Inhibiting the protein-protein interaction (PPI) between the transcription factor Nrf2 and its repressor protein Keap1 has emerged as a promising strategy to target oxidative stress in diseases, including CNS disorders. Numerous non-covalent small-molecule Keap1-Nrf2 PPI inhibitors have been reported to date, but many feature suboptimal physicochemical properties for permeating the blood-brain barrier, while others contain problematic structural moieties. Here, we present the first side-by-side assessment of all reported Keap1-Nrf2 PPI inhibitor classes using fluorescence polarization (FP), thermal shift assay (TSA), and surface plasmon resonance (SPR)-and further evaluate the compounds in an NQO1 induction cell assay and in counter tests for non-boronate ester building block 41 as the crucial carbon skeleton-building step. Synthesis of the enol triflate using the one-step NaHMDS-mediated enolization/PhNTf2-induced trapping procedure reported in the patent application 39 was not efficient in our hands, giving low yield (43% vs. 88% reported in literature 39) and significant byproduct formation. We found that using a freshly-made LiHMDS as an alternative base gave a cleaner reaction and excellent yield (quantitative). The converging SM reaction step between 38 and 41 gave several wellknown by-products, including the boronic acid, aryl boronate homo-coupling product and protodeboronation product, but could still afford the desired cross-coupling product 42 in good yield (69% vs. 33% reported in literature 39). Catalytic hydrogenation to deprotect the carboxylic acid and reduce the alkene double bond diastereoselectively furnished only the cis-cyclohexane 43 in accordance with literature; 39 this was revealed by nuclear Overhauser effect (NOE) NMR (Supporting Information Figure S1). This facial selectivity can be explained by a steric directing effect of the carboxybenzyl group. The cyclohexane carboxylic acid of 43 was finally coupled with 2-butylpyrrolidine and the pyrazole carboxylic acid deprotected to give 10 as a mixture of four stereoisomers. Attempted separation of the two diastereoisomers by preparative HPLC was unsuccessful. In the patent application, purification by HPLC is reported to give two different fractions, each containing all four stereoisomers in slightly different proportions, of which one was directly tested as a mixture. 39 Having this literature result as a reference point, we did not proceed with further purification of 10.

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The nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) multi-subunit complex is a highly abundant and central source of reactive oxygen species. NOX2 is a key enzyme of the innate immune system involved in antibacterial response, but excessive NOX2 activity is involved in oxidative stress and inflammation in many diseases. Inhibition of NOX2 has great potential as a therapeutic strategy. An intriguing pharmacological approach for inhibiting NOX2 is to target the p47phox subunit and thereby block the protein-protein interaction with p22phox, whereby assembling and activation of NOX2 is prevented. However, the shallow binding pocket of p47phox makes it difficult to develop drug-like p47phox/p22phox inhibitors. Recently, the small molecule LMH001 was reported to inhibit the p47phox/p22phox interaction, reduce endothelial NOX2 activity, and protect mice from angiotensin II-induced vascular oxidative stress. These noteworthy results could have significant impact on the field of NOX2 pharmacology, as specific and efficient inhibitors are scarce. Here, we synthesized and tested LMH001 to have it available as a positive control. We established a robust synthetic route for providing LMH001, but subsequently we experienced that LMH001 is chemically unstable in aqueous buffer. In addition, neither LMH001 nor its breakdown products were able to inhibit the p47phox/p22phox interaction in a non-cellular fluorescence polarization assay. However, LHM001 was a weak inhibitor of NOX2 in a functional cell assay, but with same low potency as one of its breakdown products. These findings question the activity and suggested mechanism of LMH001 and constitute important information for other researchers interested in chemical probes for studying NOX2 biology.

Inhibition of PSD‐95 has emerged as a promising strategy for the treatment of ischemic stroke, as shown with peptide‐based compounds that target the PDZ domains of PSD‐95. In contrast, developing potent and drug‐like small molecules against the PSD‐95 PDZ domains has so far been unsuccessful. Here, we explore the druggability of the PSD‐95 PDZ1‐2 domain and use fragment screening to investigate if this protein is prone to binding small molecules. We screened 2500 fragments by fluorescence polarization (FP) and validated the hits by surface plasmon resonance (SPR), including an inhibition counter‐test, and found four promising fragments. Three ligand efficient fragments were shown by 1H,15N HSQC NMR to bind in the small hydrophobic P0 pockets of PDZ1‐2, and one of them underwent structure‐activity relationship (SAR) studies. Overall, we demonstrate that fragment screening can successfully be applied to PDZ1‐2 of PSD‐95 and disclose novel fragments that can serve as starting points for optimization towards small‐molecule PDZ domain inhibitors.

The coronavirus pandemic known as COVID‐19 caused by severe acute respiratory syndrome coronavirus 2, threatens public health worldwide. Approval of COVID‐19 vaccines and antiviral drugs have greatly reduced the severe cases and mortality rate. However, the continuous mutations of viruses are challenging the efficacies of vaccines and antiviral drugs. A drug repurposing campaign has identified two JAK1/2 inhibitors ruxolitinib and baricitinib as potential antiviral drugs. Ruxolitinib and baricitinib exert dual antiviral effect by modulation of inflammatory response via JAK1/2 and inhibition of viral entry via AAK1 and GAK. Inspired by this, in an effort to diversify chemical space, three analogues (( R )‐ 8 , ( S )‐ 8 , and 9 ) of ruxolitinib and baricitinb were made using a scaffold hopping strategy. Compound 9 displayed potent and comparable potencies against AAK1, JAK1, and JAK2 compared to baricitinib. Notably, compound 9 showed better selectivity for AAK1, JAK1, and JAK2 over GAK. Besides, compound 9 displayed good druglikeness according to Lipinski's and Veber's rule. We thereby identified a potential lead compound 9 , which might be used for the further development of anti‐coronaviral therapy.