Pb(II) coordination to the nonclassical zinc finger tristetraprolin: retained function with an altered fold.

Tristetraprolin (TTP) is a nonclassical CCCH zinc finger (ZF) that plays a crucial role in regulating inflammation. TTP regulates cytokine mRNAs by specific binding of its two conserved ZF domains (CysX8CysX5CysX3His) to adenylate-uridylate-rich sequences (AREs) at the 3'-untranslated region, leading to degradation of the RNA. Dysregulation of TTP in animal models has demonstrated several cytokine-related syndromes, including chronic inflammation and autoimmune disorders. Exposure to Pb(II), a prevalent environmental toxin, is known to contribute to similar pathologies, in part by disruption of and/or competition with cysteine-rich metalloproteins. TTP's role during stress as a ubiquitous translational regulator of cell signaling (and dysfunction), which may underpin various phenotypes of Pb(II) toxicity, highlights the importance of understanding the interaction between TTP and Pb(II). The impact of Pb(II) binding on TTP's fold and RNA-binding function was analyzed via UV-Vis spectroscopy, circular dichroism, X-ray absorption spectroscopy, nuclear magnetic resonance spectroscopy, and fluorescence anisotropy. A construct containing the two ZF domains of TTP (TTP-2D) bound to Pb(II) with nanomolar affinity and exhibited a different geometry and fold in comparison to Zn2-TTP-2D. Despite the altered secondary structure, Pb(II)-substituted TTP-2D bound a canonical ARE sequence more selectively than Zn2-TTP-2D. Taken together, these data suggest that Pb(II) may interfere with proper TTP regulation and hinder the cell's ability to respond to inflammation.

Mechanistic Analysis of an Extracellular Signal-Regulated Kinase 2-Interacting Compound that Inhibits Mutant BRAF-Expressing Melanoma Cells by Inducing Oxidative Stress.

Constitutively active extracellular signal-regulated kinase (ERK) 1/2 signaling promotes cancer cell proliferation and survival. We previously described a class of compounds containing a 1,1-dioxido-2,5-dihydrothiophen-3-yl 4-benzenesulfonate scaffold that targeted ERK2 substrate docking sites and selectively inhibited ERK1/2-dependent functions, including activator protein-1-mediated transcription and growth of cancer cells containing active ERK1/2 due to mutations in Ras G-proteins or BRAF, Proto-oncogene B-RAF (Rapidly Acclerated Fibrosarcoma) kinase. The current study identified chemical features required for biologic activity and global effects on gene and protein levels in A375 melanoma cells containing mutant BRAF (V600E). Saturation transfer difference-NMR and mass spectrometry analyses revealed interactions between a lead compound (SF-3-030) and ERK2, including the formation of a covalent adduct on cysteine 252 that is located near the docking site for ERK/FXF (DEF) motif for substrate recruitment. Cells treated with SF-3-030 showed rapid changes in immediate early gene levels, including DEF motif-containing ERK1/2 substrates in the Fos family. Analysis of transcriptome and proteome changes showed that the SF-3-030 effects overlapped with ATP-competitive or catalytic site inhibitors of MAPK/ERK Kinase 1/2 (MEK1/2) or ERK1/2. Like other ERK1/2 pathway inhibitors, SF-3-030 induced reactive oxygen species (ROS) and genes associated with oxidative stress, including nuclear factor erythroid 2-related factor 2 (NRF2). Whereas the addition of the ROS inhibitor N-acetyl cysteine reversed SF-3-030-induced ROS and inhibition of A375 cell proliferation, the addition of NRF2 inhibitors has little effect on cell proliferation. These studies provide mechanistic information on a novel chemical scaffold that selectively regulates ERK1/2-targeted transcription factors and inhibits the proliferation of A375 melanoma cells through a ROS-dependent mechanism. SIGNIFICANCE STATEMENT: Constitutive activation of the extracellular signal-regulated kinase (ERK1/2) pathway drives the proliferation and survival of many cancer cell types. Given the diversity of cellular functions regulated by ERK1/2, the current studies have examined the mechanism of a novel chemical scaffold that targets ERK2 near a substrate binding site and inhibits select ERK functions. Using transcriptomic and proteomic analyses, we provide a mechanistic basis for how this class of compounds inhibits melanoma cells containing mutated BRAF and active ERK1/2.

