DUSP22 inhibits lung tumorigenesis by suppression of EGFR/c-Met signaling.

DUSP22, an atypical dual-specificity phosphatase enzyme, plays a significant role in regulating multiple kinase signaling pathways by dephosphorylation. Our study demonstrated that decreased DUSP22 expression is associated with shorter disease-free survival, advanced TNM (tumor, lymph nodes, and metastasis), cancer stage, and higher tumor grade in lung adenocarcinoma (LUAD) patients. Exogenous DUSP22 expression reduces the colony-forming capacity of lung cancer cells and inhibits xenograft tumor growth primarily by targeting EGFR and suppressing its activity through dephosphorylation. Knockdown of DUSP22 using shRNA enhances EGFR dependency in HCC827 lung cancer cells and increases sensitivity to gefitinib, an EGFR inhibitor. Consistently, genetic deletion of DUSP22 enhances EGFRdel (exon 19 deletion)-driven lung tumorigenesis and elevates EGFR activity. Pharmacological inhibition of DUSP22 activates EGFR, ERK1/2, and upregulates downstream PD-L1 expression. Additionally, lentiviral deletion of DUSP22 by shRNA enhances lung cancer cell migration through EGFR/c-Met and PD-L1-dependent pathways. Gefitinib, an EGFR inhibitor, mechanistically suppresses migration induced by DUSP22 deletion and inhibits c-Met activity. Furthermore, cabozantinib, a c-Met inhibitor, reduces migration and attenuates EGFR activation caused by DUSP22 deletion. Collectively, our findings support the hypothesis that loss of DUSP22 function in lung cancer cells confers a survival advantage by augmenting EGFR signaling, leading to increased activation of downstream c-Met, ERK1/2, and PD-L1 axis, ultimately contributing to the progression of advanced lung cancer.

Binding Affinity, Selectivity, and Pharmacokinetics of the Oxytocin Receptor Antagonist L-368,899 in the Coyote (Canis latrans).

L-368,899 is a selective small-molecule oxytocin receptor (OXTR) antagonist originally developed in the 1990s to prevent preterm labor. Although its utility for that purpose was limited, L-368,899 is now one of the most commonly used drugs in animal research for the selective blockade of neural OXTR after peripheral delivery. A growing number of rodent and primate studies have used L-368,899 to evaluate whether certain behaviors are oxytocin dependent. These studies have improved our understanding of oxytocin's function in the brains of rodents and monkeys, but very little work has been done in other mammals, and only a single paper in macaques has provided any evidence that L-368,899 can be detected in the CNS after peripheral delivery. The current study sought to extend those findings in a novel species: coyotes ( Canis latrans ). Coyotes are ubiquitous North American canids that form long-term monogamous pair-bonds. Although monogamy is rare in rodents and primates, all wild canid species studied to date exhibit social monogamy. Coyotes are therefore an excellent model organism for the study of oxytocin and social bonds. Our goal was to determine whether L-368,899 is a viable candidate for future use in behavioral studies in coyotes. We used captive coyotes at the USDA National Wildlife Research Center's Predator Research Facility to evaluate the pharmacokinetics of L-368,899 in blood and CSF during a 90-min time course after intramuscular injection. We then characterized the binding affinity and selectivity of L-368,899 to coyote OXTR and the structurally similar vasopressin 1a receptor. We found that L-368,899 peaked in CSF at 15 to 30 min after intramuscular injection and slowly accumulated in blood. L-368,899 was 40 times more selective for OXTR than vasopressin 1a receptors and bound to the coyote OXTR with an affinity of 12 nM. These features of L-368,899 support its utility in future studies to probe the oxytocin system of coyotes.

Mesobiliverdin IXα-enriched microalgae feed additive eliminates reliance on antibiotic tylosin to promote intestinal health of weaning piglets.

