Antibacterial activity of 1-[(2,4-dichlorophenethyl)amino]-3-phenoxypropan-2-ol against antibiotic-resistant strains of diverse bacterial pathogens, biofilms and in pre-clinical infection models

We recently described the novel anti-persister compound 1-[(2,4-dichlorophenethyl)amino]-3-phenoxypropan-2-ol (SPI009), capable of directly killing persister cells of the Gram-negative pathogen Pseudomonas aeruginosa. This compound also shows antibacterial effects against non-persister cells, suggesting that SPI009 could be used as an adjuvant for antibacterial combination therapy. Here, we demonstrate the broad-spectrum activity of SPI009, combined with different classes of antibiotics, against the clinically relevant ESKAPE pathogens Enterobacter aerogenes, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, Enterococcus faecium and Burkholderia cenocepacia and Escherichia coli. Importantly, SPI009 re-enabled killing of antibiotic-resistant strains and effectively lowered the required antibiotic concentrations. The clinical potential was further confirmed in biofilm models of P. aeruginosa and S. aureus where SPI009 exhibited effective biofilm inhibition and eradication. Caenorhabditis elegans infected with P. aeruginosa also showed a significant improvement in survival when SPI009 was added to conventional antibiotic treatment. Overall, we demonstrate that SPI009, initially discovered as an anti-persister molecule in P. aeruginosa, possesses broad-spectrum activity and is highly suitable for the development of antibacterial combination therapies in the fight against chronic infections

Modifying Rap1-signalling by targeting Pde6δ is neuroprotective in models of Alzheimer’s disease

Background: Neuronal Ca2+ dyshomeostasis and hyperactivity play a central role in Alzheimer's disease pathology arid progression. Amyloid-beta together with non-genetic risk-factors of Alzheimer's disease contributes to increased Ca2+ influx and aberrant neuronal activity, which accelerates neurodegeneration in a feed-forward fashion. As such, identifying new targets and drugs to modulate excessive Ca2+ signalling and neuronal hyperactivity, without overly suppressing them, has promising therapeutic potential. Methods: Here we show, using biochemical, electrophysiological, imaging, and behavioural tools, that pharmacological modulation of Rap1 signalling by inhibiting its interaction with Pde6 delta normalises disease associated Ca2+ aberrations and neuronal activity, conferring neuroprotection in models of Alzheimer's disease. Results: The newly identified inhibitors of the Rap1-Pde6 delta interaction counteract AD phenotypes, by reconfiguring Rapt signalling underlying synaptic efficacy, Ca2+ influx, and neuronal repolarisation, without adverse effects in-cellulo or invivo. Thus, modulation of Rap1 by Pde6 delta accommodates key mechanisms underlying neuronal activity, and therefore represents a promising new drug target for early or late intervention in neurodegenerative disorders. Conclusion: Targeting the Pde6 delta-Rap1 interaction has promising therapeutic potential for disorders characterised by neuronal hyperactivity, such as Alzheimer's disease

The Substitutions L50F, E166A, and L167F in SARS-CoV-2 3CLpro Are Selected by a Protease Inhibitor In Vitro and Confer Resistance To Nirmatrelvir.

The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of 3CLpro-inhibitors to select for drug-resistant variants needs to be established. Therefore, SARS-CoV-2 was passaged in vitro in the presence of increasing concentrations of ALG-097161, a probe compound designed in the context of a 3CLpro drug discovery program. We identified a combination of amino acid substitutions in 3CLpro (L50F E166A L167F) that is associated with a >20× increase in 50% effective concentration (EC50) values for ALG-097161, nirmatrelvir (PF-07321332), PF-00835231, and ensitrelvir. While two of the single substitutions (E166A and L167F) provide low-level resistance to the inhibitors in a biochemical assay, the triple mutant results in the highest levels of resistance (6× to 72×). All substitutions are associated with a significant loss of enzymatic 3CLpro activity, suggesting a reduction in viral fitness. Structural biology analysis indicates that the different substitutions reduce the number of inhibitor/enzyme interactions while the binding of the substrate is maintained. These observations will be important for the interpretation of resistance development to 3CLpro inhibitors in the clinical setting. IMPORTANCE Paxlovid is the first oral antiviral approved for treatment of SARS-CoV-2 infection. Antiviral treatments are often associated with the development of drug-resistant viruses. In order to guide the use of novel antivirals, it is essential to understand the risk of resistance development and to characterize the associated changes in the viral genes and proteins. In this work, we describe for the first time a pathway that allows SARS-CoV-2 to develop resistance against Paxlovid in vitro. The characteristics of in vitro antiviral resistance development may be predictive for the clinical situation. Therefore, our work will be important for the management of COVID-19 with Paxlovid and next-generation SARS-CoV-2 3CLpro inhibitors

