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.

Bacterial lipopolysaccharide augments febrile-range hyperthermia-induced heat shock protein 70 expression and extracellular release in human THP1 cells.

Sepsis, a devastating and often lethal complication of severe infection, is characterized by fever and dysregulated inflammation. While infections activate the inflammatory response in part through Toll-like receptors (TLRs), fever can partially activate the heat shock response with generation of heat shock proteins (HSPs). Since extracellular HSPs, especially HSP70 (eHSP70), are proinflammatory TLR agonists, we investigated how exposure to the TLR4 agonist, bacterial lipopolysaccharide (LPS) and febrile range hyperthermia (FRH; 39.5°C) modify HSP70 expression and extracellular release. Using differentiated THP1 cells, we found that concurrent exposure to FRH and LPS as well as TLR2 and TLR3 agonists synergized to activate expression of inducible HSP72 (HSPA1A) mRNA and protein via a p38 MAP kinase-requiring mechanism. Treatment with LPS for 6 h stimulated eHSP70 release; levels of eHSP70 released at 39.5°C were higher than at 37°C roughly paralleling the increase in intracellular HSP72 in the 39.5°C cells. By contrast, 6 h exposure to FRH in the absence of LPS failed to promote eHSP70 release. Release of eHSP70 by LPS-treated THP1 cells was inhibited by glibenclamide, but not brefeldin, indicating that eHSP70 secretion occurred via a non-classical protein secretory mechanism. Analysis of eHSP70 levels in exosomes and exosome-depleted culture supernatants from LPS-treated THP1 cells using ELISA demonstrated similar eHSP70 levels in unfractionated and exosome-depleted culture supernatants, indicating that LPS-stimulated eHSP70 release did not occur via the exosome pathway. Immunoblot analysis of the exosome fraction of culture supernatants from these cells showed constitutive HSC70 (HSPA8) to be the predominant HSP70 family member present in exosomes. In summary, we have shown that LPS stimulates macrophages to secrete inducible HSP72 via a non-classical non-exosomal pathway while synergizing with FRH exposure to increase both intracellular and secreted levels of inducible HSP72. The impact of increased macrophage intracellular HSP70 levels and augmented secretion of proinflammatory eHSP70 in the febrile, infected patient remains to be elucidated.

Polymorphisms in human heat shock factor-1 and analysis of potential biological consequences.

The stress-activated transcription factor, heat shock factor-1 (HSF1), regulates many genes including cytoprotective heat shock proteins (HSPs). We hypothesized that polymorphisms in HSF1 may alter the level or function of HSF1 protein accounting for interindividual viability in disease susceptibility or prognosis. We searched for exomic variants in HSF1 by querying human genome databases and directly sequencing DNA from 80 anonymous genomic DNA samples. Overall, HSF1 sequence was highly conserved, with no common variations. We found 31 validated deviations from a reference sequence in the dbSNP database and an additional 5 novel variants by sequencing, with allele frequencies that were 0.06 or less. Of these 36, 2 were in 5'-untranslated region (5'UTR), 10 in 3'UTR, and 24 in the coding region. The potential effects of 5'UTR on secondary structure, protein structure/function, and 3'UTR targets of microRNAs were analyzed using RNAFold, PolyPhen-2, SIFT, and MicroSNiper. One of the 5'UTR variants was predicted to strengthen secondary structure. Eight of 3'UTR variants were predicted to modify microRNA target sequences. Eight of the coding region variants were predicted to modify HSF1 structure/function. Reducing HSF1 levels in A549 cells using short hairpin RNA (shRNA) increased sensitivity to heat-induced killing demonstrating the impact that genetic variants that reduce HSF1 levels might have. Using the pmirGLO expression system, we found that the wild-type HSF1 3'UTR suppressed translation of a firefly luciferase reporter plasmid by 65 %. Introducing two of four 3'UTR single nucleotide polymorphisms (SNPs) increased HSF1 3'UTR translational suppression by 27-44 % compared with the wild-type HSF1 3'UTR sequence while a third SNP reduced suppression by 25 %. HSF1 variants may alter HSF1 protein levels or function with potential effects on cell functions, including sensitivity to stress.