Novel Noncatalytic Substrate-Selective p38α-Specific MAPK Inhibitors with Endothelial-Stabilizing and Anti-Inflammatory Activity.

The p38 MAPK family is composed of four kinases of which p38α/MAPK14 is the major proinflammatory member. These kinases contribute to many inflammatory diseases, but the currently available p38 catalytic inhibitors (e.g., SB203580) are poorly effective and cause toxicity. We reasoned that the failure of catalytic p38 inhibitors may derive from their activity against noninflammatory p38 isoforms (e.g., p38ß/MAPK11) and loss of all p38α-dependent responses, including anti-inflammatory, counterregulatory responses via mitogen- and stress-activated kinase (MSK) 1/2 and Smad3. We used computer-aided drug design to target small molecules to a pocket near the p38α glutamate-aspartate (ED) substrate-docking site rather than the catalytic site, the sequence of which had only modest homology among p38 isoforms. We identified a lead compound, UM101, that was at least as effective as SB203580 in stabilizing endothelial barrier function, reducing inflammation, and mitigating LPS-induced mouse lung injury. Differential scanning fluorimetry and saturation transfer difference-nuclear magnetic resonance demonstrated specific binding of UM101 to the computer-aided drug design-targeted pockets in p38α but not p38ß. RNA sequencing analysis of TNF-α-stimulated gene expression revealed that UM101 inhibited only 28 of 61 SB203580-inhibited genes and 7 of 15 SB203580-inhibited transcription factors, but spared the anti-inflammatory MSK1/2 pathway. We provide proof of principle that small molecules that target the ED substrate-docking site may exert anti-inflammatory effects similar to the catalytic p38 inhibitors, but their isoform specificity and substrate selectivity may confer inherent advantages over catalytic inhibitors for treating inflammatory diseases.

SARS-CoV-2 ORF3a Protein as a Therapeutic Target against COVID-19 and Long-Term Post-Infection Effects.

The COVID-19 pandemic caused by SARS-CoV-2 has posed unparalleled challenges due to its rapid transmission, ability to mutate, high mortality and morbidity, and enduring health complications. Vaccines have exhibited effectiveness, but their efficacy diminishes over time while new variants continue to emerge. Antiviral medications offer a viable alternative, but their success has been inconsistent. Therefore, there remains an ongoing need to identify innovative antiviral drugs for treating COVID-19 and its post-infection complications. The ORF3a (open reading frame 3a) protein found in SARS-CoV-2, represents a promising target for antiviral treatment due to its multifaceted role in viral pathogenesis, cytokine storms, disease severity, and mortality. ORF3a contributes significantly to viral pathogenesis by facilitating viral assembly and release, essential processes in the viral life cycle, while also suppressing the body's antiviral responses, thus aiding viral replication. ORF3a also has been implicated in triggering excessive inflammation, characterized by NF-κB-mediated cytokine production, ultimately leading to apoptotic cell death and tissue damage in the lungs, kidneys, and the central nervous system. Additionally, ORF3a triggers the activation of the NLRP3 inflammasome, inciting a cytokine storm, which is a major contributor to the severity of the disease and subsequent mortality. As with the spike protein, ORF3a also undergoes mutations, and certain mutant variants correlate with heightened disease severity in COVID-19. These mutations may influence viral replication and host cellular inflammatory responses. While establishing a direct link between ORF3a and mortality is difficult, its involvement in promoting inflammation and exacerbating disease severity likely contributes to higher mortality rates in severe COVID-19 cases. This review offers a comprehensive and detailed exploration of ORF3a's potential as an innovative antiviral drug target. Additionally, we outline potential strategies for discovering and developing ORF3a inhibitor drugs to counteract its harmful effects, alleviate tissue damage, and reduce the severity of COVID-19 and its lingering complications.

