Vitamin A homeostasis is critical to normal cellular function. Retinol-binding protein (RBP) is the sole specific carrier in the bloodstream for hydrophobic retinol, the main form in which vitamin A is transported. The integral membrane receptor STRA6 mediates cellular uptake of vitamin A by recognizing RBP-retinol to trigger release and internalization of retinol. We present the structure of zebrafish STRA6 determined to 3.9-angstrom resolution by single-particle cryo-electron microscopy. STRA6 has one intramembrane and nine transmembrane helices in an intricate dimeric assembly. Unexpectedly, calmodulin is bound tightly to STRA6 in a noncanonical arrangement. Residues involved with RBP binding map to an archlike structure that covers a deep lipophilic cleft. This cleft is open to the membrane, suggesting a possible mode for internalization of retinol through direct diffusion into the lipid bilayer.

2-aminoimidazoles (2AIs) have been documented to disrupt bacterial protection mechanisms, including biofilm formation and genetically-encoded antibiotic resistance traits. Using Acinetobacter baumannii, we provide initial insight into the mechanism of action of a 2AI-based antibiofilm agent. Confocal microscopy confirmed that the 2AI is cell permeable, while pull-down assays identified BfmR, a response regulator that is the master controller of biofilm formation, as a target for this compound. Binding assays demonstrated specificity of the 2AI for response regulators, while computational docking provided models for 2AI/BfmR interactions. The 2AI compound studied here represents a unique small molecule scaffold that targets bacterial response regulators.

Bacterial biofilms are surface-attached communities of microorganisms that are protected by an extracellular matrix of biomolecules. In the biofilm state, bacteria are significantly more resistant to external assault, including attack by antibiotics. In their native environment, bacterial biofilms underpin costly biofouling that wreaks havoc on shipping, utilities, and offshore industry. Within a host environment, they are insensitive to antiseptics and basic host immune responses. It is estimated that up to 80% of all microbial infections are biofilm-based. Biofilm infections of indwelling medical devices are of particular concern, since once the device is colonized, infection is almost impossible to eliminate. Given the prominence of biofilms in infectious diseases, there is a notable effort towards developing small, synthetically available molecules that will modulate bacterial biofilm development and maintenance. Here, we highlight the development of small molecules that inhibit and/or disperse bacterial biofilms specifically through non-microbicidal mechanisms. Importantly, we discuss several sets of compounds derived from marine sponges that we are developing in our labs to address the persistent biofilm problem. We will discuss: discovery/synthesis of natural products and their analogues—including our marine sponge-derived compounds and initial adjuvant activity and toxicological screening of our novel anti-biofilm compounds.

Bacteria have developed numerous protection strategies to ensure survival in harsh environments, with perhaps the most robust method being the formation of a protective biofilm. In biofilms, bacterial cells are embedded within a matrix that is composed of a complex mixture of polysaccharides, proteins and DNA. The Gram-positive bacterium Bacillus subtilis has become a model organism for studying regulatory networks directing biofilm formation. The phenotypic transition from a planktonic to biofilm state is regulated by the activity of the transcriptional repressor, SinR, and its inactivation by its primary antagonist, SinI. In this work, we present the first full-length structural model of tetrameric SinR using a hybrid approach combining highresolution solution NMR, chemical crosslinking, mass spectrometry, and molecular docking. We also present the solution NMR structure of the antagonist SinI dimer, and probe the mechanism behind the SinR-SinI interaction using a combination of biochemical and biophysical techniques. As a result of these findings, we propose that SinI utilizes a residue replacement mechanism to block SinR multimerization, resulting in diminished DNA binding and concomitant decreased *

