Fig 2: Cytotoxicity measurements of pure CSH71 and CSH18CN in CTCL cells. (A and B) Cytotoxicity assays and IC50s determination of newly synthesized compounds which are considered as two site binders CSH71 and CSH18CN Hut78 CTCL cells treated with increasing doses, respectively, for 48 h (A.) and 96 h. (B). F7 and ß-OG, served as one-site binder, ATP binding and lipid-binding of p38?, respectively. PBMC from healthy donors (HD) used as a control as its p38? level is at undetected level. Rest of text we focus on CSH71 for further analysis of its biological activity due to its interaction with the p38? LBS. (C) Enrichment of dual-phosphorylated p38 by Immunoprecipitation (IP). Sequence of p38 isoforms that all contain TGY motif dual activation site T180/183 GY182/185 (for p38? the motif is T183-G-Y185, all other isoforms are T180 and Y182, Left). Right: IP results, first pulling down with dual-p-p38 of p38 antibody, then detect with p38? and dual-p p38 antibody, respectively, in cytosol of Hut78 cells both untreated (o µm) and CSH71-treated (5 µm). The blue arrow points to p38? that contains dual phosphorylation p-p38 at T183-G-Y185. (D) CSH71 has only two predicted binding sites of p38? by all around docking pose commutating analysis; and the MaxGScores of ATP-binding and lipid-binding, are -6.82, and -6.18 kcal·mol-1, respectively, which also indicates the binding affinity of CSH71 in LBD is much weaker than that of ß-OG with a MaxGScore of -8.9 kcal·mol-1.

Fig 3: Lessons we learnt from p38a on LBD. (A) Consensus amino acids sequences alignment diagram in the lipid-binding domain among four p38 isoforms by clustalW2. MARP insert harbors LBD which is between two aL14 helixes, a1L14 and a2L14. L246 and I259 are two conserved residues of p38a in two L14 loops that indicated in *. Likewise, the counterparts of above two residues in p38 ? are F249 and M262 that are conserved. (B) structures of ligands bound to p38a from 253 PBD entries that have X-ray Crystallography structures. Most of the ligands are single-site binders, either on the ATP-binding site or the lipid-binding site, while some ligands bound to two sites simultaneously. Circled is the area ß-OG bound at the lipid-binding pocket. (C) Chemical structure of ß-OG (n-octyl-ß-glucopyranoside, top) and 2-dimensional (2D) interaction diagram of ß-OG in complex with p38a. The hydrophobic tail of ß-OG is surrounded by hydrophobic amino acids of p38a (bottom). (D) 17 small ligands of the p38a solely target lipid-binding domain (LBD) and 6 small molecules targets both LBD and ATP binding pocket (data summarized from X-ray crystallography). The column of TargetSites shows the number of sites for a ligand; 2-site (teal color) means that the inhibitor binds at both the lipid-binding site and ATP-binding site simultaneously, and LBD indicates the inhibitor can only bind LBS site of p38a. Only one PDB id is listed for each small molecule. (E) Two site binders of p38a with X-ray crystallography structure consist of 6 molecules: GG5 (PBD: 3hvc); p41 (PBD: 3c5u); D13 (PBD: 1ian); 279 (PBD: 2rg6); 80T (PBD: 5n65); and 6RG (PBD: 4dlj) are ligands for two sites, that is, the ATP binding and the lipid-binding of p38a. (F) ß-OG lipid binding of p38a may cause allosteric effects on ATP binding. We superimposed two public PBD datasets from RSCB, the DFG-out conformation of p38a alone (PDB 2baj) is displayed as cyan cartoons; and the inactive p38a bound ß-OG as grey cartoons (PDB 3gcv). When ß-OG bound, the structure is changed significantly near both the lipid-binding and ATP-binding sites (depicted as dotted red circles), which suggests that the binding of ß-OG at the lipid-binding site causes conformational changes near the ATP-binding site and thereby affect its kinase activity allosterically.

