant, okay? But for this reason at first we focused on neclosamide that is an anti-homintic. It is used against the warm infections in the lung, in the, sorry, in the gut, okay? And neclosamide, so we tested the neclosamide in the wild type strain in our laboratory model strain that is called PA-14 and it has no effect on growth at different very high concentrations. It inhibits the production of a trioxy-C12 signal and it also inhibits the production of the C4 signal. And this not only because the last system, the trioxy-C12 dependence system positively controls the C4 dependence system but even in the last minus mutant, the drug is effective against the C4 system. So somehow it can affect the acylohomocerin, the both, the acylohomocerin lactons dependent coronal sensing systems. And we made an eye coronary analysis and neclosamide is able to decrease the expression of more or less 80 genes and the vast majority of these genes are also genes known to be activated by coronal sensing. Therefore, there is a good overlap between the two regulants and this indicates that neclosamide has a high degree of specificity towards coronal sensing. Indeed, it is able to inhibit the expression of the major coronal virulence factors, secret virulence factors produced by pseudomonas aeruginones such as pyocyanin, rhinolipids, elastase. It inhibits swarming motility, surface motility and even if at a higher concentration it has also an effect on bio-information. So this is very nice. We also tested the effect of neclosamide in this insect model of infection. It is able to protect the larvae of Galleria Merlonella from the pseudomonas aeruginones infection and but we were unable to test the molecule in mice and this is because neclosamide has been developed to treat infections in the gut. It is usually given by oral administration. It is not absorbed. It doesn't go in the bloodstream that is very good to treat an intestinal infection by words and if you try to give it by bloodstream, by the bloodstream way, it is fastly cleared by spleen and kidney action. So we decided to go for a new formulation of the neclosamide because all these characteristics of neclosamide, so fast clearance in the bloodstream, in the body and local action are very good properties for the treatment, could be very good properties for the treatment of lung infections by inhalation therapy. So we started a collaboration with pharmacologists and the neclosamide is a very low solubility, hypermeability and they developed this nice, different formulations, these nice nanoparticles that are complex with polysorbate and mannitol and this formulation can be given by inhalatory devices both as dry powder that is the top, the most advanced therapy in cystic fibrosis. So you give directly the dry powder within the lung or it can be resuspending in saline and they use it with a normal inhalatory device. And they made a lot of pharmacokinetic studies. The drug can go, they used this system that mimics the inhalatory device. They were able to show that the drug goes very heavily, goes very deeply inside the lung quite heavily and it is not toxic for rats, inhalatory rats and it has a very low level of toxicity at least at the active concentration in cystic fibrosis lung cells. So this, and so we can, we will, okay, well now we have a system to study the effect of neclosamide in vivo, in urine models. Actually the work is in progress. We tried with mice because we already had the collaboration with a group able to make infections to mimic chronic line infections in the lung of mice, but we had problems in the administration of the drug probably because they are very tiny with respect to rats and therefore probably we will continue by using rats in the next experiments. So speaking about cystic fibrosis, the target of neclosamide, well we know that it is the signaling process because if you give the, in the mutant, in the biosensory, if you give the trioxy-C12, you still see the reductions of the effect. So it is not, for sure it is not the synthesis. It should be the activity or the expression of the laser receptor. Preliminary experiment told us that probably it is affected the transcription of the receptor, of the laser receptor, but we are still working on the mechanism of action, okay. So going back to cystic fibrosis, there is an issue about the use of anti-quarantine drugs in cystic fibrosis. Pete Grimmer mentioned it yesterday. And this is because, and also today, we had a dance talk by Roy, speaking about the evolution of pseudomonas, purcaldaria in the cystic fibrosis lung. So in particular, what happens is that cystic fibrosis patients start to acquire intermittent infections caused by different strains of pseudomonas aeruginosa since they are babies. And they are treated with very strong, aggressive antibiotic therapy, and for some years it is possible to control these intermittent infections. And you can see that many different clonal variants can infect the lung and then be cleared by the aggressive antibiotic therapy. Until at a certain point, for reasons that are still poorly understood, a single clone, and the colors represents different strains, different clonal variants. At a certain point, a single clone establish a chronic infection. From that point, that patient will carry on the infection for the rest of his or her life, and this will lead to continuous inflammation and to decline in the time of the lung function. So the clonal strain can receive, this chronic infection can be kept under control for several years, even for decades, by continuous antibiotic treatments. And in the meanwhile, the lung changes day after day because the inflammation increases. So the environment is very variable. And during this time, a lot of mutations of cures, there is an evolution phenomenon, and hypermutant strains emerge, and you can find different kinds of mutations that are supposed to be adaptive mutations, adaptive mutations to the lung of the cystic fibrosis patient, among these resistance to antibiotics, of course, but even many other functions are mutated in these chronic strains. So, and if you play to the mucous of the cystic fibrosis patients, so you will find different colony morphology, so like small hugus colony variants, hypermucoid strains producing algeidate. In the years ago, this was the hallmark for the establishment of the chronic