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Multi-target inhibitors have become increasing popular as a means to leverage the advantages of poly-pharmacology while simplifying drug delivery. Here, we describe dual inhibitors for soluble epoxide hydrolase (sEH) and fatty acid amide hydrolase (FAAH), two targets known to synergize when treating inflammatory and neuropathic pain. The structure activity relationship (SAR) study described herein initially started with t-TUCB (trans-4-[4-(3-trifluoromethoxyphenyl-l-ureido)-cyclohexyloxy]-benzoic acid), a potent sEH inhibitor that was previously shown to weakly inhibit FAAH. Inhibitors with a 6-fold increase of FAAH potency while maintaining high sEH potency were developed by optimization. Interestingly, compared to most FAAH inhibitors that inhibit through time-dependent covalent modification, t-TUCB and related compounds appear to inhibit FAAH through a time-independent, competitive mechanism. These inhibitors are selective for FAAH over other serine hydrolases. In addition, FAAH inhibition by t-TUCB appears to be higher in human FAAH over other species; however, the new dual sEH/FAAH inhibitors have improved cross-species potency. These dual inhibitors may be useful for future studies in understanding the therapeutic application of dual sEH/FAAH inhibition.

Diabetes is affecting the life of millions of people. A large proportion of diabetic patients suffer from severe complications such as neuropathic pain, and current treatments for these complications have deleterious side effects. Thus, alternate therapeutic strategies are needed. Recently, the elevation of epoxy-fatty acids through inhibition of soluble epoxide hydrolase (sEH) was shown to reduce diabetic neuropathic pain in rodents. In this report, we describe a series of newly synthesized sEH inhibitors with at least 5-fold higher potency and doubled residence time inside both the human and rodent sEH enzyme than previously reported inhibitors. These inhibitors also have better physical properties and optimized pharmacokinetic profiles. The optimized inhibitor selected from this new series displayed improved efficacy of almost 10-fold in relieving pain perception in diabetic neuropathic rats as compared to the approved drug, gabapentin, and previously published sEH inhibitors. Therefore, these new sEH inhibitors could be an attractive alternative to treat diabetic neuropathy in humans.

Specialized pro‐resolving mediators (SPM), primarily produced in innate immune cells, exert crucial bioactions for resolving inflammation. Among various lipoxygenases (LOX), 15‐LOX‐1 is key for SPM biosynthesis, but cellular activation principles of 15‐LOX‐1 are unexplored. It was shown that 3‐O‐acetyl‐11‐keto‐β‐boswellic acid (AKBA) shifts 5‐LOX regiospecificity from 5‐ to 12‐lipoxygenation products. Here, it is demonstrated that AKBA additionally activates cellular 15‐LOX‐1 via an allosteric site accomplishing robust SPM formation in innate immune cells, particularly in M2 macrophages. Compared to ionophore, AKBA‐induced LOX activation is Ca2+‐ and phosphorylation‐independent, with modest induction of 5‐LOX products. AKBA docks into a groove between the catalytic and regulatory domains of 15‐LOX‐1 interacting with R98; replacement of R98 by alanine abolishes AKBA‐induced 15‐LOX product formation in HEK293 cells. In zymosan‐induced murine peritonitis, AKBA strikingly elevates SPM levels and promotes inflammation resolution. Together, targeted allosteric modulation of LOX activities governs SPM formation and offers new concepts for inflammation resolution pharmacotherapy. [ABSTRACT FROM AUTHOR]

Research was conducted to examine the crystal structure of retinal dehydrogenase type II complexed with nicotinamide adenine dinucleotide at 2.7 angstroms resolution using X-ray crystallography. The molecular replacement using the crystal structure of a mitochondrial aldehyde dehydrogenase (ADLDH2) as a model solved the structure. The enzyme apparently uses a disordered stretch of approximately 20 amino acids to limit access to the catalytic site by smaller aldehydes.