Weaning is a critical period in raising pigs. Novel animal feed additives that promote gut health and regulate immune function of piglets without antibiotics are needed. In this study, we aimed to test the ability of mesobiliverdin IXα-enriched microalgae (MBV IXα-enriched microalgae) to eliminate reliance on antibiotics to promote intestinal health in piglets. Eighty 28-day-old weaned piglets were randomly allocated to four groups each with four replicate pens and five piglets per pen. The dietary treatments were a basal diet as control (NC), basal diet plus 0.05% tylosin (PC), basal diet plus 0.1% or 0.5% MBV IXα-enriched microalgae as low (MBV-SP1) or high (MBV-SP2) dose respectively. All treated animals showed no significant differences in live weight, average daily gain and feed efficiency compared to control animals. Histological examination showed that MBV-SP1 and particularly MBV-SP2 increased the ratio of villus height to crypt depth in the jejunum and ileum compared to NC (p < 0.05). Similarly, tylosin treatment also increased villi lengths and the ratio of villus height to crypt depth in the jejunum and ileum compared to the NC (p < 0.05). MBV-SP1 and particularly MBV-SP2 reduced the levels of inflammatory cytokines interleukin-6 and tumour necrosis factor-alpha in the small intestine. MBV-SP2 and tylosin similarly reduced the lipid peroxidation marker (TBARS value) in the duodenum and ileum. In conclusion, feed supplementation with MBV IXα-enriched microalgae improved gut health by villus height and production of immunomodulators that correlated with down-regulated secretion of inflammatory cytokines.

KI04 an Aminoglycosides-Derived Molecule Acts as an Inhibitor of Human Connexin46 Hemichannels Expressed in HeLa Cells.

BACKGROUND: Connexins (Cxs) are proteins that help cells to communicate with the extracellular media and with the cytoplasm of neighboring cells. Despite their importance in several human physiological and pathological conditions, their pharmacology is very poor. In the last decade, some molecules derived from aminoglycosides have been developed as inhibitors of Cxs hemichannels. However, these studies have been performed in E. coli, which is a very simple model. Therefore, our main goal is to test whether these molecules have similar effects in mammalian cells. METHODS: We transfected HeLa cells with the human Cx46tGFP and characterized the effect of a kanamycin-derived molecule (KI04) on Cx46 hemichannel activity by time-lapse recordings, changes in phosphorylation by Western blot, localization by epifluorescence, and possible binding sites by molecular dynamics (MD). RESULTS: We observed that kanamycin and KI04 were the most potent inhibitors of Cx46 hemichannels among several aminoglycosides, presenting an IC50 close to 10 µM. The inhibitory effect was not associated with changes in Cx46 electrophoretic mobility or its intracellular localization. Interestingly, 5 mM DTT did not reverse KI04 inhibition, but the KI04 effect completely disappeared after washing out KI04 from the recording media. MD analysis revealed two putative binding sites of KI04 in the Cx46 hemichannel. RESULTS: These results demonstrate that KI04 could be used as a Cx46 inhibitor and could help to develop future selective Cx46 inhibitors.

Amphiphilic aminoglycosides: Modifications that revive old natural product antibiotics.

Widely-used Streptomyces-derived antibacterial aminoglycosides have encountered challenges because of antibiotic resistance and toxicity. Today, they are largely relegated to medicinal topical applications. However, chemical modification to amphiphilic aminoglycosides can revive their efficacy against bacterial pathogens and expand their targets to other pathogenic microbes and disorders associated with hyperactive connexin hemichannels. For example, amphiphilic versions of neomycin and neamine are not subject to resistance and have expanded antibacterial spectra, and amphiphilic kanamycins are effective antifungals and have promising therapeutic uses as connexin hemichannel inhibitors. With further research and discoveries aimed at improved formulations and delivery, amphiphilic aminoglycosides may achieve new horizons in pharmacopeia and agriculture for Streptomyces aminoglycosides beyond just serving as topical antibacterials.

Mesobiliverdin IXα ameliorates osteoporosis via promoting osteogenic differentiation of mesenchymal stem cells.