Synthesis, Structure–Activity Relationships, and Antiviral Profiling of 1-Heteroaryl-2-Alkoxyphenyl Analogs as Inhibitors of SARS-CoV-2 Replication

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has led to a pandemic, that continues to be a huge public health burden. Despite the availability of vaccines, there is still a need for small-molecule antiviral drugs. In an effort to identify novel and drug-like hit matter that can be used for subsequent hit-to-lead optimization campaigns, we conducted a high-throughput screening of a 160 K compound library against SARS-CoV-2, yielding a 1-heteroaryl-2-alkoxyphenyl analog as a promising hit. Antiviral profiling revealed this compound was active against various beta-coronaviruses and preliminary mode-of-action experiments demonstrated that it interfered with viral entry. A systematic structure–activity relationship (SAR) study demonstrated that a 3- or 4-pyridyl moiety on the oxadiazole moiety is optimal, whereas the oxadiazole can be replaced by various other heteroaromatic cycles. In addition, the alkoxy group tolerates some structural diversity

Elucidation of the Mode of Action of a New Antibacterial Compound Active against Staphylococcus aureus and Pseudomonas aeruginosa.

Nosocomial and community-acquired infections caused by multidrug resistant bacteria represent a major human health problem. Thus, there is an urgent need for the development of antibiotics with new modes of action. In this study, we investigated the antibacterial characteristics and mode of action of a new antimicrobial compound, SPI031 (N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol), which was previously identified in our group. This compound exhibits broad-spectrum antibacterial activity, including activity against the human pathogens Staphylococcus aureus and Pseudomonas aeruginosa. We found that SPI031 has rapid bactericidal activity (7-log reduction within 30 min at 4x MIC) and that the frequency of resistance development against SPI031 is low. To elucidate the mode of action of SPI031, we performed a macromolecular synthesis assay, which showed that SPI031 causes non-specific inhibition of macromolecular biosynthesis pathways. Liposome leakage and membrane permeability studies revealed that SPI031 rapidly exerts membrane damage, which is likely the primary cause of its antibacterial activity. These findings were supported by a mutational analysis of SPI031-resistant mutants, a transcriptome analysis and the identification of transposon mutants with altered sensitivity to the compound. In conclusion, our results show that SPI031 exerts its antimicrobial activity by causing membrane damage, making it an interesting starting point for the development of new antibacterial therapies

Development of new dsDNA ligands using combinatorial chemistry and solution phase screening

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Comparison of library screening techniques used in the development of dsDNA ligands

The gel retardation and FID (fluorescent intercalator displacement) techniques have been compared for the selection of dsDNA binding ligands out of library mixtures. The selection procedure involves the synthesis and screening of unnatural oligopeptide libraries based on an iterative deconvolution procedure. Both methods yield comparable selection results and binding constants for the selected compounds, meaning that they can be considered as complementary in the discovery process of new antigene compounds. Furthermore, a quinazolin-2,4-dione amino acid has been identified as possessing interesting properties for interaction with dsDNA.status: publishe

In search of Broad Applicable, Small Molecule Inhibitors of Salmonella Biofilm Formation

Salmonella is one of the most important causes of foodborne infections worldwide. A major difficulty in the battle against Salmonella is the fact that Salmonella can form biofilms on various biotic and abiotic surfaces. Biofilms are multicellular structures, in which the bacteria live in a self-produced gel-like matrix. Within these biofilms, Salmonella is protected against a wide range of environmental influences such as desiccation, antibiotics, disinfectants and the immune system. As such, biofilm formation is an important survival strategy of Salmonella, both in- and outside the host. Therefore, the prevention and/or eradication of these biofilms could be an effective way to limit the spread of Salmonella. Our laboratory has previously identified a potent group of Salmonella biofilm inhibitors. A drawback of these compounds is that they only seem to be active at temperatures below 25 °C. Therefore, we are currently conducting a high-throughput screening (using the ‘Calgary Biofilm device’) of a compound library, consisting of 17.000 small molecules, in search for Salmonella biofilm inhibitors which are active at a temperature ranging from 16 °C till 37 °C, and therefore have potential to be used both in- and outside the host. The library contains a broad range of compounds, selected on their possible drug ability. The compounds have a molecular weight between 200 and 500 dalton and the screening is executed both at 16 °C and 37 °C. We aim at identifying compounds that inhibit biofilm formation, but that do not kill the bacteria. In this way, the development of resistance, which is a major drawback of classical antibiotics, is less likely. As such, these biofilm inhibitors could be a sustainable alternative in the production of safe and healthy food. After the screening of the first ±1800 compounds, we already found 47 possible biofilm inhibitors, of which 37 are active at 16 °C and 10 at 37 °C. None of those inhibitors are active at both temperatures but these preliminary results indicate it will probably be possible to indentify new broad applicable Salmonella biofilm inhibitors using this assay.Posterstatus: publishe