Screening of Small-Molecule Libraries Using SARS-CoV-2-Derived Sequences Identifies Novel Furin Inhibitors.

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, IC50 = 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.

Exploring the potential of bis(thiazol-5-yl)phenylmethane derivatives as novel candidates against genetically defined multidrug-resistant Staphylococcus aureus.

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.

The Effect of 4-(Dimethylamino)phenyl-5-oxopyrrolidines on Breast and Pancreatic Cancer Cell Colony Formation, Migration, and Growth of Tumor Spheroids.

A series of hydrazones, azoles, and azines bearing a 4-dimethylaminophenyl-5-oxopyrrolidine scaffold was synthesized. Their cytotoxic effect against human pancreatic carcinoma Panc-1 and triple-negative breast cancer MDA-MB-231 cell lines was established by MTT assay. Pyrrolidinone derivatives 3c and 3d, with incorporated 5-chloro and 5-methylbenzimidazole fragments; hydrazone 5k bearing a 5-nitrothien-2-yl substitution; and hydrazone 5l with a naphth-1-yl fragment in the structure significantly decreased the viability of both cancer cell lines. Compounds 3c and 5k showed the highest selectivity, especially against the MDA-MB-231 cancer cell line. The EC50 values of the most active compound 5k against the MDA-MB231 cell line was 7.3 ± 0.4 µM, which were slightly higher against the Panc-1 cell line (10.2 ± 2.6 µM). Four selected pyrrolidone derivatives showed relatively high activity in a clonogenic assay. Compound 5k was the most active in both cell cultures, and it completely disturbed MDA-MB-231 cell colony growth at 1 and 2 µM and showed a strong effect on Panc-1 cell colony formation, especially at 2 µM. The compounds did not show an inhibitory effect on cell line migration by the 'wound-healing' assay. Compound 3d most efficiently inhibited the growth of Panc-1 spheroids and reduced cell viability in MDA-MB-231 spheroids. Considering these different activities in biological assays, the selected pyrrolidinone derivatives could be further tested to better understand the structure-activity relationship and their mechanism of action.

Synthesis of 3-((4-Hydroxyphenyl)amino)propanoic Acid Derivatives as Promising Scaffolds for the Development of Antimicrobial Candidates Targeting Multidrug-Resistant Bacterial and Fungal Pathogens.

Infections caused by multidrug-resistant bacterial and fungal pathogens represent a significant global health concern, contributing to increased morbidity and mortality rates. Therefore, it is crucial to develop novel compounds targeting drug-resistant microbial strains. Herein, we report the synthesis of amino acid derivatives bearing an incorporated 4-hydroxyphenyl moiety with various substitutions. The resultant novel 3-((4-hydroxyphenyl)amino)propanoic acid derivatives 2-37 exhibited structure-dependent antimicrobial activity against both ESKAPE group bacteria and drug-resistant Candida species. Furthermore, these derivatives demonstrated substantial activity against Candida auris, with minimum inhibitory concentrations ranging from 0.5 to 64 µg/mL. Hydrazones 14-16, containing heterocyclic substituents, showed the most potent and broad-spectrum antimicrobial activity. This activity extended to methicillin-resistant Staphylococcus aureus (MRSA) with MIC values ranging from 1 to 8 µg/mL, vancomycin-resistant Enterococcus faecalis (0.5-2 µg/mL), Gram-negative pathogens (MIC 8-64 µg/mL), and drug-resistant Candida species (MIC 8-64 µg/mL), including Candida auris. Collectively, these findings underscore the potential utility of the novel 3-((4-hydroxyphenyl)amino)propanoic acid scaffold for further development as a foundational platform for novel antimicrobial agents targeting emerging and drug-resistant bacterial and fungal pathogens.

Screening, Synthesis and Biochemical Characterization of SARS-CoV-2 Protease Inhibitors.

The severe acute respiratory syndrome-causing coronavirus 2 (SARS-CoV-2) papain-like protease (PLpro) and main protease (Mpro) play an important role in viral replication events and are important targets for anti-coronavirus drug discovery. In search of these protease inhibitors, we screened a library of 1300 compounds using a fluorescence thermal shift assay (FTSA) and identified 53 hits that thermally stabilized or destabilized PLpro. The hit compounds structurally belonged to two classes of small molecules: thiazole derivatives and symmetrical disulfide compounds. Compound dissociation constants (Kd) were determined using an enzymatic inhibition method. Seven aromatic disulfide compounds were identified as efficient PLpro inhibitors with Kd values in the micromolar range. Two disulfides displayed six-fold higher potency for PLpro (Kd = 0.5 µM) than for Mpro. The disulfide derivatives bound covalently to both proteases, as confirmed through mass spectrometry. The identified compounds can serve as lead compounds for further chemical optimization toward anti-COVID-19 drugs.