Heme oxygenase-1 (HO-1) expression promotes osteogenesis, but the mechanisms remain unclear and therapeutic strategies using it to target bone disorders such as osteoporosis have not progressed. Mesobiliverdin IXα is a naturally occurring bilin analog of HO-1 catalytic product biliverdin IXα. Inclusion of mesobiliverdin IXα in the feed diet of ovariectomized osteoporotic mice was observed to increase femur bone volume, trabecular thickness and osteogenesis serum markers osteoprotegrin and osteocalcin and to decrease bone resorption serum markers cross-linked N-teleopeptide and tartrate-resistant acid phosphatase 5b. Moreover, in vitro exposure of human bone marrow mesenchymal stem cells to mesobiliverdin IXα enhanced osteogenic differentiation efficiency by two-fold over non-exposed controls. Our results imply that mesobiliverdin IXα promotes osteogenesis in ways that reflect the potential therapeutic effects of induced HO-1 expression in alleviating osteoporosis.

A Glimpse into the Diverse Cellular Immunity against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific cellular immune response has been shown to play a critical role in preventing severe illness or death in patients infected with SARS-CoV-2 or its variants. Given the multiple T-cell epitopes shared by wild-type virus and its variants, we hypothesized that vaccines that target multiple T-cell epitopes of SARS-CoV-2 may provide a "universal protection" against the wild-type virus as well as its variants, even the heavily mutated ones. To test this, we assessed SARS-CoV-2-specific T-cell precursors in healthy individuals using overlapping peptide pools of SARS-CoV-2 structural and functional proteins, including spike (S), membrane (M), envelope (E), nucleocapsid (N), and protease (P) proteins as target antigens. Diverse T-cell precursor frequencies specific to these viral antigens were detected in healthy individuals, including high, medium, low, and no responders. This was further confirmed by efficient induction of anti-SARS-CoV-2 T-cell immune responses using ex vivo dendritic cell (DC)/T cell coculture. The results demonstrated T-cell responses consistent with the precursor frequencies of each of the individuals tested. Importantly, the combination of all five viral peptide pools induced the strongest cellular immune response, and further, after a DC-peptides re-stimulation, even the no responders developed an increased anti-viral T-cell response. These analyses recapitulate the presence of a broad anti-SARS-CoV-2 cellular immunity even in an immune naïve population, which could be enhanced by antigen presenting cells presenting the overlapping antigenic peptides. Given the critical role of cellular immunity in COVID-19 protection, these results have important implications for vaccine design and immunotherapy in fighting SARS-CoV-2 and its variants.

Connexin hemichannel inhibitors with a focus on aminoglycosides.

Connexins are membrane proteins involved directly in cell-to-cell communication through the formation of gap-junctional channels. These channels result from the head-to-head docking of two hemichannels, one from each of two adjacent cells. Undocked hemichannels are also present at the plasma membrane where they mediate the efflux of molecules that participate in autocrine and paracrine signaling, but abnormal increase in hemichannel activity can lead to cell damage in disorders such as cardiac infarct, stroke, deafness, cataracts, and skin diseases. For this reason, connexin hemichannels have emerged as a valid therapeutic target. Know small molecule hemichannel inhibitors are not ideal leads for the development of better drugs for clinical use because they are not specific and/or have toxic effects. Newer inhibitors are more selective and include connexin mimetic peptides, anti-connexin antibodies and drugs that reduce connexin expression such as antisense oligonucleotides. Re-purposed drugs and their derivatives are also promising because of the significant experience with their clinical use. Among these, aminoglycoside antibiotics have been identified as inhibitors of connexin hemichannels that do not inhibit gap-junctional channels. In this review, we discuss connexin hemichannels and their inhibitors, with a focus on aminoglycoside antibiotics and derivatives of kanamycin A that inhibit connexin hemichannels, but do not have antibiotic effect.

Advances in the development of connexin hemichannel inhibitors selective toward Cx43.

