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Mineralocorticoid receptor (MR) antagonists continue to be a prevalent area of research in the pharmaceutical industry. Herein we report the discovery of various spirooxindole and dibenzoxazepine constructs as potent MR antagonists. SAR analysis of our spirooxindole hit led to highly potent compounds containing polar solubilizing groups, which interact with the helix-11 region of the MR ligand binding domain (LBD). Various dibenzoxazepine moieties were also prepared in an effort to replace a known dibenzoxepane system which interacts with the hydrophobic region of the MR LBD. In addition, an X-ray crystal structure was obtained from a highly potent compound which was shown to exhibit both partial agonist and antagonist modes of action against MR.

Liver X receptor (LXR) agonists have been reported to lower brain amyloid beta (Aβ) and thus to have potential for the treatment of Alzheimer's disease. Structure and property based design led to the discovery of a series of orally bioavailable, brain penetrant LXR agonists. Oral administration of compound 18 to rats resulted in significant upregulation of the expression of the LXR target gene ABCA1 in brain tissue, but no significant effect on Aβ levels was detected.

Quorum-sensing bacteria communicate via small molecules called autoinducers to coordinate collective behaviors. Because quorum sensing controls virulence factor expression in many clinically relevant pathogens, membrane-permeable quorum sensing antagonists that prevent population-wide expression of virulence genes offer a potential route to novel antibacterial therapeutics. Here, we report a strategy for inhibiting quorum-sensing receptors of the widespread LuxR family. Structure-function studies with natural and synthetic ligands demonstrate that the dimeric LuxR-type transcription factor CviR from Chromobacterium violaceum is potently antagonized by molecules that bind in place of the native acylated homoserine lactone autoinducer, provided that they stabilize a closed conformation. In such conformations, each of the two DNA-binding domains interacts with the ligand-binding domain of the opposing monomer. Consequently, the DNA-binding helices are held apart by ~60 Å, twice the ~30 Å separation required for operator binding. This approach may represent a general strategy for the inhibition of multi-domain proteins.

Many Gram-negative pathogens utilize type III secretion systems (T3SS) to translocate effector proteins into eukaryotic host cells. Expression of T3SS genes is highly regulated and is often coupled to type III secretory activity. Transcription of the Pseudomonas aeruginosa T3SS genes is coupled to secretion by a cascade of interacting regulatory proteins (ExsA, ExsD, ExsC, and ExsE). ExsA is an activator of type III gene transcription, ExsD binds ExsA to inhibit transcription, ExsC inhibits ExsD activity, and ExsE inhibits ExsC activity. The entire process is coupled to secretion by virtue of the fact that ExsE is a secreted substrate of the T3SS. Changes in the intracellular concentration of ExsE are thought to govern formation of the ExsC-ExsE, ExsC-ExsD, and ExsD-ExsA complexes. Whereas formation of the ExsC-ExsE complex allows ExsD to bind ExsA and transcription of the T3SS is repressed, formation of the ExsC-ExsD complex sequesters ExsD from ExsA and transcription of the T3SS is induced. In this study, we characterized the self-association states of ExsC, ExsD, and ExsE and the binding interactions of ExsC with ExsE and ExsD. ExsC exists as a homodimer and binds one molecule of ExsE substrate. Dimeric ExsC also interacts directly with ExsD to form a heterotetrameric complex. The difference in binding affinities between the ExsC-ExsE (K(d) 1 nm) and ExsC-ExsD (K(d) 18 nm) complexes supports a model in which ExsC preferentially binds cytoplasmic ExsE, resulting in the inhibition of T3SS gene transcription.