The synthesis and antibiofilm activities of sulfonamide, urea, and thiourea oroidin analogues are described. The most active derivative was able to selectively inhibit P. aeruginosa biofilm development and is also shown to be non-toxic upwards of 1 mM to the development of C. elegans in comparison to other similar isosteric analogues and the natural product oroidin.Bacterial biofilm formation is often described as a developmental process initiated when free floating (planktonic) bacteria adhere to a surface suitable for growth and initiate the formation of a microcolony. 1 Occupation of a biofilm growth state confers to the bacteria a unique set of phenotypic traits which include resistance to microbicides and antibiotics that would often lead to eradication. [2][3][4] In a medical setting, biofilms pose a serious threat to individuals who suffer from a myriad of diseases. Recent estimates have attributed biofilm-associated infections as being responsible for upwards of 75% of microbial infections in the human body. 5 This problem is further exacerbated by the increased spread of antibiotic resistance. Additionally, biofilms are known to infect patients with indwelling medical devices (IMDs a ) such as catheters and heart stents. 6-8 Remediation of biofilm infected IMDs is traditionally accomplished by device removal due to the lack of antibiotic efficacy.As the medical community works towards new approaches aimed at combating the deleterious effects of biofilms, one area that has garnered significant attention is the identification of nonmicrobicidal modulators of biofilm growth and maintenance (Figure 1). 9-13 By not directly killing bacteria, it is postulated that development of resistance to these molecules would be mitigated or significantly impaired. Implementation of remediation therapies which focus on the co-dosing of an antibiofilm modulator with an antibiotic also provides an attractive avenue for treatment. One of the few naturally occurring scaffolds shown to possess non-microbicidal antibiofilm properties are compounds derived from the oroidin class of natural products. Oroidin 5 has been reported to be a moderate inhibitor of Pseudomonas aeruginosa PAO1/ PA14 biofilm growth (PAO1 IC 50 = 190 μM, PA14 IC 50 = 166 μM). 14 *To whom correspondence should be addressed. We have recently begun to focus on the development of methodologies to access oroidin analogues for antibiofilm screening. [15][16][17] Of these approaches, development of conditions for a generic reductive acylation reaction has allowed access to previously unattainable oroidin derivatives through the use of acid chlorides, anhydrides, succinimide esters, and trichloromethylketone pyrroles. 18 In a continued effort, we sought to further apply this approach in generating isosteric analogues possessing sulfonamide, urea and thiourea functionalities to further probe the structure-activity relationships (SAR) of the oroidin family in the context of antibiofilm activity and preliminary toxicity.The Gram-negative γ-proteobacteria Pseu...

Acinetobacter baumannii has quickly become one of the most insidious and prevalent nosocomial infections. Recently, the reverse-amide class of 2-aminoimidazole compounds (RA-2AI) was found both to prevent A. baumannii biofilm formation and also to disperse preexisting formations, putatively through interactions with cytosolic response regulators. Here we focus on how this class of antibiofilm agent traverses cellular membranes. Following the discovery of dosage-dependent growth rate changes, the cellular effects of RA-2AI were investigated using a combination of molecular assays and microscopic techniques. It was found that RA-2AI exposure has measureable effects on the bacterial membranes, resulting in a period of increased permeability and visible structural aberrations. Based on these results, we propose a model that describes how the structure of RA-2AI allows it to insert itself into and disrupt the fluidity of the membrane, creating an opportunity for increased molecular permeability.

Biofilm formation is a ubiquitous bacterial defense mechanism and has been shown to be a primary element in the antibiotic resistance of many human diseases, especially in the case of nosocomial infections. Recently, we have developed several compound libraries that are extremely effective at both dispersing preexisting biofilms and also inhibiting their initial formation. In addition to their antibiofilm properties, some of these molecules are able to resensitize resistant bacterial strains to previously ineffective antibiotics and are being assessed as adjuvants. In this study, we evaluated the toxic effects of three of our most effective 2-aminoimidazole compounds (dihydrosventrin, RA, and SPAR) using a rapid pipeline that combines a series of assays. A methylthiazolyldiphenyl-tetrazolium assay, using the HaCaT keratinocyte cell line was used to determine epidermal irritants and was combined with Caenorhabditis elegans fecundity assays that demonstrated the effects of environmental exposure to various concentrations of these molecules. In each case, the assays showed that the compounds did not exhibit toxicity until they reached well above their current biofilm dispersion/inhibition concentrations. The most effective antibiofilm compound also had significant effects when used in conjunction with several standard antibiotics against resistant bacteria. Consequently, it was further investigated using the C. elegans assay in combination with different antibiotics and was found to maintain the same low level of toxicity as when acting alone, bolstering its candidacy for further testing as an adjuvant.

Bacillus subtilis is a soil-dwelling Gram-positive bacterial species that has been extensively studied as a model of biofilm formation and stress-induced cellular differentiation. The tetrameric protein, SinR, has been identified as a master regulator for biofilm formation and linked to the regulation of the early transition states during cellular stress response, such as motility and biofilm-linked biosynthetic genes. SinR is a 111-residue protein that is active as a dimer of dimers, composed of two distinct domains, a DNA-binding helix-turn-helix N-terminus domain and a C-terminal multimerization domain. In order for biofilm formation to proceed, the antagonist, SinI, must inactivate SinR. This interaction results in a dramatic structural rearrangement of both proteins. Here we report the full-length backbone and side chain chemical shift values in addition to the experimentally derived secondary structure predictions as the first step towards directly studying the complex interaction dynamics between SinR and SinI.