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

Abstract

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., p38β/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 p38β. 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.

A temperature-dependent conformational shift in p38α MAPK substrate–binding region associated with changes in substrate phosphorylation profile

“…926-88070) were obtained from LI-COR. All plasmids containing N-terminal His-tagged coding sequences were transformed in E. coli BL21, grown in lysogeny broth medium, induced with 1 mM isopropyl 1-thio-␤-D-galactopyranoside (Thermo Fisher Scientific) for 4 h, and proteins were purified using cobalt columns (TALON TM ; Clontech) according to the manufacturer's protocol, and purified proteins were confirmed by SDS-PAGE and immunoblotting as described previously (71). N-terminal His/HA-dually tagged p38␣ and p38␤ were expressed from pRSetA plasmid (Thermo Fisher Scientific) as described previously (71).…”

“…All plasmids containing N-terminal His-tagged coding sequences were transformed in E. coli BL21, grown in lysogeny broth medium, induced with 1 mM isopropyl 1-thio-␤-D-galactopyranoside (Thermo Fisher Scientific) for 4 h, and proteins were purified using cobalt columns (TALON TM ; Clontech) according to the manufacturer's protocol, and purified proteins were confirmed by SDS-PAGE and immunoblotting as described previously (71). N-terminal His/HA-dually tagged p38␣ and p38␤ were expressed from pRSetA plasmid (Thermo Fisher Scientific) as described previously (71). Human dually phosphorylated N-terminal His-tagged p38␣ was expressed from pETDuet plasmid (EMD-Millipore) containing the untagged sequence for the constitutively active human MAPK kinase-6 (MKK6) mutant (S207G/T211G) in the second multicloning site as described (pETDuet-p38␣) (71).…”

“…N-terminal His/HA-dually tagged p38␣ and p38␤ were expressed from pRSetA plasmid (Thermo Fisher Scientific) as described previously (71). Human dually phosphorylated N-terminal His-tagged p38␣ was expressed from pETDuet plasmid (EMD-Millipore) containing the untagged sequence for the constitutively active human MAPK kinase-6 (MKK6) mutant (S207G/T211G) in the second multicloning site as described (pETDuet-p38␣) (71). We created a similar expression plasmid for dually phosphorylated p38␤ by exchanging the p38␤-coding sequence for the p38␣-coding sequence in pETDuet-p38␣ to create pETDuet-p38␤.…”

Avoiding or Co-Opting ATP Inhibition: Overview of Type III, IV, V, and VI Kinase Inhibitors

“…Shah et al used computational approaches to identify compounds that target a pocket adjacent to the DRS of p38α MAP kinase [ 59 ]. Unique to these compounds were their isoform preference for interactions with p38α over p38β MAP kinase, which may help mitigate excess toxicity observed with the ATP-competitive inhibitors tested previously [ 60 , 61 ].…”

Treatments for Pulmonary Ricin Intoxication: Current Aspects and Future Prospects

“…It has previously been demonstrated that the inhibition of p38 protects cells from ricin-induced effects [ 22 ]; however, p38 inhibitors are often toxic and therefore exhibit very limited success in clinical trials. Recently, using computer-aided drug design, UM101, a novel inhibitor of p38α, the major isoform responsible for the proinflammatory effects of this MAPK, was evaluated [ 124 ]. This compound was more potent than the non-selective p38 inhibitor SB203580 in stabilizing endothelial barrier function and reducing inflammation in the course of LPS-induced murine acute lung injury.…”