Photoinduced ring opening reactions and subsequent thermal ring closure reactions in the thymine-ammonia system and related systems.

The photo-induced ring opening reactions of thymine (Thy) and 1-methylthymine (MeT) with ammonia and with methylamine (MA) at basic pH, and the subsequent ring closure reactions of the resulting adducts, have been studied. In the photo-induced reaction of Thy with ammonia, the dominant product is the E form of N-carbamoyl-3-amino-2-methylacrylamide (IIIa). Heating or acid treatment of aqueous IIIa results in rapid formation of Thy as final product, while allowing IIIa to stand at 4 degrees C produces Thy and two isomeric Thy hydrates, namely trans- and cis-6-hydroxy-5,6-dihydrothymine (Ia and IIa). The main products in the other reactions have analogous structures and undergo analogous ring closure reactions. Incubation of IIIa (and similar adducts) in phosphate buffer near pH 7 significantly enhances the rate of hydrate formation; other weak acid/conjugate base buffer systems also increase the rate of hydrate formation. A mechanism leading from opened ring adduct (e.g.IIIa) to hydrates (e.g.Ia and IIa) is proposed; ring closure leads initially to a dihydrothymine intermediate containing a 6-amino moiety, while further reaction with water produces the observed hydrates. The results described may be relevant to understanding the fate of cross-links generated by photo-induced reaction of thymine moieties in DNA with lysine residues in nuclear proteins (e.g. histones) when they are formed in a cellular environment. Decomposition of such cross-links, catalyzed by cellular phosphate, could lead to production of thymine hydrates attached to lysine residues contained in the protein partner and concurrent generation of an apyrimidinic site in the DNA partner.

Gallium(III)-Salophen as a Dual Inhibitor of Pseudomonas aeruginosa Heme Sensing and Iron Acquisition.

Pseudomonas aeruginosa is an opportunistic bacterium that causes life-threatening infections in immunocompromised patients. In infection, it uses heme as a primary iron source and senses the availability of exogenous heme through the heme assimilation system (Has), an extra cytoplasmic function σ-factor system. A secreted hemophore HasAp scavenges heme and, upon interaction with the outer-membrane receptor HasR, activates a signaling cascade, which in turn creates a positive feedback loop critical for sensing and adaptation within the host. The ability to sense and respond to heme as an iron source contributes to virulence. Consequently, the inhibition of this system will lead to a disruption in iron homeostasis, decreasing virulence. We have identified a salophen scaffold that successfully inhibits the activation of the Has signaling system while simultaneously targeting iron uptake via xenosiderophore receptors. We propose this dual mechanism wherein free Ga3+-salophen reduces growth through uptake and iron mimicry. A dual mechanism targeting extracellular heme signaling and uptake together with Ga3+-induced toxicity following active Ga3+salophen uptake provides a significant therapeutic advantage while reducing the propensity to develop resistance.

Synthesis and Structural Elucidation of a Pyranomorphinan Opioid and in Vitro Studies.

During optimization of the synthesis of the mixed µ opioid agonist/δ opioid antagonist 5-(hydroxymethyl)oxymorphone (UMB425) for scale-up, it was unexpectedly discovered that the 4,5-epoxy bridge underwent rearrangement on treatment with boron tribromide (BBr3) to yield a novel opioid with a little-studied pyranomorphinan skeleton. This finding opens the pyranomorphinans for further investigations of their pharmacological profiles and represents a novel drug class with the dual profile (µ vs δ) predicted to yield lower tolerance and dependence. The structure was assigned with the help of 1D, 2D NMR and the X-ray crystal structure.

Cranberry Xyloglucan Structure and Inhibition of Escherichia coli Adhesion to Epithelial Cells.