Fig 4: NMR demonstrates a lipid‐binding site on p38γ for β‐OG. (A) 2D NMR CSP experiment locates the p38γ residues that significantly shifted due to addition of β‐OG, indicated in orange. NMR CSPs data by monitoring 1H‐15N HSQC and 1H‐13C HMQC spectra upon the addition of β‐OG alone and p38γ to β‐OG with a molar ratio of 1 : 10. The red hollow arrows indicate three residues, G243, T244, and D295 that are conserved among species from human to yeast (See also Figure 2D, red bracket). (B) The structures of p38γ in complex with β‐OG. A 3D structural model presenting where β‐OG bound to LBD of p38γ, based on NMR CSP titration data. The p38γ crystallography structure coordinate datasets were downloaded from RCSB PDB [PDB id 1cm8 [39]]. (C) β‐OG binds p38 γ in the MKI region of in the lipid‐binding domain and residues which greatly perturbed residues in NMR CSP assay are W200, M262, G243, D295, and E 297. K259 and K263 are consistent with that of alpha p38 which are perturbed by B‐OG in Figure 1F. (D) ClustalW2 consensus alignment diagram of amino acid sequences in the lipid‐binding domain of p38γ among indicated species. β‐OG NMR CSP perturbed residues of human p38γ: G243, T244, and D295 (in red brackets) are conserved among species including yeast. Three residues E297, K259, and M262 share homology with mouse.

Fig 5: CSH71 inducing alternative p38 and NFAT activation. (A) Illustration of conserved segments of the DEF site interaction pocket (top), including a EF and a F loop, G-Helix and MARP insert (MKI) which are structurally conserved among four p38 isoforms, which participates the formation of DEF pocket, many residues are perturbed by CSH71 binding underscored with *. The segments at bottom are p38s alternative activation site Y323/Y326 by ClustalW2 Consensus alignment diagram analysis with p38? residue numbering. Residues ETPL is colored pink as PDZ binding Motif that solely existed in p38?. (B) Western blot analysis to detect protein levels changes upon treatments of CSH71 (NCI) of four isoforms of p38 and two phosphorylated forms dual and alternative p38 together with GAPDH as protein internal loading control. (C) Confocal fluorescence microscopy analysis of untreated Hut78 cells indicates the subcellular location of alternative p38 activation by immunofluorescent staining using the specific antibody Y323 p-p38. Co-staining with antibodies against DLGH1 (red) and IL17RA (green) was used to identify downstream targets. DAPI was used to stain the nucleus. Alternative p38 activation is seen on the newly synthesized nuclear envelopes that surround anaphase chromatins (red arrow) and also within the nucleus (yellow arrow). (D) Hut 78 cell cytosolic and nuclear fractions lysates treated with indicated doses of CSH71 (250 nm and 5 µm) for 24 or 48 h are subject to WB analysis, using anti-p38?, anti-p-p38 at Y323 (alternative), anti-p-p38 (dual), anti-PTPN3 and anti-NFATC1, anti NFATC4 antibodies. Anti-Lamin A/C antibody for the Lamin protein as the loading control for nuclear proteins and GAPDH for cytosol proteins. (E) Proposed model of interaction between NFAT with the DEF site of p38? when the alternative site at Y326 is activated (phosphorylated). Left, 3D p38? with residues reflect allosteric network from LBD to ATP binding site. Right, NFAT Interaction interact with p38? via two docking motifs—FxF and LxL sequences; p38? is also known as ERK6, which implicates similarity with the ERK family; in the REL domain of NFATc4, which contains an FxF motif (F681, F683) at the COOH-terminal that allows interaction with the DEF pocket. Other NFAT members (NFATC1, NFATC2, and NFATC3) have FxY motifs in which possibly allows interaction with ERK. The LxL sequence is specific for other MAP kinases, such as c-Jun NH2-terminal protein kinase (JNK).