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SARS-CoV-2 is the pathogen responsible for the most recent global pandemic, which has claimed hundreds of thousands of victims worldwide. Despite remarkable efforts to develop an effective vaccine, concerns have been raised about the actual protection against novel variants. Thus, researchers are eager to identify alternative strategies to fight against this pathogen. Like other opportunistic entities, a key step in the SARS-CoV-2 lifecycle is the maturation of the envelope glycoprotein at the RARR685↓ motif by the cellular enzyme Furin. Inhibition of this cleavage greatly affects viral propagation, thus representing an ideal drug target to contain infection. Importantly, no Furin-escape variants have ever been detected, suggesting that the pathogen cannot replace this protease by any means. Here, we designed a novel fluorogenic SARS-CoV-2-derived substrate to screen commercially available and custom-made libraries of small molecules for the identification of new Furin inhibitors. We found that a peptide substrate mimicking the cleavage site of the envelope glycoprotein of the Omicron variant (QTQTKSHRRAR-AMC) is a superior tool for screening Furin activity when compared to the commercially available Pyr-RTKR-AMC substrate. Using this setting, we identified promising novel compounds able to modulate Furin activity in vitro and suitable for interfering with SARS-CoV-2 maturation. In particular, we showed that 3-((5-((5-bromothiophen-2-yl)methylene)-4-oxo-4,5 dihydrothiazol-2-yl)(3-chloro-4-methylphenyl)amino)propanoic acid ( P3 , IC 50 = 35 μM) may represent an attractive chemical scaffold for the development of more effective antiviral drugs via a mechanism of action that possibly implies the targeting of Furin secondary sites (exosites) rather than its canonical catalytic pocket. Overall, a SARS-CoV-2-derived peptide was investigated as a new substrate for in vitro high-throughput screening (HTS) of Furin inhibitors and allowed the identification of compound P3 as a promising hit with an innovative chemical scaffold. Given the key role of Furin in infection and the lack of any Food and Drug Administration (FDA)-approved Furin inhibitor, P3 represents an interesting antiviral candidate.

Antimicrobial resistance (AMR) represents an alarming global challenge to public health. Infections caused by multidrug-resistant Staphylococcus aureus (S. aureus) pose an emerging global threat. Therefore, it is crucial to develop novel compounds with promising antimicrobial activity against S. aureus especially those with challenging resistance mechanisms and biofilm formation. Series of bis(thiazol-5-yl)phenylmethane derivatives were evaluated against drug-resistant Gram-positive bacteria. The screening revealed an S. aureus-selective mechanism of bis(thiazol-5-yl)phenylmethane derivatives (MIC 2-64 μg/mL), while significantly lower activity was observed with vancomycin-resistant Enterococcus faecalis (MIC 64 μg/mL) (p<0.05). The most active phenylmethane-based (p-tolyl) derivative, 23a, containing nitro and dimethylamine substituents, and the naphthalene-based derivative, 28b, harboring fluorine and nitro substituents, exhibited strong, near MIC bactericidal activity against S. aureus with genetically defined resistance phenotypes such as MSSA, MRSA, and VRSA and their biofilms. The in silico modeling revealed that most promising compounds 23a and 28b were predicted to bind S. aureus MurC ligase. The 23a and 28b formed bonds with MurC residues at binding site, specifically Ser12 and Arg375, indicating consequential interactions essential for complex stability. The in vitro antimicrobial activity of compound 28b was not affected by the addition of 50% serum. Finally, all tested bis(thiazol-5-yl)phenylmethane derivatives showed favorable cytotoxicity profiles in A549 and THP-1-derived macrophage models. These results demonstrated that bis(thiazol-5-yl)phenylmethane derivatives 23a and 28b could be potentially explored as scaffolds for the development of novel candidates targeting drug-resistant S. aureus. Further studies are also warranted to understand in vivo safety, efficacy, and pharmacological bioavailability of bis(thiazol-5-yl)phenylmethane derivatives., Competing Interests: The authors have declared that no competing interests exist, (Copyright: © 2024 Kavaliauskas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The emergence of drug-resistant Staphylococcus aureus is responsible for high morbidity and mortality worldwide. New therapeutic options are needed to fight the increasing antimicrobial resistance among S. aureus in the clinical setting. We, therefore, characterized the in silico absorption, distribution, metabolism, elimination, and toxicity (ADMET) and in vitro antimicrobial activity of 5-nitro-2-thiophenecarbaldehyde N-((E)-(5-nitrothienyl)methylidene)hydrazone (KTU-286) against drug-resistant S. aureus strains with genetically defined resistance mechanisms. The antimicrobial activity of KTU-286 was determined by CLSI recommendations. The ADMET properties were estimated by using in silico modeling. The activity on biofilm integrity was examined by crystal violet assay. KTU-286 demonstrated low estimated toxicity and low skin permeability. The highest antimicrobial activity was observed among pan-susceptible (Pan-S) S. aureus (minimal inhibitory concentration (MIC) 0.5-2.0 µg/mL, IC 50 = 0.460 µg/mL), followed by vancomycin resistant S. aureus (VRSA) (MIC 4.0 µg/mL, IC 50 = 1.697 µg/mL) and methicillin-resistant S. aureus (MRSA) (MIC 1.0-16.0 µg/mL, IC 50 = 2.282 µg/mL). KTU-286 resulted in significant ( p < 0.05) loss of S. aureus biofilm integrity in vitro. Further studies are needed for a better understanding of safety, synergistic relationship, and therapeutic potency of KTU-286.