Cryo-EM Structures of the Klebsiella pneumoniae AcrB Multidrug Efflux Pump.

The continued challenges of the COVID-19 pandemic combined with the growing problem of antimicrobial-resistant bacterial infections has severely impacted global health. Specifically, the Gram-negative pathogen Klebsiella pneumoniae is one of the most prevalent causes of secondary bacterial infection in COVID-19 patients, with approximately an 83% mortality rate observed among COVID-19 patients with these bacterial coinfections. K. pneumoniae belongs to the ESKAPE group of pathogens, a group that commonly gives rise to severe infections that are often life-threatening. Recently, K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae has drawn wide public attention, as the mortality rate for this infection can be as high as 71%. The most predominant and clinically important multidrug efflux system in K. pneumoniae is the acriflavine resistance B (AcrB) multidrug efflux pump. This pump mediates resistance to different classes of structurally diverse antimicrobial agents, including quinolones, ß-lactams, tetracyclines, macrolides, aminoglycosides, and chloramphenicol. We here report single-particle cryo-electron microscopy (cryo-EM) structures of K. pneumoniae AcrB, in both the absence and the presence of the antibiotic erythromycin. These structures allow us to elucidate specific pump-drug interactions and pinpoint exactly how this pump recognizes antibiotics. IMPORTANCE Klebsiella pneumoniae has emerged as one of the most problematic and highly antibiotic-resistant pathogens worldwide. It is the second most common causative agent involved in secondary bacterial infection in COVID-19 patients. K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae is a major concern in global public health because of the high mortality rate of this infection. Its drug resistance is due, in a significant part, to active efflux of these bactericides, a major mechanism that K. pneumoniae uses to resist to the action of multiple classes of antibiotics. Here, we report cryo-electron microscopy (cryo-EM) structures of the prevalent and clinically important K. pneumoniae AcrB multidrug efflux pump, in both the absence and the presence of the erythromycin antibiotic. These structures allow us to understand the action mechanism for drug recognition in this pump. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens.

Acriflavine and proflavine hemisulfate as potential antivirals by targeting Mpro.

The evolving SARS-CoV-2 epidemic buffets the world, and the concerted efforts are needed to explore effective drugs. M^pro is an intriguing antiviral target for interfering with viral RNA replication and transcription. In order to get potential anti-SARS-CoV-2 agents, we established an enzymatic assay using a fluorogenic substrate to screen the inhibitors of M^pro. Fortunately, Acriflavine (ACF) and Proflavine Hemisulfate (PRF) with the same acridine scaffold were picked out for their good inhibitory activity against M^pro with IC₅₀ of 5.60 ± 0.29 µM and 2.07 ± 0.01 µM, respectively. Further evaluation of MST assay and enzymatic kinetics experiment in vitro showed that they had a certain affinity to SARS-CoV-2 M^pro and were both non-competitive inhibitors. In addition, they inhibited about 90 % HCoV-OC43 replication in BHK-21 cells at 1 µM. Both compounds showed nano-molar activities against SARS-CoV-2 virus, which were superior to GC376 for anti-HCoV-43, and equivalent to the standard molecule remdesivir. Our study demonstrated that ACF and PRF were inhibitors of M^pro, and ACF has been previously reported as a PL^pro inhibitor. Taken together, ACF and PRF might be dual-targeted inhibitors to provide protection against infections of coronaviruses.

Acriflavine, an Acridine Derivative for Biomedical Application: Current State of the Art.

Acriflavine (ACF) has been known for years as an antibacterial drug. The identification of key oncogenic mechanisms has brought, in recent years, a significant increase in studies on ACF as a multipurpose drug that would improve the prognosis for cancer patients. ACF interferes with the expression of the hypoxia inducible factor, thus acting on metastatic niches of tumors and significantly enhancing the effects of other anticancer therapies. It has been recognized as the most potent HIF-1 inhibitor out of the 336 drugs approved by the FDA. This work presents up-to-date knowledge about the mechanisms of action of ACF and its related prodrug systems in the context of anticancer and SARS-CoV-2 inhibitory properties. It explains the multitask nature of this drug and suggests mechanisms of ACF's action on the coronavirus. Other recent reports on ACF-based systems as potential antibacterial and antiviral drugs are also described.

Zirconium-Based Metal-Organic Frameworks as Acriflavine Cargos in the Battle against Coronaviruses─A Theoretical and Experimental Approach.

In this study, we present a complementary approach for obtaining an effective drug, based on acriflavine (ACF) and zirconium-based metal-organic frameworks (MOFs), against SARS-CoV-2. The experimental results showed that acriflavine inhibits the interaction between viral receptor-binding domain (RBD) of spike protein and angiotensin converting enzyme-2 (ACE2) host receptor driving viral cell entry. The prepared ACF@MOF composites exhibited low (MOF-808 and UiO-66) and high (UiO-67 and NU-1000) ACF loadings. The drug release profiles from prepared composites showed different release kinetics depending on the local pore environment. The long-term ACF release with the effective antiviral ACF concentration was observed for all studied ACF@MOF composites. The density functional theory (DFT) calculations allowed us to determine that π-π stacking together with electrostatic interaction plays an important role in acriflavine adsorption and release from ACF@MOF composites. The molecular docking results have shown that acriflavine interacts with several possible binding sites within the RBD and binding site at the RBD/ACE2 interface. The cytotoxicity and ecotoxicity results have confirmed that the prepared ACF@MOF composites may be considered potentially safe for living organisms. The complementary experimental and theoretical results presented in this study have confirmed that the ACF@MOF composites may be considered a potential candidate for the COVID-19 treatment, which makes them good candidates for clinical trials.