Gap-junction channels formed by two connexin hemichannels play diverse and pivotal roles in intercellular communication and regulation. Normally hemichannels at the plasma membrane participate in autocrine and paracrine signaling, but abnormal increase in their activity can lead or contribute to various diseases. Selective inhibitors toward connexin hemichannels are of great interest. Among more than 20 identified isoforms of connexins, connexin 43 (Cx43) attracts the most interest due to its prevalence and link to cell damage in many disorders or diseases. Traditional antibacterial kanamycin decorated with hydrophobic groups yields amphiphilic kanamycins that show low cytotoxicity and prominent inhibitory effect against Cx43. This review focuses on the development of amphiphilic kanamycins as connexin hemichannel inhibitors and their future perspective.

Label-free discrimination and quantitative analysis of oxidative stress induced cytotoxicity and potential protection of antioxidants using Raman micro-spectroscopy and machine learning.

Diesel exhaust particles (DEPs) are major constituents of air pollution and associated with numerous oxidative stress-induced human diseases. In vitro toxicity studies are useful for developing a better understanding of species-specific in vivo conditions. Conventional in vitro assessments based on oxidative biomarkers are destructive and inefficient. In this study, Raman spectroscopy, as a non-invasive imaging tool, was used to capture the molecular fingerprints of overall cellular component responses (nucleic acid, lipids, proteins, carbohydrates) to DEP damage and antioxidant protection. We apply a novel data visualization algorithm called PHATE, which preserves both global and local structure, to display the progression of cell damage over DEP exposure time. Meanwhile, a mutual information (MI) estimator was used to identify the most informative Raman peaks associated with cytotoxicity. A health index was defined to quantitatively assess the protective effects of two antioxidants (resveratrol and mesobiliverdin IXα) against DEP induced cytotoxicity. In addition, a number of machine learning classifiers were applied to successfully discriminate different treatment groups with high accuracy. Correlations between Raman spectra and immunomodulatory cytokine and chemokine levels were evaluated. In conclusion, the combination of label-free, non-disruptive Raman micro-spectroscopy and machine learning analysis is demonstrated as a useful tool in quantitative analysis of oxidative stress induced cytotoxicity and for effectively assessing various antioxidant treatments, suggesting that this framework can serve as a high throughput platform for screening various potential antioxidants based on their effectiveness at battling the effects of air pollution on human health.

Amphiphilic aminoglycosides with increased selectivity for inhibition of connexin 43 (Cx43) hemichannels.

Gap junction channels formed by the association of connexin hemichannels play a crucial role in intercellular communication. Connexin 43 (Cx43) is expressed in a variety of tissues and organs, including heart and brain, and abnormal sustained opening of undocked "free" hemichannels contributes to the cell damage in cardiac infarcts and stroke. Selective inhibitors of Cx43 hemichannels for clinical use are then desirable. Here, we synthesized and tested new aminoglycosides for their connexin inhibitory activity towards Cx26 and Cx43 hemichannels. The lead compounds displayed enhanced Cx43/Cx26 selectivity for hemichannel inhibition when compared to the parent kanamycin A and other commercially available aminoglycosides. These lead compounds are not cytotoxic to mammalian cells and show promise for the treatment of ischemic damage of the heart, brain, and kidneys. We identified a new compound as a promising lead based on its good selectivity for Cx43 hemichannels inhibition and the simplicity and affordability of its production.

Nanoconfinement of metal oxide MgO and ZnO in zeolitic imidazolate framework ZIF-8 for CO2 adsorption and regeneration.

