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

Role of RND Efflux Pumps in Drug Resistance of Cystic Fibrosis Pathogens

Drug resistance represents a great concern among people with cystic fibrosis (CF), due to the recurrent and prolonged antibiotic therapy they should often undergo. Among Multi Drug Resistance (MDR) determinants, Resistance-Nodulation-cell Division (RND) efflux pumps have been reported as the main contributors, due to their ability to extrude a wide variety of molecules out of the bacterial cell. In this review, we summarize the principal RND efflux pump families described in CF pathogens, focusing on the main Gram-negative bacterial species (Pseudomonas aeruginosa, Burkholderia cenocepacia, Achromobacter xylosoxidans, Stenotrophomonas maltophilia) for which a predominant role of RND pumps has been associated to MDR phenotypes

The cell division protein FtsZ as a cellular target to hit cystic fibrosis pathogens

Cystic fibrosis is a rare genetic disease characterized by the production of dehydrated mucus in the lung able to trap bacteria and rendering their proliferation particularly dangerous, thus leading to chronic infections. Among these bacteria, Staphylococcus aureus and Pseudomonas aeruginosa play a major role while, within emerging pathogens, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, Burkholderia cepacia complex species, as well as non-tuberculous mycobacteria are listed. Since a common feature of these bacteria is the high level of drug resistance, cell division, and in particular FtsZ, has been explored as a novel therapeutic target for the design of new molecules with antibacterial properties. This review summarizes and provides insight into recent advances in the discovery of compounds targeting FtsZ: the majority of them exhibit anti-staphylococcal activity, while a few were directed against the cystic fibrosis Gram negative pathogens

Molecular Characterization of the Burkholderia cenocepacia dcw Operon and FtsZ Interactors as New Targets for Novel Antimicrobial Design

The worldwide spread of antimicrobial resistance highlights the need of new druggable cellular targets. The increasing knowledge of bacterial cell division suggested the potentiality of this pathway as a pool of alternative drug targets, mainly based on the essentiality of these proteins, as well as on the divergence from their eukaryotic counterparts. People suffering from cystic fibrosis are particularly challenged by the lack of antibiotic alternatives. Among the opportunistic pathogens that colonize the lungs of these patients, Burkholderia cenocepacia is a well-known multi-drug resistant bacterium, particularly difficult to treat. Here we describe the organization of its division cell wall (dcw) cluster: we found that 15 genes of the dcw operon can be transcribed as a polycistronic mRNA from mraZ to ftsZ and that its transcription is under the control of a strong promoter regulated by MraZ. B. cenocepacia J2315 FtsZ was also shown to interact with the other components of the divisome machinery, with a few differences respect to other bacteria, such as the direct interaction with FtsQ. Using an in vitro sedimentation assay, we validated the role of SulA as FtsZ inhibitor, and the roles of FtsA and ZipA as tethers of FtsZ polymers. Together our results pave the way for future antimicrobial design based on the divisome as pool of antibiotic cellular targets

Biochemical Characterization of Glutamate Racemase-A New Candidate Drug Target against Burkholderia cenocepacia Infections

The greatest obstacle for the treatment of cystic fibrosis patients infected with the Burkholderia species is their intrinsic antibiotic resistance. For this reason, there is a need to develop new effective compounds. Glutamate racemase, an essential enzyme for the biosynthesis of the bacterial cell wall, is an excellent candidate target for the design of new antibacterial drugs. To this aim, we recombinantly produced and characterized glutamate racemase from Burkholderia cenocepacia J2315. From the screening of an in-house library of compounds, two Zn (II) and Mn (III) 1,3,5-triazapentadienate complexes were found to efficiently inhibit the glutamate racemase activity with IC50 values of 35.3 and 10.0 μM, respectively. Using multiple biochemical approaches, the metal complexes have been shown to affect the enzyme activity by binding to the enzyme-substrate complex and promoting the formation of an inhibited dimeric form of the enzyme. Our results corroborate the value of glutamate racemase as a good target for the development of novel inhibitors against Burkholderia

Identification by Reverse Vaccinology of Three Virulence Factors in <i>Burkholderia cenocepacia</i> That May Represent Ideal Vaccine Antigens