Synthesis of 1-(2-Hydroxyphenyl)- and (3,5-Dichloro-2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic Acid Derivatives as Promising Scaffolds for the Development of Novel Antimicrobial and Anticancer Agents.

Increasing antimicrobial resistance among Gram-positive pathogens and pathogenic fungi remains one of the major public healthcare threats. Therefore, novel antimicrobial candidates and scaffolds are critically needed to overcome resistance in Gram-positive pathogens and drug-resistant fungal pathogens. In this study, we explored 1-(2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic acid and its 3,5-dichloro-2-hydroxyphenyl analogue for their in vitro antimicrobial activity against multidrug-resistant pathogens. The compounds showed structure-dependent antimicrobial activity against Gram-positive pathogens (S. aureus, E. faecalis, C. difficile). Compounds 14 and 24b showed promising activity against vancomycin-intermediate S. aureus strains, and favorable cytotoxic profiles in HSAEC-1 cells, making them attractive scaffolds for further development. 5-Fluorobenzimidazole, having a 3,5-dichloro-2-hydroxyphenyl substituent, was found to be four-fold, and hydrazone, with a thien-2-yl fragment, was two-fold stronger than clindamycin against methicillin resistant S. aureus TCH 1516. Moreover, hydrazone, bearing a 5-nitrothien-2-yl moiety, showed promising activity against three tested multidrug-resistant C. auris isolates representing major genetic lineages (MIC 16 µg/mL) and azole-resistant A. fumigatus strains harboring TR34/L98H mutations in the CYP51A gene. The anticancer activity characterization demonstrated that the 5-fluorobenzimidazole derivative with a 3,5-dichloro-2-hydroxyphenyl substituent showed the highest anticancer activity in an A549 human pulmonary cancer cell culture model. Collectively these results demonstrate that 1-(2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic acid derivatives could be further explored for the development of novel candidates targeting Gram-positive pathogens and drug-resistant fungi.

Synthesis of Novel Benzenesulfonamide-Bearing Functionalized Imidazole Derivatives as Novel Candidates Targeting Multidrug-Resistant Mycobacterium abscessus Complex.

Infections caused by drug-resistant (DR) Mycobacterium abscessus (M. abscessus) complex (MAC) are an important public health concern, particularly when affecting individuals with various immunodeficiencies or chronic pulmonary diseases. Rapidly growing antimicrobial resistance among MAC urges us to develop novel antimicrobial candidates for future optimization. Therefore, we have designed and synthesized benzenesulfonamide-bearing functionalized imidazole or S-alkylated derivatives and evaluated their antimicrobial activity using multidrug-resistant M. abscessus strains and compared their antimycobacterial activity using M. bovis BCG and M. tuberculosis H37Ra. Benzenesulfonamide-bearing imidazole-2-thiol compound 13, containing 4-CF3 substituent in benzene ring, showed strong antimicrobial activity against the tested mycobacterial strains and was more active than some antibiotics used as a reference. Furthermore, an imidazole-bearing 4-F substituent and S-methyl group demonstrated good antimicrobial activity against M. abscessus complex strains, as well as M. bovis BCG and M. tuberculosis H37Ra. In summary, these results demonstrated that novel benzenesulfonamide derivatives, bearing substituted imidazoles, could be further explored as potential candidates for the further hit-to-lead optimization of novel antimycobacterial compounds.

Synthesis and Development of N-2,5-Dimethylphenylthioureido Acid Derivatives as Scaffolds for New Antimicrobial Candidates Targeting Multidrug-Resistant Gram-Positive Pathogens.

The growing antimicrobial resistance to last-line antimicrobials among Gram-positive pathogens remains a major healthcare emergency worldwide. Therefore, the search for new small molecules targeting multidrug-resistant pathogens remains of great importance. In this paper, we report the synthesis and in vitro antimicrobial activity characterisation of novel thiazole derivatives using representative Gram-negative and Gram-positive strains, including tedizolid/linezolid-resistant S. aureus, as well as emerging fungal pathogens. The 4-substituted thiazoles 3h, and 3j with naphthoquinone-fused thiazole derivative 7 with excellent activity against methicillin and tedizolid/linezolid-resistant S. aureus. Moreover, compounds 3h, 3j and 7 showed favourable activity against vancomycin-resistant E. faecium. Compounds 9f and 14f showed broad-spectrum antifungal activity against drug-resistant Candida strains, while ester 8f showed good activity against Candida auris which was greater than fluconazole. Collectively, these data demonstrate that N-2,5-dimethylphenylthioureido acid derivatives could be further explored as novel scaffolds for the development of antimicrobial candidates targeting Gram-positive bacteria and drug-resistant pathogenic fungi.

