Nature-Inspired Peptide Antifouling Biocide: Coating Compatibility, Field Validation, and Environmental Stability.

This study reports the development of a class of eco-friendly antifouling biocides based on a cyclic dipeptide scaffold, 2,5-diketopiperazine (2,5-DKP). The lead compound cyclo(N-Bip-l-Arg-N-Bip-l-Arg) (1) was synthesized in gram amounts and used to assess the compatibility with an ablation/hydration coating, efficacy against biofouling, and biodegradation. Leaching of 1 from the coating into seawater was assessed via a rotating drum method, revealing relatively stable and predictable leaching rates under dynamic shear stress conditions (36.1 ± 19.7 to 25.2 ± 9.1 ng-1 cm-2 day-1) but low or no leaching under static conditions. The coatings were further analyzed using time-of-flight secondary ion mass spectrometry (ToF-SIMS), with 1 seen to localize at the surface of the coating in a surfactant-like fashion. When coatings were deployed in the ocean, detectable reductions in biofouling development were measured for up to 11 weeks. After this time, biofouling overwhelmed the performance of the coating, consistent with leaching kinetics. Biodegradation of 1 in seawater was assessed using theoretical oxygen demand and analytical quantification. Masking effects were observed at higher concentrations of 1 due to antimicrobial properties, but half-lives were calculated ranging from 13.4 to 16.2 days. The results can rationally inform future development toward commercial antifouling products.

Disruption of Metallostasis in the Anaerobic Human Pathogen Fusobacterium nucleatum by the Zinc Ionophore PBT2.

The Gram-negative anaerobe Fusobacterium nucleatum is an opportunistic human pathogen, most frequently associated with periodontal disease through dental biofilm formation and, increasingly, with colorectal cancer development and progression. F. nucleatum infections are routinely treated by broad-spectrum ß-lactam antibiotics and metronidazole. However, these antibiotics can negatively impact the normal microflora. Therefore, the development of novel narrow-spectrum antimicrobials active against anaerobic pathogens is of great interest. Here, we examined the antimicrobial Zn ionophore PBT2, an 8-hydroxyquinoline analogue with metal chelating properties, against a single type isolate F. nucleatum ATCC 25586. PBT2-Zn was a potent inhibitor of growth and exhibited synergistic bactericidal (>3-log10 killing) activity at 5× MIC in planktonic cells, and at the MIC in biofilms grown in vitro. Physiological and transcriptional analyses uncovered a strong cellular response relating to Zn and Fe homeostasis in PBT2-Zn treated cells across subinhibitory and inhibitory concentrations. At 1× MIC, PBT2 alone induced a 3.75-fold increase in intracellular Zn, whereas PBT2-Zn challenge induced a 19-fold accumulation of intracellular Zn after 2 h. A corresponding 2.1-fold loss of Fe was observed at 1× MIC. Transcriptional analyses after subinhibitory PBT2-Zn challenge (0.125 µg/mL and 200 µM ZnSO4) revealed significant differential expression of 15 genes at 0.5 h, and 12 genes at 1 h. Upregulated genes included those with roles in Zn homeostasis (e.g., a Zn-transporting ATPase and the Zn-sensing transcriptional regulator, smtB) and hemin transport (hmuTUV) to re-establish Fe homeostasis. A concentration-dependent protective effect was observed for cells pretreated with hemin (50 µg/mL) prior to PBT2-Zn challenge. The data presented here supports our proposal that targeting the disruption of metallostasis by Zn-translocating ionophores is a strategy worth investigating further for the treatment of Gram-negative anaerobic pathogens.

'Tethering' fragment-based drug discovery to identify inhibitors of the essential respiratory membrane protein type II NADH dehydrogenase.

Energy generation is a promising area of drug discovery for both bacterial pathogens and parasites. Type II NADH dehydrogenase (NDH-2), a vital respiratory membrane protein, has attracted attention as a target for the development of new antitubercular and antimalarial agents. To date, however, no potent, specific inhibitors have been identified. Here, we performed a site-directed screening technique, tethering-fragment based drug discovery, against wild-type and mutant forms of NDH-2 containing engineered active-site cysteines. Inhibitory fragments displayed IC50 values between 3 and 110 µM against NDH-2 mutants. Possible binding poses were investigated by in silico modelling, providing a basis for optimisation of fragment binding and improved potency against NDH-2.

Combretastatin-like chalcones as inhibitors of microtubule polymerization. Part 1: synthesis and biological evaluation of antivascular activity.

The alpha-methyl chalcone SD400 is a potent inhibitor of tubulin assembly and possesses potent anticancer activity. Various chalcone analogues were synthesized and evaluated for their cell growth inhibitory properties against the K562 human chronic myelogenous leukemia cell line (SD400, IC(50) 0.21nM; combretastatin A4 CA4, IC(50) 2.0nM). Cell cycle analysis by flow cytometry indicated that these agents are antimitotic (SD400, 83% of the cells are in G(2)/M phase; CA4 90%). They inhibit tubulin assembly at low concentration (SD400, IC(50) 0.46microM; CA4, 0.10microM) and compete with [(3)H]colchicine for binding to tubulin (8% [(3)H]colchicine remained bound to tubulin after competition with SD400 or CA4). Upon treatment with SD400, remarkable cell shape changes were elicited in HUVEC cells, consistent with vasculature damaging activity.

The total synthesis of an aurone isolated from Uvaria hamiltonii: aurones and flavones as anticancer agents.

The naturally occurring aurone 1, isolated from Uvaria hamiltonii, and a series of aurones analogues based structurally on known tubulin binding agents were prepared and evaluated for anticancer activity. Aurone 20 was the most active (IC(50) K562 50 nM) and caused significant G2/M cell-cycle arrest.