New Genetically Engineered Derivatives of Antibacterial Darobactins Underpin Their Potential for Antibiotic Development.

Biosynthetic engineering of bicyclic darobactins, selectively sealing the lateral gate of the outer membrane protein BamA, leads to active analogues, which are up to 128-fold more potent against Gram-negative pathogens compared to native counterparts. Because of their excellent antibacterial activity, darobactins represent one of the most promising new antibiotic classes of the past decades. Here, we present a series of structure-driven biosynthetic modifications of our current frontrunner, darobactin 22 (D22), to investigate modifications at the understudied positions 2, 4, and 5 for their impact on bioactivity. Novel darobactins were found to be highly active against critical pathogens from the WHO priority list. Antibacterial activity data were corroborated by dissociation constants with BamA. The most active derivatives D22 and D69 were subjected to ADMET profiling, showing promising features. We further evaluated D22 and D69 for bioactivity against multidrug-resistant clinical isolates and found them to have strong activity.

Advanced Glycation End Products as a Potential Target for Restructuring the Ovarian Cancer Microenvironment: A Pilot Study.

Ovarian cancer is the sixth leading cause of cancer-related death in women, and both occurrence and mortality are increased in women over the age of 60. There are documented age-related changes in the ovarian cancer microenvironment that have been shown to create a permissive metastatic niche, including the formation of advanced glycation end products, or AGEs, that form crosslinks between collagen molecules. Small molecules that disrupt AGEs, known as AGE breakers, have been examined in other diseases, but their efficacy in ovarian cancer has not been evaluated. The goal of this pilot study is to target age-related changes in the tumor microenvironment with the long-term aim of improving response to therapy in older patients. Here, we show that AGE breakers have the potential to change the omental collagen structure and modulate the peritoneal immune landscape, suggesting a potential use for AGE breakers in the treatment of ovarian cancer.

Identification of Staphylococcus aureus Penicillin Binding Protein 4 (PBP4) Inhibitors.

Methicillin-resistant Staphylococcus aureus (MRSA) is a global healthcare concern. Such resistance has historically been attributed to the acquisition of mecA (or mecC), which encodes an alternative penicillin binding protein, PBP2a, with low ß-lactam affinity. However, recent studies have indicated that penicillin binding protein 4 (PBP4) is also a critical determinant of S. aureus methicillin resistance, particularly among community-acquired MRSA strains. Thus, PBP4 has been considered an intriguing therapeutic target as corresponding inhibitors may restore MRSA ß-lactam susceptibility. In addition to its role in antibiotic resistance, PBP4 has also recently been shown to be required for S. aureus cortical bone osteocyte lacuno-canalicular network (OLCN) invasion and colonization, providing the organism with a niche for re-occurring bone infection. From these perspectives, the development of PBP4 inhibitors may have tremendous impact as agents that both reverse methicillin resistance and inhibit the organism's ability to cause chronic osteomyelitis. Accordingly, using a whole-cell high-throughput screen of a 30,000-member small molecule chemical library and secondary assays we identified putative S. aureus PBP4 inhibitors. Quantitative reverse transcriptase mediated PCR and PBP4 binding assays revealed that hits could be further distinguished as compounds that reduce PBP4 expression versus compounds that are likely to affect the protein's function. We also showed that 6.25 µM (2.5 µg/mL) of the lead candidate, 9314848, reverses the organism's PBP4-dependent MRSA phenotype and inhibits its ability to traverse Microfluidic-Silicon Membrane-Canalicular Arrays (µSiM-CA) that model the OLCN orifice. Collectively, these molecules may represent promising potential as PBP4-inhibitors that can be further developed as adjuvants for the treatment of MRSA infections and/or osteomyelitis prophylactics.

Novel Nitro-Heteroaromatic Antimicrobial Agents for the Control and Eradication of Biofilm-Forming Bacteria.

