Covalent Inhibitors for Treatment of Inflammation and Neurodegeneration

Background and Unmet Need

The JNK signaling cascade plays an integral role in the pathology of several neurodegenerative diseases, including Parkinson’s and Alzheimer’s disease, as well as in inflammatory and autoimmune responses. Due to its extensive activity profile, its direct inhibition would likely have toxic effects. At the same time, inhibition of its activating kinases, MKK7 and MKK4, via ATP binding site antagonists, is challenging due to the close homology between its ATP binding and that of other kinases. To date, no selective MKK7/4-specific inhibitors have been reported.

The Solution

A selective MKK7 inhibitor which covalently targets a native, non-conserved cysteine residue.

Technology Essence

The designed inhibitor, identified using a proprietary covalent virtual screening platform, targets cysteine 218, found in an identical context in only 10 other protein kinases. The MKK7 inhibitor led to reduced JNK phosphorylation (Figure 1A) and subsequently to reduce phosphorylation of its downstream targets c-Jun and ATF2 in cultured cells in the single-digit micromolar range. In addition, it demonstrated a dose-dependent effect on B cell response to lipopolysaccharide challenge1 (Figure 1B). The inhibitor proved highly selective, with no inhibitory effect on p38 or ERK, two protein kinases involved in a broad range of cellular processes. Similarly, it had no effect on phosphorylated JNK levels in MKK7-knockout cell lines.

Figure 1 – (A) In-cell western assay demonstrating inhibition of JNK phosphorylation in HEK293 cells after 2 hr preincubation with MKK7 inhibitors and sorbitol. (B) Inhibition of Primary mouse B cells activation with MKK7 inhibitors (as assessed by CD86 staining). Except for the negative control MKK7-NEG-1, all compounds show >60% inhibition with MKK7-COV-9 and MKK7-COV 12 showing similar levels of inhibition to the positive control (JNKIN-8)

Applications and Advantages

• Selective and context-specific inhibition
• Prolonged, potent inhibitory effect with therapeutic potential

Metabolically stable

• Suitability for a number of inflammatory and neurological pathologies
• Likely blood brain barrier-penetrant
• Unique chemical tool, e.g., to define the role of MKK7 versus MKK4

Development Status

Cellular proof of inhibition has been achieved. The phenotype associated with its activity is currently being assessed, as well as its effect in disease-relevant models. In parallel, optimization of inhibitor potency, selectivity and metabolic stability is being pursued to enhance its suitability for in vivo applications.

References

Shraga A, Olshvang E, Davidzohn N, et al. Covalent Docking Identifies a Potent and Selective MKK7 Inhibitor. Cell Chem Biol. 2019;26(1):98-108.e5. doi:10.1016/j.chembiol.2018.10.011 [1]