Microporous materials exhibit fast CO2 adsorption rate with possible sacrificed capacity, while CO2 chemisorption on metal oxides is remarkable but kinetics and reactive area are critical. In order to adopt the advantages of both microporous sorbent zeolitic imidazolate framework (ZIF) and metal oxide (MO), in this research, magnesium oxide (MgO) and zinc oxide (ZnO) were doped to ZIF-8 (MO@ZIF) using infiltration and calcination processes. The powder X-ray diffraction patterns showed retained ZIF-8 integrity after MO addition. Broad MgO peaks implied well-dispersed nanoparticles, while sharp ZnO diffractions indicated oxide agglomeration, supported by the field emission transmission electron microscope images. ZIF pore size was expanded due to confined MgO without sacrificing the framework porosity. Because of nanoconfinement, the MgO@ZIF-8 room temperature CO2 adsorption, as well as the adsorption rate constant in pseudo-second order model, were two-fold higher than expectation. In addition, the decarbonation temperature in MgO@ZIF-8 was reduced by 40 degrees. In general, it was found that metal oxide nanoconfinement in microporous zeolitic imidazolate frameworks performed improved CO2 uptake, facilitated adsorption kinetics at ambient temperature, and lowered regeneration temperature to release CO2.

Effects of Mesobiliverdin IXα-Enriched Microalgae Feed on Gut Health and Microbiota of Broilers.

Gut inflammatory bowel diseases (IBDs) links to animal medicinal feed and antibiotic-resistance are fueling major economic impacts in the agricultural livestock industry. New animal feeds that promote livestock gut health and control of IBDs without antibiotics are needed. This study investigates the effects of mesobiliverdin IXα (MBV)-enriched microalgae spirulina extracts on the growth performance, blood parameters, intestinal morphology, and gut microbiota of broilers. A total of 288 1-day-old broiler chicks (Arbor Acres) were randomly allotted to six dietary treatments (4 pens/treatment and 12 birds/pen). The dietary treatments comprised a basal diet as control (CON), basal diet plus 0.05 and 0.1% microalgae extract as low and high dose, respectively (SP1 and SP2), basal diet plus 0.05 and 0.1% MBV-enriched microalgae extract as low and high dose, respectively (MBV-SP1 and MBV-SP2), and basal diet plus 0.1% amoxicillin (AMX). All treated animals showed no significant differences in live weight, average daily gain, and feed efficiency compared to control animals. Histological examination showed that AMX treatment decreased the villi lengths of the duodenum and ileum below control villi length (P < 0.05) while MBV-SP1 and particularly MBV-SP2 increased villi lengths in the duodenum, jejunum, and ileum above AMX -treatment lengths (P < 0.05). The Firmicutes/Bacteroidetes ratio increased in the cecum of broilers fed AMX (P < 0.05) while SP2, MBV-SP1, and MBV-SP2-fed animals showed (in order) increasing ratios up to the AMX level. The abundance of bacterial species of the genus Lactobacillus increased in MBV-SP1 and MBV-SP2-fed groups including a striking increase in Lactobacillus salivarius abundance with MBV-SP2 (P < 0.05). Feeding MBV-SP1 and MBV-SP2 decreased the level of pro-inflammatory cytokine IL-6 in plasma of broilers to a greater extent than SP1 and SP2. These results reveal that MBV-enriched microalgae extracts improve the intestinal health and beneficial microflora composition of broilers.

DUSP22 suppresses prostate cancer proliferation by targeting the EGFR-AR axis.