Cranberry juice has been recognized as a treatment for urinary tract infections on the basis of scientific reports of proanthocyanidin anti-adhesion activity against Escherichia coli as well as from folklore. Xyloglucan oligosaccharides were detected in cranberry juice and the residue remaining following commercial juice extraction that included pectinase maceration of the pulp. A novel xyloglucan was detected through tandem mass spectrometry analysis of an ion at m/z 1055 that was determined to be a branched, three hexose, four pentose oligosaccharide consistent with an arabino-xyloglucan structure. Two-dimensional nuclear magnetic resonance spectroscopy analysis provided through-bond correlations for the α-L-Araf (1→2) α-D-Xylp (1→6) ß-D-Glcp sequence, proving the S-type cranberry xyloglucan structure. Cranberry xyloglucan-rich fractions inhibited the adhesion of E. coli CFT073 and UTI89 strains to T24 human bladder epithelial cells and that of E. coli O157:H7 to HT29 human colonic epithelial cells. SSGG xyloglucan oligosaccharides represent a new cranberry bioactive component with E. coli anti-adhesion activity and high affinity for type 1 fimbriae.

Enhanced silencing and stabilization of siRNA polyplexes by histidine-mediated hydrogen bonds.

Branched peptides containing histidines and lysines (HK) have been shown to be effective carriers for DNA and siRNA. We anticipate that elucidation of the binding mechanism of HK with siRNA will provide greater insight into the self-assembly and delivery of the HK:siRNA polyplex. Non-covalent bonds between histidine residues and nucleic acids may enhance the stability of siRNA polyplexes. We first compared the polyplex biophysical properties of a branched HK with those of branched asparagine-lysine peptide (NK). Consistent with siRNA silencing experiments, gel electrophoresis demonstrated that the HK siRNA polyplex maintained its integrity with prolonged incubation in serum, whereas siRNA in complex with NK was degraded in a time-dependent manner. Isothermal titration calorimetry of various peptides binding to siRNA at pH 7.3 showed that branched polylysine, interacted with siRNA was initially endothermic, whereas branched HK exhibited an exothermic reaction at initial binding. The exothermic interaction indicates formation of non-ionic bonds between histidines and siRNA; purely electrostatic interaction is entropy-driven and endothermic. To investigate the type of non-ionic bond, we studied the protonation state of imidazole rings of a selectively (15)N labeled branched HK by heteronuclear single quantum coherence NMR. The peak of Nδ1-H tautomers of imidazole shifted downfield (in the direction of deprotonation) by 0.5-1.0 ppm with addition of siRNA, providing direct evidence that histidines formed hydrogen bonds with siRNA at physiological pH. These results establish that histidine-rich peptides form hydrogen bonds with siRNA, thereby enhancing the stability and biological activity of the polyplex in vitro and in vivo.

Photohydrate-mediated reactions of uridine, 2'-deoxyuridine and 2'-deoxycytidine with amines at near neutral pH.

Photohydrates are formed in high yield when uridine (Urd), 2'-deoxyuridine (dUrd), cytidine (Cyd) and 2'-deoxycytidine (dCyd) are irradiated with UVC in aqueous solution. The thermal reactions of the photohydrates of Urd with amines at pH values near pH 7.5 have been studied using UV spectroscopy, HPLC, mass spectrometry and, in some cases, NMR. It has been found that a number of amines (i.e. ethylenediamine, N,N'-dimethylethylenediamine, glycine, glycinamide, glycylglycine, glycylgylcylglycine, putrescine, spermidine and spermine) react thermally with such hydrates to form products with UV spectra characteristic of opened ring uridine-amine adducts. In general, these products display a strong absorption peak with λmax in the range between 288 and 310 nm. Mass spectral studies of a number of the products indicate that they contain one molecule of parent nucleoside and one molecule of reactant amine. Upon standing in water these products revert to parent hydrate, while heating produces parent nucleoside. Less comprehensive studies indicate that photohydrates of dUrd and dCyd undergo analogous thermal reactions. Preliminary results suggest that UV-irradiated polyuridylic acid and polycytidylic acid undergo similar reactions. These results may have relevance for obtaining a complete understanding of the biological effects of producing Urd and dCyd photohydrates in a cellular environment.