Synthesis and Biological Activity Characterization of Novel 5-Oxopyrrolidine Derivatives with Promising Anticancer and Antimicrobial Activity.

The 1-(4-acetamidophenyl)-5-oxopyrrolidine carboxylic acid was applied for synthesizing derivatives bearing azole, diazole, and hydrazone moieties in the molecule. Modification of an acetamide fragment to the free amino group afforded compounds with two functional groups, which enabled to provide a series of 4-substituted-1-(4-substituted phenyl)pyrrolidine-2-ones. The resulted compounds 2 and 4-22 were subjected to the in vitro anticancer and antimicrobial activity determination. The compounds 18-22 exerted the most potent anticancer activity against A549 cells. Furthermore, compound 21 bearing 5-nitrothiophene substituents demonstrated promising and selective antimicrobial activity against multidrug-resistant Staphylococcus aureus strains, including linezolid and tedizolid-resistant S. aureus. These results demonstrate that 5-oxopyrolidine derivatives are attractive scaffolds for the further development of anticancer and antimicrobial compounds targeting multidrug-resistant Gram-positive pathogens.

Synthesis of Novel Aminothiazole Derivatives as Promising Antiviral, Antioxidant and Antibacterial Candidates.

It is well-known that thiazole derivatives are usually found in lead structures, which demonstrate a wide range of pharmacological effects. The aim of this research was to explore the antiviral, antioxidant, and antibacterial activities of novel, substituted thiazole compounds and to find potential agents that could have biological activities in one single biomolecule. A series of novel aminothiazoles were synthesized, and their biological activity was characterized. The obtained results were compared with those of the standard antiviral, antioxidant, antibacterial and anticancer agents. The compound bearing 4-cianophenyl substituent in the thiazole ring demonstrated the highest cytotoxic properties by decreasing the A549 viability to 87.2%. The compound bearing 4-trifluoromethylphenyl substituent in the thiazole ring showed significant antiviral activity against the PR8 influenza A strain, which was comparable to the oseltamivir and amantadine. Novel compounds with 4-chlorophenyl, 4-trifluoromethylphenyl, phenyl, 4-fluorophenyl, and 4-cianophenyl substituents in the thiazole ring demonstrated antioxidant activity by DPPH, reducing power, FRAP methods, and antibacterial activity against Escherichia coli and Bacillus subtilis bacteria. These data demonstrate that substituted aminothiazole derivatives are promising scaffolds for further optimization and development of new compounds with potential influenza A-targeted antiviral activity. Study results could demonstrate that structure optimization of novel aminothiazole compounds may be useful in the prevention of reactive oxygen species and developing new specifically targeted antioxidant and antibacterial agents.

Synthesis, Biological Activity, and Molecular Modelling Studies of Naphthoquinone Derivatives as Promising Anticancer Candidates Targeting COX-2.

Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-associated mortalities worldwide. Therefore, it is crucial to develop a novel therapeutic option targeting localized and metastatic NSCLC. In this paper, we describe the synthesis and biological activity characterization of naphthoquinone derivatives bearing selective anticancer activity to NSCLC via a COX-2 mediated pathway. The biological evaluation of compounds 9−16 showed promising structure-dependent anticancer activity on A549 cells in 2D and 3D models. Compounds were able to significantly (p < 0.05) reduce the A549 viability after 24 h of treatment in comparison to treated control. Compounds 9 and 16 bearing phenylamino and 4-hydroxyphenylamino substituents demonstrated the most promising anticancer activity and were able to induce mitochondrial damage and ROS formation. Furthermore, most promising compounds showed significantly lower cytotoxicity to non-cancerous Vero cells. The in silico ADMET properties revealed promising drug-like properties of compounds 9 and 16. Both compounds demonstrated favorable predicted GI absorption values, while only 16 was predicted to be permeable through the blood−brain barrier. Molecular modeling studies identified that compound 16 is able to interact with COX-2 in arachidonic acid site. Further studies are needed to better understand the safety and in vivo efficacy of compounds 9 and 16.

