Pharmacokinetic Optimization of Small Molecule Janus Kinase 3 Inhibitors to Target Immune Cells

Laux, Julian; Förster, Michael; Riexinger, Laura; Schwamborn, Anna; Guezguez, Jamil; Pokoj, Christina; Kudolo, Mark; Berger, Lena M.; Knapp, Stefan; Schollmeyer, Dieter; Guse, Jan; Burnet, Michael; Laufer, Stefan

doi:10.1021/acsptsci.2c00054

Cited by 7 publications

(46 citation statements)

References 72 publications

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“…3 is an example of the macrolide conjugate class with promising in vitro efficacy coupled with appropriate in vivo pharmacokinetics (oral availability and extended tissue half-life). 25 Oral In subsequent studies, 3 was replaced by 4, a similar conjugate compound with improved in vitro potency and pharmacokinetics (see Supporting Information, Figure S5 and Table S1). The other test compounds, 1 and 2, were selected based on pharmacokinetic properties: 1 has adequate JAK3 potency and distributes well into tissues including the CNS.…”

Section: Jak3 Inhibitors Differentially Affect the Cytokine Response ...mentioning

confidence: 99%

“…23,24 We recently reported the synthesis and characterization of a series of JAK3 inhibitors based on its scaffold (Figures 3 and 4). 25 1−4 are structurally related analogues of 8. Their inhibitory potencies for JAK3 are within the same order of magnitude as 8 or slightly lower (see Supporting Information, Table S1), while possessing different pharmacokinetic profiles and in vivo stabilities.…”

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confidence: 99%

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Selective Inhibitors of Janus Kinase 3 Modify Responses to Lipopolysaccharides by Increasing the Interleukin-10-to-Tumor Necrosis Factor α Ratio

Laux

Martorelli

Späth

et al. 2023

ACS Pharmacol. Transl. Sci.

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Janus kinase (JAK) inhibitors act at low doses (e.g., tofacitinib, 0.2–0.4 μmol/kg bid) in clinical use, suggesting an efficient underlying mode of action. We hypothesized that their effectiveness is due to their ability to raise the ratio of IL-10 to TNFα. Unlike other JAK isoforms, JAK3 is expressed mainly in hematopoietic cells and is essential for immune function. We used JAK3 selective inhibitors with preferential distribution to immune cells. Inhibition of JAK3 in human leukocytes reduced TNFα and IL-6 but maintained levels of IL-10, while pan-JAK inhibitors increased TNFα, IL-6, and IL-10. JAK1 is required for IL-10 receptor signaling, which suggests that, at exposure above the IC50 (55 nM for tofacitinib on JAK1), there is less feedback control of TNFα levels. This leads to self-limiting effects of JAK1 inhibitors and could place an upper limit on appropriate doses. In vivo, treating mice with JAK3 inhibitors before LPS administration decreased plasma TNFα and increased IL-10 above vehicle levels, suggesting that JAK3 inhibition may limit TNFα release by increasing IL-10 while leaving the IL-10 receptor functional. This mechanism should have general utility in controlling autoimmune diseases and can be conveniently observed by measuring the ratio of IL-10 to TNFα. In summary, our targeted, “leukotropic” inhibitors more effectively increased IL-10/TNFα ratios than unselective control compounds and could, therefore, be ideal for autoimmune therapy.

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Section: Jak3 Inhibitors Differentially Affect the Cytokine Response ...mentioning

confidence: 99%

mentioning

confidence: 99%

Selective Inhibitors of Janus Kinase 3 Modify Responses to Lipopolysaccharides by Increasing the Interleukin-10-to-Tumor Necrosis Factor α Ratio

Laux

Martorelli

Späth

et al. 2023

ACS Pharmacol. Transl. Sci.

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“…Due to the highly conserved structural features of the ATP binding pocket, it has been challenging to achieve high selectivity among the JAK family. Many recent developments of JAK3 inhibitors have been focused on a JAK3 unique cysteine residue (CYS909) by forming a covalent bond with JAK3 inhibitors [ 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. The idea of developing an inhibitor that can covalently bind to cysteine 909 was from other covalent drugs such as afatinib, osimertinib, and ibrutinib ( Figure 2 ).…”

Section: Introductionmentioning

confidence: 99%

“…Three drugs in Figure 2 shared a common α,β-unsaturated amide moiety to allow the Michael addition to the target protein via covalent binding. A number of JAK3 covalently bound inhibitors have been reported and some have been studied under various stages of clinical trials [ 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ].…”

Section: Introductionmentioning

confidence: 99%

Docking and Selectivity Studies of Covalently Bound Janus Kinase 3 Inhibitors

Zhong

Almahmoud

2023

IJMS

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The Janus kinases (JAKs) are a family of non-receptor cytosolic protein kinases critical for immune signaling. Many covalently bound ligands of JAK3 inhibitors have been reported. To help design selective JAK inhibitors, in this paper, we used five model proteins to study the subtype selectivity of and the mutational effects on inhibitor binding. We also compared the Covalent Dock programs from the Schrodinger software suite and the MOE software suite to determine which method to use for the drug design of covalent inhibitors. Our results showed that the docking affinity from 4Z16 (JAK3 wild-type model), 4E4N (JAK1), 4D1S (JAK2), and 7UYT (TYK2) from the Schrödinger software suite agreed well with the experimentally derived binding free energies with small predicted mean errors. However, the data from the mutant 5TTV model using the Schrödinger software suite yielded relatively large mean errors, whereas the MOE Covalent Dock program gave small mean errors in both the wild-type and mutant models for our model proteins. The docking data revealed that Leu905 of JAK3 and the hydrophobic residue at the same position in different subtypes (Leu959 of JAK1, Leu932 of JAK2, and Val981 of TYK2) is important for ligand binding to the JAK proteins. Arg911 and Asp912 of JAK3, Asp939 of JAK2, and Asp988 of TYK2 can be used for selective binding over JAK1, which contains Lys965 and Glu966 at the respective positions. Asp1021, Asp1039, and Asp1042 can be utilized for JAK1-selective ligand design, whereas Arg901 and Val981 may help guide TYK2-selective molecule design.