Small molecule antivirulents targeting the iron-regulated heme oxygenase (HemO) of P. aeruginosa.

Bacteria require iron for survival and virulence and employ several mechanisms including utilization of the host heme containing proteins. The final step in releasing iron is the oxidative cleavage of heme by HemO. A recent computer aided drug design (CADD) study identified several inhibitors of the bacterial HemOs. Herein we report the near complete HN, N, CO, Cα, and Cß chemical shift assignment of the P. aeruginosa HemO in the absence and presence of inhibitors (E)-3-(4-(phenylamino)phenylcarbamoyl)acrylic acid (3) and (E)-N'-(4-(dimethylamino)benzylidene) diazenecarboximidhydrazide (5). The NMR data confirm that the inhibitors bind within the heme pocket of HemO consistent with in silico molecular dynamic simulations. Both inhibitors and the phenoxy derivative of 3 have activity against P. aeruginosa clinical isolates. Furthermore, 5 showed antimicrobial activity in the in vivo C. elegans curing assay. Thus, targeting virulence mechanisms required within the host is a viable antimicrobial strategy for the development of novel antivirulants.

Synthesis and Characterization of a Novel Diels - Alder Adduct of Codeine.

The Diels - Alder reaction was applied to 4,5-epoxymorphinan opioids to generate a novel aromatic cycloadduct at C(7) - C(8): Thermolytic cleavage of sultine 8 produced the reactive diene o-quinodimethane 7 which condensed favorably with codeine (11), but not with codeinone (9) or 14- hydroxycodeinone (10), producing the desired tetrahydronaphtho adduct 12 with (7R,8R) geometry (Scheme). The configuration of the cycloadduct was determined by 1D- and 2D-NMR experiments. The unanticipated reactivity of these codeine derivatives was investigated by quantum-mechanical calculations, and it was determined that steric effects of the 6-keto and 14-hydroxy group likely precluded condensation by raising the molecular energy of their respective transition states.

NMR metabolomic analysis of caco-2 cell differentiation.

The high-resolution (1)H NMR spectra as applied to Caco-2 cells during their differentiation into enterocyte like cells are presented. The data clearly reveal differences in the metabolic profiles over time as the Caco-2 cells differentiate. In the (1)H NMR spectra, the aliphatic regions from 4.5 to 1.0 ppm are dominated by peaks from myo-inositol, creatine, taurine, glutamine, glutamate, phosphatidylcholine, choline, alanine and lactate. While a majority of metabolites are present at both the early undifferentiated state and the late differentiated states, the levels of certain metabolites are seen to change dramatically, and in particular, the ratio of myo-inositol and taurine. The NMR spectrum from 10 to 5 ppm shows the aromatic amino acids (Phe, Tyr), NAD, ATP and ribose signals. The appearance of glucose resonances in the differentiated cells (30 days old) spectra suggests that these cells become gluconeogenic. Our study represents a novel method to analyze the differentiation of Caco-2 cells using a metabolomic approach. The results indicate, for the first time, that taurine and glucose biosynthesis occurs in these cells and thus by extension may occur in the intestine. This metabolomic approach can therefore be used to detect novel biological pathways as well as yield useful markers for differentiation.

NMR assignments of cd-HO, a 24 kDa heme oxygenase from Corynebacterium diphtheria.

We are employing a number of selective in vitro and in vivo methods including NMR to screen compounds that bind to heme oxygenases from pathogenic bacteria. We report the nearly complete HN, N, CO, Calpha and Cbeta chemical shift assignments of a 215-amino acid HO from Corynebacterium diphtheria in three forms, apo cd-HO-G135A, apo cd-HO and CO-bound ferrous holo cd-HO; these assignments will enable us to identify residues on cd-HO that are perturbed upon binding to selected compounds, and to help with the development of inhibitors specific to the bacterial proteins.