Discovery of new diketopiperazines inhibiting Burkholderia cenocepacia quorum sensing in vitro and in vivo

Burkholderia cenocepacia, an opportunistic respiratory pathogen particularly relevant for cystic fibrosis patients, is difficult to eradicate due to its high level of resistance to most clinically relevant antimicrobials. Consequently, the discovery of new antimicrobials as well as molecules capable of inhibiting its virulence is mandatory. In this regard quorum sensing (QS) represents a good target for anti-virulence therapies, as it has been linked to biofilm formation and is important for the production of several virulence factors, including proteases and siderophores. Here, we report the discovery of new diketopiperazine inhibitors of the B. cenocepacia acyl homoserine lactone synthase CepI, and report their anti-virulence properties. Out of ten different compounds assayed against recombinant CepI, four were effective inhibitors, with IC50 values in the micromolar range. The best compounds interfered with protease and siderophore production, as well as with biofilm formation, and showed good in vivo activity in a Caenorhabditis elegans infection model. These molecules were also tested in human cells and showed very low toxicity. Therefore, they could be considered for in vivo combined treatments with established or novel antimicrobials, to improve the current therapeutic strategies against B. cenocepacia

Characterization of the dispirotripiperazine derivative PDSTP as antibiotic adjuvant and antivirulence compound against Pseudomonas aeruginosa

Pseudomonas aeruginosa is a major human pathogen, able to establish difficult-to-treat infections in immunocompromised and people with cystic fibrosis (CF). The high rate of antibiotic treatment failure is due to its notorious drug resistance, often mediated by the formation of persistent biofilms. Alternative strategies, capable of overcoming P. aeruginosa resistance, include antivirulence compounds which impair bacterial pathogenesis without exerting a strong selective pressure, and the use of antimicrobial adjuvants that can resensitize drug-resistant bacteria to specific antibiotics. In this work, the dispirotripiperazine derivative PDSTP, already studied as antiviral, was characterized for its activity against P. aeruginosa adhesion to epithelial cells, its antibiotic adjuvant ability and its biofilm inhibitory potential. PDSTP was effective in impairing the adhesion of P. aeruginosa to various immortalized cell lines. Moreover, the combination of clinically relevant antibiotics with the compound led to a remarkable enhancement of the antibiotic efficacy towards multidrug-resistant CF clinical strains. PDSTP-ceftazidime combination maintained its efficacy in vivo in a Galleria mellonella infection model. Finally, the compound showed a promising biofilm inhibitory activity at low concentrations when tested both in vitro and using an ex vivo pig lung model. Altogether, these results validate PDSTP as a promising compound, combining the ability to decrease P. aeruginosa virulence by impairing its adhesion and biofilm formation, with the capability to increase antibiotic efficacy against antibiotic resistant strains

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Burkholderia cenocepacia Infections in Cystic Fibrosis Patients: Drug Resistance and Therapeutic Approaches

Burkholderia cenocepacia is an opportunistic pathogen particularly dangerous for cystic fibrosis (CF) patients. It can cause a severe decline in CF lung function possibly developing into a life-threatening systemic infection known as cepacia syndrome. Antibiotic resistance and presence of numerous virulence determinants in the genome make B. cenocepacia extremely difficult to treat. Better understanding of its resistance profiles and mechanisms is crucial to improve management of these infections. Here, we present the clinical distribution of B. cenocepacia described in the last 6 years and methods for identification and classification of epidemic strains. We also detail new antibiotics, clinical trials, and alternative approaches reported in the literature in the last 5 years to tackle B. cenocepacia resistance issue. All together these findings point out the urgent need of new and alternative therapies to improve CF patients’ life expectancy

Differential roles of RND efflux pumps in antimicrobial drug resistance of sessile and planktonic Burkholderia cenocepacia cells

Burkholderia cenocepacia is notorious for causing respiratory tract infections in people with cystic fibrosis. Infections with this organism are particularly difficult to treat due to its high level of intrinsic resistance to most antibiotics. Multidrug resistance in B. cenocepacia can be ascribed to different mechanisms, including the activity of efflux pumps and biofilm formation. In the present study, the effects of deletion of the 16 operons encoding resistance-nodulation-cell division (RND)-type efflux pumps in B. cenocepacia strain J2315 were investigated by determining the MICs of various antibiotics and by investigating the antibiofilm effect of these antibiotics. Finally, the expression levels of selected RND genes in treated and untreated cultures were investigated using reverse transcriptase quantitative PCR (RT-qPCR). Our data indicate that the RND-3 and RND-4 efflux pumps are important for resistance to various antimicrobial drugs (including tobramycin and ciprofloxacin) in planktonic B. cenocepacia J2315 populations, while the RND-3, RND-8, and RND-9 efflux systems protect biofilm-grown cells against tobramycin. The RND-8 and RND-9 efflux pumps are not involved in ciprofloxacin resistance. Results from the RT-qPCR experiments on the wild-type strain B. cenocepacia J2315 suggest that there is little regulation at the level of mRNA expression for these efflux pumps under the conditions tested

Investigating the Mechanism of Action of Diketopiperazines Inhibitors of the Burkholderia cenocepacia Quorum Sensing Synthase CepI: A Site-Directed Mutagenesis Study

Quorum sensing (QS) is a bacterial intercellular communication process which controls the production of major virulence factors, such as proteases, siderophores, and toxins, as well as biofilm formation. Since the inhibition of this pathway reduces bacterial virulence, QS is considered a valuable candidate drug target, particularly for the treatment of opportunistic infections, such as those caused by Burkholderia cenocepacia in cystic fibrosis patients. Diketopiperazine inhibitors of the acyl homoserine lactone synthase CepI have been recently described. These compounds are able to impair the ability of B. cenocepacia to produce proteases, siderophores, and to form biofilm, being also active in a Caenorhabditis elegans infection model. However, the precise mechanism of action of the compounds, as well as their effect on the cell metabolism, fundamental for candidate drug optimization, are still not completely defined. Here, we performed a proteomic analysis of B. cenocepacia cells treated with one of these inhibitors, and compared it with a cepI deleted strain. Our results demonstrate that the effects of the compound are similar to the deletion of cepI, clearly confirming that these molecules function as inhibitors of the acyl homoserine lactone synthase. Moreover, to deepen our knowledge about the binding mechanisms of the compound to CepI, we exploited previously published in silico structural insights about this enzyme structure and validated different candidate binding pockets on the enzyme surface using site-directed mutagenesis and biochemical analyses. Our experiments identified a region near the predicted S-adenosylmethionine binding site critically involved in interactions with the inhibitor. These results could be useful for future structure-based optimization of these CepI inhibitors