Were Series 2 compounds run through a kinase screen?

[mattodd]

The MoA results from Lee (#15) are suggesting kinases as potential mammalian targets, and this may suggest reasons for the tox we're seeing. Some suggested targets are kinases (@danielgedder will summarise separately). This raises the question: were OSA2 compounds (or a representative compound) run through a kinase panel?

I'm assuming yes. In the proposal we said "The diarylimidazole SB-400868 was originally prepared in an effort targeting the human kinase TGFβR (Figure 1).8 SB-400868 was included in the Published Kinase Inhibitor Set (PKIS), a collection of kinase inhibitors made openly available to the research community.9" where the refs are 8) J. Med. Chem. 2002, 45, 999–1001 9) Nat. Biotechnol. 2016, 34, 95–103

I note we had a preliminary discussion about this in #2. SB-400868 was not originally submitted for MRSA testing, despite it being the inspiration for the series!

So @danielgedder can you please

• [x] check which OSA compound SB-400868 is? I'm assuming we re-made it... What's the MIC vs MRSA?
• [ ] get hold of the kinase data on this compound. It must be somewhere. If we can't find it, we can ask David Drewry and Bill Zuercher.
• [ ] Correlate what's known of this compound's activity with what Lee is finding.

Ping @AlvaroLor @TwistedSwisster

[danielgedder]

Initial data provided by Lee M. Graves from the University of North Carolina (UNC) Chapel Hill, using the MIB-MS method with MRSA cells incubated with active compound OSA_822, showed some indications that NEK2, NEK3, CSNK1E and TGFRB1 could potentially be the target for this series of compounds. Figure 1 shows the structure of OSA_822, one of the most potent compounds against MRSA and formed the basis of our SAR and MoA studies of this chemotype.

Figure 1. Structure of representative diarylimidazole OSA_822 with a) SAR features of interest highlighted and b) initial MoA studies.

Moreover, MoA studies are still in progress with improved compounds and we are investigating this first evidence to validate our findings. In parallel, to validate our MoA studies performed by Lee, searches on ChEMBL databases using OSA_822 analogue as a filter, resulted in a library containing about 9 thousand molecules (NEK2 – 3324 compounds; NEK3 – 817 compounds; CSNK1E – 1207; TGFRB1 – 3536) with bioactivities against for the targets identified in MIB-MS technique. After applying the appropriate filters, we found compounds containing similar scaffolds to the OSA_822 derivative. Most of the compounds bearing pyrrolo[1,2-a]imidazole fused ring were inactive against serine/threonine-protein kinases (NEK2, NEK3), as example 1 (Figure 2). (1)

Figure 2. Results of a search for similar OSA-like compounds in the ChEMBL database with examples of the most significant compounds with their respective bioactivities.

Scaffolds related to OSA_822 have been tested against CSNK1E (Casein Kinase 1 Epsilon), a set of 109 pyrrolo[1,2-a]pyrazine derivatives showed potency against this target. (2) Eight compounds showed IC50 of 1 nM. Example 2 (3) displayed in Figure 2 represents one of the promising inhibitor of this series of compounds. We found 103 compounds containing pyrrolo[1,2-b]pyrazole, that showed activity against the transforming growth factor TGF-β1 (Example 3). Galunisertib (LY2157299) is an oral and selective TGF-β1 kinase inhibitor with an IC50 of 56 nM and inactive against NEK2. (4) During phase II Trial, Galunisertib was discontinued due the progress of malignant glioma disease in patients enrolled in this study. (5) One of the OSA analogue is known to be a potential kinase inhibitor (OSA_1053; ALK IC50 = 5.8 uM, p38 = inactive) (6) and a possible starting point for the design new TGF-β1 kinase inhibitors. A screening of GlaxoSmithKline internal library collection for inhibitors of the transforming growth factor β1 (TGF-β1) type I receptor (ALK5) identified 2-diarylimidazoles as a promising hit (OSA_812/SB-400868, IC50 of 0.46 µM against ALK5 and non inhibition against p38 kinase). ALK5 is the receptor of TGF-β and plays a pivotal role in the canonical TGF-β signaling pathway. Compound OSA_812 inhibits TGF- β1- induced fibronectin (FN) mRNA (IC50 of 0.50 µM) in A498 cells. Further optimizations (pyrrolo moiety replaced for benzamide group) lead to selective inhibitor SB-431542 (IC50 of 94 nM against ALK5 and non inhibition against p38 kinase, LD50 > 30 uM), which inhibits TGF-β1-induced fibronectin mRNA (IC50 of 0.05 µM) formation while displaying no measurable cytotoxicity in the 48 h XTT assay. (7) Using genetic analysis,(8) a publication showed that Lb21-mediated (Lactobacillus spp.) MRSA resistance is dependent on the DBL-1 ligand of the TGF-β signaling pathway in C. elegans. This response is evolutionarily conserved as that Lb21 also induces the TGF-β pathway in porcine epithelial cells. In addition, the anti-inflammatory cytokine transforming growth factor β1 (TGF-β1) plays an important role in Chagas disease (CD), by inducing the synthesis of extracellular matrix components and decreasing its degradation and turn-over.(9) CD patients with higher TGF-β1 serum level show a worse clinical outcome, implicating a predictive value of serum TGF-β as a surrogate biomarker of clinical relevance. (10) A previous study showed that SB-431542, an inhibitor of the TGF-β1 receptor (ALK5), inhibits T. cruzi-induced activation of the TGF-β1 pathway in epithelial cells and in cardiomyocytes. It reduces cardiomyocyte invasion by T. cruzi. SB-431542 treatment significantly reduces the number of parasites per infected cell and trypomastigote differentiation and release. SB-431542 is able to arrest parasite multiplication and release. This study demonstrated that small inhibitors of the TGF-β1 signalling pathway might be potential pharmacological tools for the treatment of Chagas disease. (11) These findings from the literature (12) reaffirm that inhibition of the TGF-β signaling pathway could be considered an important alternative strategy for the treatment of infectious diseases.

References:

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7. Callahan JF, Burgess JL, Fornwald JA, Gaster LM, Harling JD, Harrington FP, et al. Identification of novel inhibitors of the transforming growth factor beta1 (TGF-beta1) type 1 receptor (ALK5). J Med Chem. 2002;45(5):999-1001.
8. Mørch MGM, Møller KV, Hesselager MO, Harders RH, Kidmose CL, Buhl T, et al. The TGF-β ligand DBL-1 is a key player in a multifaceted probiotic protection against MRSA in C. elegans. Scientific Reports. 2021;11(1):10717.
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11. Waghabi MC, Keramidas M, Calvet CM, Meuser M, Soeiro MdNC, Mendonça-Lima L, et al. SB-431542, a Transforming Growth Factor β Inhibitor, Impairs Trypanosoma cruzi Infection in Cardiomyocytes and Parasite Cycle Completion. Antimicrobial Agents and Chemotherapy. 2007;51(8):2905-10.
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[danielgedder]

Hi @mattodd, SB-400868 is OSA_812 in Figure 2.