Dual-specificity phosphatases (DUSPs) regulate the activity of various downstream kinases through serine or threonine or tyrosine dephosphorylation. Loss of function and aberrant expression of DUSPs has been implicated in cancer progression and poor survival, yet the function of DUSP22 in prostate cancer (PCa) cells is not clear. Gene Expression Omnibus and cBioPortal microarray database analyses showed that DUSP22 expression was lower in PCa tissues than normal prostate tissues, and altered DUSP22 expression was associated with shorter progression-free and disease-free survival of patients with PCa. Exogenous DUSP22 expression in LNCaP, PC3, and C4-2B PCa cells inhibited cellular proliferation and colony formation, supporting a growth inhibitory role for DUSP22 in PCa cells. DUSP22 expression significantly attenuated epidermal growth factor (EGF) receptor (EGFR) and its downstream ERK1/2 signaling by dephosphorylation. However, DUSP22 failed to suppress the growth of CWR22Rv1 and DU145 cells with elevated phosphorylated (p-)ERK1/2 levels. A serine-to-alanine mutation at position 58, a potential ERK1/2-targeted phosphorylation site in DUSP22, was sufficient to suppress growth of CWR22Rv1 cells with elevated p-ERK1/2 levels, suggesting a mutually antagonistic relationship between DUSP22 and ERK1/2 dependent on phosphorylation status. We showed that DUSP22 can suppress prostate-specific antigen gene expression through phosphatase-dependent pathways, suggesting that DUSP22 is an important regulator of the androgen receptor (AR) in PCa cells. Mechanistically, DUSP22 can interact with AR as a regulatory partner and interfere with EGF-induced AR phosphorylation at Tyr534, suggesting that DUSP22 serves as a crucial suppressor of both EGFR and AR-dependent signaling in PCa cells via dephosphorylation. Our findings indicate that loss of function of DUSP22 in PCa cells leads to aberrant activation of both EGFR-ERKs and AR signaling and ultimately progression of PCa, supporting the potential for novel therapeutic design of harnessing DUSP22 in the treatment of PCa.-Lin, H.-P., Ho, H.-M., Chang, C.-W., Yeh, S.-D., Su, Y.-W., Tan, T.-H., Lin, W.-J. DUSP22 suppresses prostate cancer proliferation by targeting the EGFR-AR axis.

Scalable and cost-effective tosylation-mediated synthesis of antifungal and fungal diagnostic 6″-Modified amphiphilic kanamycins.

Amphiphilic kanamycins bearing hydrophobic modifications at the 6″ position have attracted interest due to remarkable antibacterial-to-antifungal switches in bioactivity. In this report, we investigate a hurdle that hinders practical applications of these amphiphilic kanamycins: a cost-effective synthesis that allows the incorporation of various connecting functionalities to which the hydrophobic moieties are connected to the kanamycin core. A cost-effective tosylation enables various modifications at the 6″ position, which is scalable to a 90-g scale. The connecting functionalities, such as amine and thiol, were not the dominant factor for biological activity. Instead, the linear chain length played the decisive role. Amphiphilic kanamycin attached with tetradecyl (C14) or hexadecyl (C16) showed strong antifungal and modest antibacterial activities than with shorter chains (C6-C10). However, increases in chain length were closely correlated with an increase in HeLa cell toxicity. Thus, a compromise between the antimicrobial activities and cytotoxicities, for optimal efficacy of amphiphilic kanamycins may contain chain lengths between C8 and C12. Finally, the described synthetic protocol also allows the preparation of a fluorescent amphiphilic kanamycin selective toward fungi.

Cationic Anthraquinone Analogs as Selective Antimicrobials.

Development of new antibiotics is always needed in the fight against growing threat from multiple drug-resistant bacteria, such as resistant Gram-negative (G-) Escherichia coli and Klebsiella pneumoniae. While the development of broad-spectrum antibiotics has attracted great attention, careful administration of these antibiotics is important to avoid adverse effects, like Clostridium difficile infection (CDI). The use of broad-spectrum antibiotics, for example, quinolones, can increase the risk of CDI by eradicating the protective bacteria in intestine and encouraging C difficile spore germination. Many common intestine bacteria are G- or anaerobic, including Enterococcus faecalis, Bacteroides fragilis, and E coli. Hence, it may be advantageous in certain therapeutic practices to employ selective antimicrobials. For instance, Gram-positive (G+) methicillin-resistant Staphylococcus aureus (MRSA) that can cause life-threatening sepsis can be controlled with the use of selective antibiotic, vancomycin. Nevertheless, its effectiveness has been limited with the emerging of vancomycin-resistant Staphylococcus aureus (VRSA). A recent report on antimicrobial cationic anthraquinone analogs (CAAs) that show tunable activity and selectivity may provide new hope in the search for selective antimicrobials. In particular, the lead CAA displays prominent activity against MRSA while manifesting low activity against E coli and low cytotoxicity toward normal mammalian cells.