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“…Generally, protein kinases are grouped, depending on their amino acid residue they phosphorylate -as serine/ threonine kinases, tyrosine kinases or dual-specificity kinases. The total number of protein kinases included in the kinome is still under debate, since some kinases phosphorylate non-protein substrates or are so called atypical kinases 7 . The subgroups defined by Manning et al are defined as such: AGC family containing PKA, PKG and PKC, calcium/ calmodulin dependent kinase (CAMK), casein kinase1 (CK1), a group of cyclin dependent kinases, MAP kinases, glycogen synthase kinase and casein kinases (CMGC), sterile 20-like kinases (STE), tyrosine kinases (TK) and tyrosine kinase like (TKL), and others 6 .…”

Section: Zusammenfassung (Germanmentioning

confidence: 99%

Cellular selectivity and kinetics of clinical kinase inhibitors

Berger¹

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Protein kinases are key signalling molecules and transduce intracellular signals via the post-translational phosphorylation of substrate proteins, often other protein kinases. Dysregulation of this protein family has been linked to many diseases including neurodegenerative diseases, inflammation and cancer and amplifications of kinases play important roles as diagnostic biomarkers in a variety of cancers. Various strategies have been developed to treat dysregulated protein kinases. Most commonly, chemical small molecule inhibitors are used to modulate protein kinase activity in cancer cells. Many inhibitor and general research efforts have focused only on a small subset of protein kinases, resulting in a large portion of the kinome, the so-called “dark” kinome, remaining largely unexplored. As part of the strategy to develop inhibitors, it is crucial to understand the structure-activity-relationships (SAR) of small molecules to the activity towards the targets based on understanding small molecule-target affinities as determined by biophysical, biochemical, and cellular methods. However, not always do in vitro determined affinities, which are frequently used as basis for SAR considerations, correlate with the cellular affinity. For protein kinases in particular, it has been shown that the cellular concentration of the natural substrate adenosine-triphosphate (ATP) plays a critical role for the resulting small molecule affinity, as substrate and inhibitor frequently compete for the same binding site of the protein kinase. The cellular target engagement assay NanoBRET is a versatile assay that overcomes this problem and can be used to assess binding of a compound to the full-length protein kinase, in the presence of natural binding partners. Another important factor in inhibitor optimization is the selectivity of the molecule within the family of protein kinases. When comparing the selectivity profiles of small molecule kinase inhibitors in vitro and in cells, different profiles can be observed. Frequently, a compound, binds fewer protein kinases with high affinity in cells, indicating that cellular profiling of protein kinase inhibitors is necessary to understand the selectivity profile of an inhibitor. The goal of this work was to understand cellular SARs of inhibitors for kinases and dark kinases in medicinal chemistry projects, and to understand the selectivity profiles of existing small molecules in cells, including already approved drugs and clinically used kinases inhibitors. The cellular potency and selectivity aspects guided optimization of the inhibitors towards selective small molecules ‘chemical probes’ or highly validated inhibitors with a narrow selectivity profile as part of ‘chemogenomic libraries’. One strategy to improve selectivity has been to use sterically restricted cyclic small molecules, called macrocycles, that allow fewer conformations of the molecule than their non-cyclic parent compound. In this thesis the dark kinase STK17A was investigated. Macrocyclization was used to develop a selective chemical probe molecule that is also selective in the cellular context. For another kinase, SIK2, a rational design approach was used to exclude off-targets bound by the lead structure, resulting in a chemical probe that selectively targets the SIK1/2/3 proteins. Assessing cellular potency of another series of inhibitors, a probe was developed for the PCTAIRE subfamily of the CDK kinases. This required co-expression of the binding partners of CDKs, the cyclins, in cells to obtain a functional assay. To identify new candidates for the neglected family of splicing kinases comprising the CLK, SRPK, DYRK and HIPK protein kinase subfamilies, a literature review was conducted, and the best small molecule candidates were compared for their target engagement in cells. This led to a series of small molecule inhibitors that may be used as a set or single agents to target the CLK proteins and SRPK proteins or in combination to target the remaining proteins. In search of new starting points for this subfamily of kinases, an initial screen with NanoBRET technology was performed using a library of over 2000 inhibitors, and new starting points were identified. Additionally, a set of clinical and approved small molecule kinase inhibitors was assessed for their selectivity in cells. Several highly selective molecules were identified that were much less selective in in vitro approaches. The set of data allowed for a comprehensive comparison of cellular potencies with published data using in vitro binding, in vitro activity and data obtained from cell lysates and identified several protein kinases that would need to be investigated in cells...

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Pharmacokinetic Optimization of Small Molecule Janus Kinase 3 Inhibitors to Target Immune Cells

Cited by 7 publications

References 72 publications

Selective Inhibitors of Janus Kinase 3 Modify Responses to Lipopolysaccharides by Increasing the Interleukin-10-to-Tumor Necrosis Factor α Ratio

Selective Inhibitors of Janus Kinase 3 Modify Responses to Lipopolysaccharides by Increasing the Interleukin-10-to-Tumor Necrosis Factor α Ratio

Docking and Selectivity Studies of Covalently Bound Janus Kinase 3 Inhibitors

Cellular selectivity and kinetics of clinical kinase inhibitors

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