The synthesis and biological activity of several novel nitrothiazole, nitrobenzothiazole, and nitrofuran containing antimicrobial agents for the eradication of biofilm-forming Gram-negative and Gram-positive pathogens is described. Nitazoxanide (NTZ), nitrofurantoin, and furazolidone are commercial antimicrobials which were used as models to show how structural modification improved activity toward planktonic bacteria via minimum inhibitory concentration (MIC) assays and biofilms via minimum biofilm eradication concentration (MBEC) assays. Structure-activity relationship (SAR) studies illustrate the ways in which improvements have been made to the aforementioned antimicrobial agents. It is of particular interest in this regard that the introduction of a chloro substituent at the 5-position of NTZ (analog 1b) resulted in marked activity enhancement, as did the replacement of the 2-acetoxy substituent in the latter compound with a basic amine group (analog 7b). It is also of importance that analog 4a, which is a simple methacrylamide, displayed noteworthy activity against S. epidermidis biofilms. These lead compounds identified to have high activity towards biofilms provide promise as starting points in future pro-drug studies.

Novel phenolic antimicrobials enhanced activity of iminodiacetate prodrugs against biofilm and planktonic bacteria.

Prodrugs are pharmacologically attenuated derivatives of drugs that undergo bioconversion into the active compound once reaching the targeted site, thereby maximizing their efficiency. This strategy has been implemented in pharmaceuticals to overcome obstacles related to absorption, distribution, and metabolism, as well as with intracellular dyes to ensure concentration within cells. In this study, we provide the first examples of a prodrug strategy that can be applied to simple phenolic antimicrobials to increase their potency against mature biofilms. The addition of (acetoxy)methyl iminodiacetate groups increases the otherwise modest potency of simple phenols. Biofilm-forming bacteria exhibit a heightened tolerance toward antimicrobial agents, thereby accentuating the need for new antibiotics as well as those, which incorporate novel delivery strategies to enhance activity toward biofilms.

Sulfenate Esters of Simple Phenols Exhibit Enhanced Activity against Biofilms.

The recalcitrance exhibited by microbial biofilms to conventional disinfectants has motivated the development of new chemical strategies to control and eradicate biofilms. The activities of several small phenolic compounds and their trichloromethylsulfenyl ester derivatives were evaluated against planktonic cells and mature biofilms of Staphylococcus epidermidis and Pseudomonas aeruginosa. Some of the phenolic parent compounds are well-studied constituents of plant essential oils, for example, eugenol, menthol, carvacrol, and thymol. The potency of sulfenate ester derivatives was markedly and consistently increased toward both planktonic cells and biofilms. The mean fold difference between the parent and derivative minimum inhibitory concentration against planktonic cells was 44 for S. epidermidis and 16 for P. aeruginosa. The mean fold difference between the parent and derivative biofilm eradication concentration for 22 tested compounds against both S. epidermidis and P. aeruginosa was 3. This work demonstrates the possibilities of a new class of biofilm-targeting disinfectants deploying a sulfenate ester functional group to increase the antimicrobial potency toward microorganisms in biofilms.

Antimicrobial Activity of Naturally Occurring Phenols and Derivatives Against Biofilm and Planktonic Bacteria.

Biofilm-forming bacteria present formidable challenges across diverse settings, and there is a need for new antimicrobial agents that are both environmentally acceptable and relatively potent against microorganisms in the biofilm state. The antimicrobial activity of three naturally occurring, low molecular weight, phenols, and their derivatives were evaluated against planktonic and biofilm Staphylococcus epidermidis and Pseudomonas aeruginosa. The structure activity relationships of eugenol, thymol, carvacrol, and their corresponding 2- and 4-allyl, 2-methallyl, and 2- and 4-n-propyl derivatives were evaluated. Allyl derivatives showed a consistent increased potency with both killing and inhibiting planktonic cells but they exhibited a decrease in potency against biofilms. This result underscores the importance of using biofilm assays to develop structure-activity relationships when the end target is biofilm.