Antifungal Activities of 4″,6″-Disubstituted Amphiphilic Kanamycins.

Amphiphilic kanamycins derived from the classic antibiotic kanamycin have attracted interest due to their novel bioactivities beyond inhibition of bacteria. In this study, the recently described 4″,6″-diaryl amphiphilic kanamycins reported as inhibitors of connexin were examined for their antifungal activities. Nearly all 4″,6″-diaryl amphiphilic kanamycins tested had antifungal activities comparable to those of 4″,6″-dialkyl amphiphilic kanamycins, reported previously against several fungal strains. The minimal growth inhibitory concentrations (MICs) correlated with the degree of amphiphilicity (cLogD) of the di-substituted amphiphilic kanamycins. Using the fluorogenic dyes, SYTOXTM Green and propidium iodide, the most active compounds at the corresponding MICs or at 2×MICs caused biphasic dye fluorescence increases over time with intact cells. Further lowering the concentrations to half MICs caused first-order dye fluorescence increases. Interestingly, 4×MIC or 8×MIC levels resulted in fluorescence suppression that did not correlate with the MIC and plasma membrane permeabilization. The results show that 4″,6″-diaryl amphiphilic kanamycins are antifungal and that amphiphilicity parameter cLogD is useful for the design of the most membrane-active versions. A cautionary limitation of fluorescence suppression was revealed when using fluorogenic dyes to measure cell-permeation mechanisms with these antifungals at high concentrations. Finally, 4″,6″-diaryl amphiphilic kanamycins elevate the production of cellular reactive oxygen species as other reported amphiphilic kanamycins.

Development of Fungal Selective Amphiphilic Kanamycin: Cost-Effective Synthesis and Use of Fluorescent Analogs for Mode of Action Investigation.

Amphiphilic aminoglycosides have attracted interest due to their novel antifungal activities. A crucial but often neglected factor for drug development in academia is cost of production. Herein is reported a one-step, inexpensive synthesis of amphiphilic alkyl kanamycins constituted with only natural components. The synthetic methodology also enabled the preparation of a series fluorescent amphiphilic aryl kanamycins for direct structure-activity mode of action studies. The lead compounds showed prominent antifungal activities against a panel of fungi, including Fusarium graminearum, Cryptococcus neoformans, and several Candida sp., and also significant antibacterial activities. With fluorescence-based whole cell assays, the aryl amphiphilic kanamycins were observed to permeabilize fungal surface membranes at faster rates than bacterial surface membranes. Also, the antifungal action of the amphiphilic kanamycins was observed to occur in a biphasic mode with an initial fast phase correlated with rapid membrane permeabilization at subminimal inhibitory concentrations and a slower phase membrane permeabilization that elevates the reactive oxygen species production leading to cell death. Inactive hydrophobic amphiphilic kanamycins displayed no membrane permeabilization. The results offer cost-effective methods for producing amphiphilic kanamycins and reveal insights into how nonfungal specific amphiphilic kanamycins can be employed for fungal specific diagnostic and therapeutic applications.

Antifungal amphiphilic kanamycins: new life for an old drug.

Classical aminoglycoside antibiotics are obsolete or hampered by the emergence of drug resistant bacteria. Recent discoveries of antifungal amphiphilic kanamycins offer new strategies for reviving and repurposing these old drugs. A simple structural modification turns the clinically obsolete antibacterial kanamycin into an antifungal agent. Structure-activity relationship studies have led to the production of K20, an antifungal kanamycin that can be mass-produced for uses in agriculture as well as in animals. This review delineates the path to the discovery of K20 and other related antifungal amphiphilic kanamycins, determination of its mode of action, and findings in greenhouse and field trials with K20 that could lead to crop disease protection strategies.