Evaluation of N-aryl-ß-alanine derivatives as anticancer agents in triple-negative breast cancer and glioblastoma in vitro models.

Synthesis of ß-amino acid derivatives containing hydrazone and azole moieties is described. For this purpose, the appropriate hydrazide was treated with aromatic aldehydes, ketones and phenyl iso(thio)cyanates to obtain the desired outcome. The synthesized target compounds were evaluated for their anticancer properties. The assay displayed 3,3'-((2,6-diethylphenyl)azanediyl)bis(N'-(benzylidene)propanehydrazide) to possess the convincing anticancer effect against triple-negative breast cancer cells in vitro. To further study the anticancer properties of compounds containing a hydrazone moiety in breast cancer, series of previously and newly prepared dihydrazones were investigated. It was determined that derivatives with the bis(N'-(4-bromobenzylidene) fragment in the structure are exclusively cytotoxic to cancer cells. The most active compounds against both cell lines were those containing electron withdrawing 4-BrPh or 4-ClPh moieties, together with either chlorine, bromine or iodine groups in para position of phenyl ring. Selected two representative compounds showed migrastatic activity in MDA-MB-231 cell line, where both of them reduced the growth of breast cancer and glioblastoma cell 3D cultures and inhibited cell colony formation. 2009 Elsevier Ltd. All rights reserved.

Synthesis and Antibacterial Activity of New Azole, Diazole and Triazole Derivatives Based on p-Aminobenzoic Acid.

The p-aminobenzoic acid was applied for the synthesis of substituted 1-phenyl-5-oxopyrrolidine derivatives containing benzimidazole, azole, oxadiazole, triazole, dihydrazone, and dithiosemicarbazide moieties in the structure. All the obtained compounds were evaluated for their in vitro antimicrobial activity against Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Salmonella enteritidis, Escherichia coli, and Pseudomonas aeruginosa by using MIC and MBC assays. This study showed a good bactericidal activity of γ-amino acid and benzimidazoles derivatives. The antimicrobial activity of the most promising compounds was higher than ampicillin. Furthermore, two benzimidazoles demonstrated good antimicrobial activity against L. monocytogenes (MIC 15.62 µg/mL) that was four times more potent than ampicillin (MIC 65 µg/mL). Further studies are needed to better understand the mechanism of the antimicrobial activity as well as to generate antimicrobial compounds based on the 1-phenyl-5-oxopyrrolidine scaffold.

Synthesis of Novel Thiazole Derivatives Bearing ß-Amino Acid and Aromatic Moieties as Promising Scaffolds for the Development of New Antibacterial and Antifungal Candidates Targeting Multidrug-Resistant Pathogens.

Rapidly growing antimicrobial resistance among clinically important bacterial and fungal pathogens accounts for high morbidity and mortality worldwide. Therefore, it is critical to look for new small molecules targeting multidrug-resistant pathogens. Herein, in this paper we report a synthesis, ADME properties, and in vitro antimicrobial activity characterization of novel thiazole derivatives bearing ß-amino acid, azole, and aromatic moieties. The in silico ADME characterization revealed that compounds 1-9 meet at least 2 Lipinski drug-like properties while cytotoxicity studies demonstrated low cytotoxicity to Vero cells. Further in vitro antimicrobial activity characterization showed the selective and potent bactericidal activity of 2a-c against Gram-positive pathogens (MIC 1-64 µg/mL) with profound activity against S. aureus (MIC 1-2 µg/mL) harboring genetically defined resistance mechanisms. Furthermore, the compounds 2a-c exhibited antifungal activity against azole resistant A. fumigatus, while only 2b and 5a showed antifungal activity against multidrug resistant yeasts including Candida auris. Collectively, these results demonstrate that thiazole derivatives 2a-c and 5a could be further explored as a promising scaffold for future development of antifungal and antibacterial agents targeting highly resistant pathogenic microorganisms.

Synthesis, ADMET Properties, and In Vitro Antimicrobial and Antibiofilm Activity of 5-Nitro-2-thiophenecarbaldehyde N-((E)-(5-Nitrothienyl)methylidene)hydrazone (KTU-286) against Staphylococcus aureus with Defined Resistance Mechanisms.

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, IC50 = 0.460 µg/mL), followed by vancomycin resistant S. aureus (VRSA) (MIC 4.0 µg/mL, IC50 = 1.697 µg/mL) and methicillin-resistant S. aureus (MRSA) (MIC 1.0-16.0 µg/mL, IC50 = 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.