infection. Now there are other markers. Mutants producing autolysis plaques okay, I don't know the pronunciation in colony biofilm, and it is not uncommon to isolate mutants in the last chronic sensing system, in the system depending upon trioxycyl-12 as a signal molecule. So we question whether... Sorry. Okay, so can you hear me? So we question whether chronic sensing, and in particular the last chronic sensing system could be a good target for anti-virginance therapy in cystic fibrosis. And we collected a number of strains, we constructed a library of cystic fibrosis isolates, 100 strains divided in different categories. So more or less half of the strains are first isolates, so they are not chronic strains, they are not isolated from patients with chronic infection, they could be intermittent infections. Then 25 strains isolated from patients isolated from patients from one to three years, 25 strains from patients isolated from three to seven years, and a small number of strains just then for isolated from patients with more than 15 years. And we did this on purpose because we already knew from the literature that in patients with more than 10, 15 years of infection, the number of lasar mutants is quite high, the frequency of the last mutant is quite high. While we were interested in the first, in the intermittent strains and in the first phase of the chronic infections, we measured the levels of trioxy-C12 produced by these strains, and we didn't find any significant difference in trioxy-C12 levels among these three categories. This, I mean, is not indicative of the last category because we had just few strains. Okay. And we then want to see if these strains were, so these strains, 79 strains are trioxy-C12 producers. So this means that in principle, these strains should be susceptible to a chronic sensing inhibitor. Very nice, very good. So we went to test Nikolausonite. And the results were quite disappointing because we found that Nikolausonite was able, the effect of the susceptibility to Nikolausonite is very variable over our law and unrelated to the duration of the chronic infection. And these red dots or triangles are the laboratory strains. The laboratory strain that is susceptible to Nikolausonite, so you can, as you can see, the susceptibility to Nikolausonite is not very high in the cystic fibrosis clinical isolate, even if they are trioxy-C12 producers. So they are, in principle, susceptible, but in the real, in the reality, they are not. And if you go to see the virulence factors, the level of susceptibility of the strain is still lower, always with respect to our model strain. So very fastly I will go to, so we didn't let us be discouraged by these results. We focused on a different quantum sensing system as a target, the PQS system, because in cystic fibrosis it is not so common to find newtons unable to produce this signal molecule. So we used the same approach, we constructed a specific biosensor, we made exactly the same screening, but this time specific for kilonal productions. We identified, in this case, an antibiotic, but this antibiotic is not used, Pseudomonas aeruginosa is not sensitive at any concentration to this antibiotic. It is used by against gram-positive bacteria and it is promising because it is used by the suppository form to treat high-waste, to treat a high-waste infection. So it has a good kinetics toward the lung. And it has a good, it does not affect growth in any concentration, it affects the production of the signal, it affects virulence, secret virulence factors production and storm immortality, and it protects Galeria melonella larvae from the infection. This, in this case, we used a different model strain that is named PU1, the most famous. We are now characterizing the effect of clofoctol on the cystic fibrosis isolates. In this case, right now we have analyzed only 20 strains, 19 are PQS producers, and this is the model strain PU1. So as you can see, there is overall, the alkyl kilonome productions is fairly inhibited in a fair number of strains. So let's hope that this will work better with respect to necrosomite. And so I will conclude making a take-home message that is about anti-virulence drugs targeting Pseudomonas aeruginosa coron sensing. There are several advantages. No, there are targets are already known and well characterized in many cases. There are already published adequate screening systems available and in principle, there should be a low selective pressure for dimensions of resistance. The drawbacks are that at least in cystic fibrosis infection, there is a within us evolution of resistance strains in the ears long lasting infection, but perhaps even the very early isolates are already resistant to necrosomite. We don't know why we would like to know it. We will go to study this. And about drug repurposing, there are commercial drug libraries available so it is quite easy to purchase them and make the screening. The toxicity and pharmacokinetics studies are already done. So in principle, these molecules are not toxic, should be not very toxic and already with good pharmacological properties. And the drawbacks are that as in the example of necrosomite, if the administration wrote change, you need to do again all the toxicity and pharmacokinetics studies. The other problem could be the primary activity of the drug because we are using a drug, an old drug for the treatment of a new illness, but the primary activity should be taken into consideration. I wouldn't use an anti-cancer to treat a bacterial infection, of course. And there are challenges for intellectual properties, but if you are lucky, you can find a drug like necrosomite or croffotol, which are very old drugs and the patent is already expired. So it would be easier to go on with the studies and with the application on the drug. In conclusion, finally, I would like to thank all the people, all our collaborators. First of all, my colleague, Jordano Rampuni, who is over there, Paolo Vizca, Francesco Imperi, and the pharmacologists, Francesca Ungaro and Raffaella Solentino, Paul Williams and Miguel Camara, for which collaborate with us for the PQS and croffotol story. I forgot the hospital, Bambino Gesù and Ercilia Fiscarelli, who prepared the library of 65 brosis strains. And this is our granting foundation, the Italian 65 brosis research foundation. Thank you for your attention.