The crystollographic structure of human retinol-binding protein (RBP) and transthyretin (TTR) was examined via the use of four amino acids: Val-20, Arg-21, Ile-84 and Leu-82. The results revealed an interaction, which involves Leu-183 and -182 of RBP, of the carboxy terminus of RBP at the protein-protein recognition interface. Prevention of the extensive loss of RBP via glomerular filtration was attributed to complex formation. Moreover, a decrease affinity of RBP for TTR was attributed to the loss of Leu-182 and -183.

A study examined the interaction of a hydrogen bond donor and an aromatic ring receptor in a highly homologous pair of proteins, cellular retinol-binding protein (CRBP) and cellular retinol-binding protein, type II (CRBP II). Both proteins attach the ligand all-trans-retinal with corresponding affinities, but CRBP has more or less 100-fold higher affinity for all-trans-retinol. The greater affinity of CRBP is due to the presence of an amino-aromatic hydrogen bond, which is not found in CRBP(II). Furthermore, the mutant and wild-type CRBP show no preferences for binding all-trans-retinol versus all-trans-retinal.

Two intracellular retinoid-binding proteins, a cellular retinol-binding protein type II, or CRBP(II), and cellular retinol-binding protein (CRBP) react with microsomal enzymes in different ways. The CRBP and CRBP(II) are compact, small and tightly-folded and deliver retinoids to microsomal metabolic enzymes. The difference in the reactions are caused by variation in the structure of the proteins.

Retinoic acid-binding protein from rat epididymis (E-RABP) isomer specificity which was shown by the form of electron density of the ligand in a X-ray crystallographic study, was proved by an experiment. A technique through which the protein-binding region can detect the all-trans- and 9-cis-retinoic acid while sustaining various similar protein-ligand contacts, was indicated by the 'folding' of the all-trans-retinoic acid isoprene tail. Retinoic acids adopt a shape in which the tail is 'folded' when it binds to E-RABP.

The characterization and high resolution structure of a protein that exhibits 60% sequence identity to the plasmid-encoded FosA but is encoded in the genome of the opportunistic pathogen Pseudomonas aeruginosa, is described. The structure is solved exploiting the monovalent cation-binding site for multi-wavelength anomalous diffraction (MAD) phasing with thallium(I).

The article studies 1.85 [angstrom] resolution structure of an 8R-lipoxygenase from Plexaura homomalla, an enzyme with a sequence [approximate]40% identical to that of human 5-LOX. Findings reveal that mutations within the channel significantly affect enzyme activity.

The article explains the functions of the naturally occurring bifunctional protein covalent linker obtained from Plexaura homomalla that links the sequential catalytic activities in an oxylipin biosynthetic complex. The absence of the discussed linker is shown to lead to the formation of a complex that recapitulates the structure of the bifunctional protein.

The structure and mechanisms of action of the genomically encoded FosX enzyme, the fosfomycin resistance protein obtained from Listeria monocytogenes, are described. The results have indicated that both the EGD and ATCC strains of Listeria monocytogenes have an intrinsic resistance to the antibiotic provided by the FosX enzyme.

The structure of the homodimeric state of the Prp19 U-box is determined by X-ray crystallography and it is shown that the U-box domain of Prp19 exists as a dimer within the context of the Prp19 tetramer. The comparison of the Prp19 U-box homodimer with the heterodimeric complex of BRCA1/ BARD1 RING-finger domains shows a common architecture for a family of oligomeric U-box and RING-finder E3 ubiquitin ligases.

Fosfomycin is a simple phosphonate found to have antibacterial activity against both Gram-positive and Gram-negative microorganisms. The study of the FosA-catalyzed reaction reveals that the early transition state and phosphonoformate acts as a minimal transition state analogue inhibitor.

2.3 Armstrong resolution crystal structure of Escherichia coli IscA, the protein responsible for iron-sulfur cluster assembly, is explained. Descriptions reveal that the great majority of amino acids that are conserved in IscA homologues are located in elements that constitute a well-ordered fold.

The crystal structure of the FosX protein from M. loti and Listeria is determined to explain the mechanistic basis of the hydration reaction of fosfomycin. Comparison of antibiotic resistance of FosX, FosA, and Fos B enzymes suggests evolution of fosfomycin resistance protein in Gram-positive and Gram-negative microorganisms.