The flagellum and injectisome of gram-negative bacteria are large multiprotein organelles spanning the inner and outer membranes (12). Whereas flagella are involved in motility, injectisomes function by injecting toxins into eukaryotic host cells. The biogenesis of both organelles requires a dedicated protein export system referred to as a type III secretion system (T3SS). Many of the individual protein constituents of flagella and injectisomes are first secreted via the T3SS before assembly into the complete organelles (10). The flagellar T3SS is located at the base of the basal body in the inner membrane, where it secretes proteins required for assembly of the flagellar rod, hook, and filament (12). The T3SS associated with the injectisome is also located at the base of a basal body-like structure and secretes proteins required for injectisome assembly as well as the toxins destined for delivery to host cells (10). An interesting regulatory feature of both the flagellum and injectisome systems is the direct coupling of transcription with secretion (16). The prototypical example of this regulatory mechanism is seen in Salmonella spp., where genes required for assembly of the flagellar basal body, hook, and filament are expressed in a temporal hierarchy (1). The late genes, required for the final stage of flagellar biogenesis, are not expressed until the basal body is completely assembled. Prior to completion of the basal body complex, the FlgM anti-σ factor accumulates in the cytoplasm, where it binds to and inhibits the FliA σ factor required for late gene transcription. Upon completion of the basal body complex, however, there is a switch in the substrate specificity of the T3SS, FlgM is secreted from cells, and FliA-dependent transcription of late genes ensues. The coupling of transcription to secretion provides an elegant means of coordinating gene expression with the different stages of the flagellar assembly process. Secretion competence also serves as an inducing signal for transcription in the injectisome system (16). Unlike the case in the flagellar system, however, where FlgM secretion is linked to basal body assembly, secretion competence in many injectisome systems is controlled by environmental signals (2, 14, 15, 18, 19, 27). Through a poorly understood mechanism, these environmental signals convert the type III secretion machinery from a closed (secretion incompetent) conformation to an open (secretion competent) conformation. Conversion to the open state triggers a regulatory cascade resulting in transcriptional activation. Mechanistically, these regulatory cascades fall into one of the following three general categories: (i) secretion of a negative regulatory factor, seen in Yersinia spp.; (ii) sequestration of a coactivator, as reported for Salmonella and Shigella spp.; and (iii) sequestration of a negative regulatory factor, as recently described for Pseudomonas aeruginosa (6, 7, 13, 14, 19-21). Transcription of the P. aeruginosa T3SS is induced under Ca2+-limiting growth conditions or following contact of the bacterium with a host cell (9, 22). Although the mechanism of activation by host cell contact is unclear, the low-Ca2+ signal converts the T3SS machinery from a secretion-incompetent to a secretion-competent state (14). Recent studies have demonstrated that transcription of the T3SS is intimately linked to the activity of the type III secretion machinery. Transcription is repressed when the T3SS machinery is secretion incompetent and is derepressed when the machinery is secretion competent (7, 14, 21). The mechanism of coupling transcription to secretion involves a cascade of four interacting regulatory proteins (ExsA, ExsD, ExsC, and ExsE). ExsA is a positive activator of T3SS transcription, while ExsD functions as an antiactivator by binding to and inhibiting ExsA activity (11, 14, 25, 26). ExsC functions as an anti-antiactivator by binding to and inhibiting the negative regulatory activity of ExsD and also as a type III-specific chaperone for ExsE (7, 21). Finally, ExsE binds to ExsC and inhibits its activity (20, 21). A key feature of the system is that ExsE is secreted from cells under low-Ca2+ conditions. Based on these findings, the following model has been proposed to account for the coupling of transcription with secretion (7, 21). Under high-Ca2+ conditions, intracellular ExsE binds to and sequesters ExsC, and ExsD is preferentially bound to ExsA, resulting in the inhibition of ExsA-dependent transcription. Under low-Ca2+ conditions, however, ExsE is secreted from cells. This results in a shift in the binding equilibrium whereby ExsD is preferentially bound to ExsC, and liberated ExsA is made available to activate transcription of the T3SS. In the present study, we characterize the ExsC-ExsD binding interaction. Both ExsC and ExsD were found to form self-associated multimeric complexes. ExsC and ExsD readily form a complex when coexpressed in vivo or when individually purified and mixed in vitro. Biochemical analyses suggest that formation of the complex is thermodynamically favorable and consists of multiple copies of ExsC and ExsD. Finally, an ExsD mutant unable to interact with ExsC was isolated and found to have a hyperrepressive phenotype.

The quorum-sensing transcriptional activator TraR of Agrobacterium tumefaciens, which controls the replication and conjugal transfer of the tumour-inducing (Ti) virulence plasmid, is inhibited by the TraM antiactivator. The crystal structure of TraM reveals this protein to form a homodimer in which the monomer primarily consists of two long coiled alpha-helices, and one of the helices from each monomer also bundles to form the dimeric interface. The importance of dimerization is addressed by mutational studies in which disruption of the hydrophobic dimer interface leads to aggregation of TraM. Biochemical studies confirm that TraM exists as a homodimer in solution in equilibrium with the monomeric form, and also establish that the TraM-TraR complex is a heterodimer. Thus, the TraM homodimer undergoes dissociation in forming the antiactivation complex. Combined with the structure of TraR (Zhang et al., 2002, Nature 417: 971-974; Vannini et al., 2002, EMBO J 21: 4393-4401), our structural analysis suggests overlapping interactive surfaces in homodimeric TraM with those in the TraM-TraR complex and a mechanism for TraM inhibition on TraR.

Bacteria can communicate via diffusible signal molecules they generate and release to coordinate their behavior in response to the environment. Signal molecule concentration is often proportional to bacterial population density, and when this reaches a critical concentration, reflecting a bacterial quorum, specific behaviors including virulence, symbiosis, and horizontal gene transfer are activated. Quorum-sensing regulation in many Gram-negative bacteria involves acylated homoserine lactone signals that are perceived through binding to LuxR-type, acylated-homoserine-lactone-responsive transcription factors. Bacteria of the rhizobial group employ the LuxR-type transcriptional activator TraR in quorum sensing, and its activity is further regulated through interactions with the TraM antiactivator. In this study, we have crystallographically determined the 3D structure of the TraR–TraM antiactivation complex from Rhizobium sp. strain NGR234. Unexpectedly, the antiactivator TraM binds to TraR at a site distinct from its DNA-binding motif and induces an allosteric conformational change in the protein, thereby preventing DNA binding. Structural analysis reveals a highly conserved TraR–TraM interface and suggests a mechanism for antiactivation complex formation. This structure may inform alternative strategies to control quorum-sensing-regulated microbial activity including amelioration of infectious disease and antibiotic resistance. In addition, the structural basis of antiactivation presents a regulatory interaction that provides general insights relevant to the field of transcription regulation and signal transduction.