The Burkholderia cepacia complex comprises environmental and clinical Gram-negative bacteria that infect particularly debilitated people, such as those with cystic fibrosis. Their high level of antibiotic resistance makes empirical treatments often ineffective, increasing the risk of worst outcomes and the diffusion of multi-drug resistance. However, the discovery of new antibiotics is not trivial, so an alternative can be the use of vaccination. Here, the reverse vaccinology approach has been used to identify antigen candidates, obtaining a short-list of 24 proteins. The localization and different aspects of virulence were investigated for three of them—BCAL1524, BCAM0949, and BCAS0335. The three antigens were localized in the outer membrane vesicles confirming that they are surface exposed. We showed that BCAL1524, a collagen-like protein, promotes bacteria auto-aggregation and plays an important role in virulence, in the Galleria mellonella model. BCAM0949, an extracellular lipase, mediates piperacillin resistance, biofilm formation in Luria Bertani and artificial sputum medium, rhamnolipid production, and swimming motility; its predicted lipolytic activity was also experimentally confirmed. BCAS0335, a trimeric adhesin, promotes minocycline resistance, biofilm organization in LB, and virulence in G. mellonella. Their important role in virulence necessitates further investigations to shed light on the usefulness of these proteins as antigen candidates

Bactericidal and Anti-Biofilm Activity of the FtsZ Inhibitor C109 against Acinetobacter baumannii

In the last few years, Acinetobacter baumannii has ranked as a number one priority due to its Multi Drug Resistant phenotype. The different metabolic states, such as the one adopted when growing as biofilm, help the bacterium to resist a wide variety of compounds, placing the discovery of new molecules able to counteract this pathogen as a topic of utmost importance. In this context, bacterial cell division machinery and the conserved protein FtsZ are considered very interesting cellular targets. The benzothiadiazole compound C109 is able to inhibit bacterial growth and to block FtsZ GTPase and polymerization activities in Burkholderia cenocepacia, Pseudomonas aeruginosa, and Staphylococcus aureus. In this work, the activity of C109 was tested against a panel of antibiotic sensitive and resistant A. baumannii strains. Its ability to inhibit biofilm formation was explored, together with its activity against the A. baumannii FtsZ purified protein. Our results indicated that C109 has good MIC values against A. baumannii clinical isolates. Moreover, its antibiofilm activity makes it an interesting alternative treatment, effective against diverse metabolic states. Finally, its activity was confirmed against A. baumannii FtsZ

Chemical, Metabolic, and Cellular Characterization of a FtsZ Inhibitor Effective Against Burkholderia cenocepacia

International audienceThere is an urgent need for new antimicrobials to treat the opportunistic Gram-negative Burkholderia cenocepacia, which represents a problematic challenge for cystic fibrosis patients. Recently, a benzothiadiazole derivative, C109, was shown to be effective against the infections caused by B. cenocepacia and other Gram-negative and-positive bacteria. C109 has a promising cellular target, the cell division protein FtsZ, and a recently developed PEGylated formulation make it an attractive molecule to counteract Burkholderia infections. However, the ability of efflux pumps to extrude it out of the cell represents a limitation for its use. Here, more than 50 derivatives of C109 were synthesized and tested against Gram-negative species and the Gram-positive Staphylococcus aureus. In addition, their activity was evaluated on the purified FtsZ protein. The chemical, metabolic and cellular stability of C109 has been assayed using different biological systems, including quantitative single-cell imaging. However, no further improvement on C109 was achieved, and the role of efflux in resistance was further confirmed. Also, a novel nitroreductase that can inactivate the compound was characterized, but it does not appear to play a role in natural resistance. All these data allowed a deep characterization of the compound, which will contribute to a further improvement of its properties

Antimicrobial activity of six essential oils against Burkholderia cepacia complex: Insights into mechanism(s) of action

AIM: To investigate the activity and mechanisms of action of six essential oils (EOs) against Burkholderia cepacia complex, opportunistic human pathogens highly resistant to antibiotics. MATERIALS & METHODS: Minimal inhibitory concentration of EOs alone, plus antibiotics or efflux pump inhibitors was determined. RESULTS: Origanum vulgare, Thymus vulgaris and Eugenia caryophyllata EOs resulted to be more active than the other EOs. EOs did not enhance antibiotic activity against the model strain B. cenocepacia J2315. EOs resulted more active in the presence of an efflux pump inhibitor acting on Resistance-Nodulation Cell Division efflux pumps and against B. cenocepacia J2315 Resistance-Nodulation Cell Division knocked-out mutants. CONCLUSION: EOs showed intracellular mechanisms of action and, thus, the efflux pumps inhibitor addition could boost their activity