Progressing from 2-methoxy (15a) to 2-ethoxy (25a) to 2-isoproxy (26a) resulted in a gradual decrease in cellular Tel-ALK potency.
The SAR exploration of 3,5-diamino-1,2,4-triazole urea scaffold revealed that the one carbon spacer of the urea side chain (n = 1), 1-acyl substitution, and the 2-methoxy group of the aniline side chain with N-methylpyparazine were key structural features required to achieve potent cellular activity against Tel-ALK and EML4-ALK.
To quantify the potency of LY2784544 and four other clinical JAK2 inhibitors in TEL-JAK cellular systems, more quantitative Alpha-Screen SureFire pSTAT5 assays were utilized to determine the IC50.
This allows the SOCS protein to operate as an adaptor to trigger ubiquitination and degradation of proteins involved in cellular signaling including JAK [ 11], TEL-JAK2 [ 12], IRS-1/2 [ 13], FAK [ 14], Vav [ 15] and Mal [ 16].
To rule out any off-target effect, we performed screening of BAF3 cells expressing Tel-JAK3 (having a cellular IC50 value of 2300 nm) at 2000 and 3000 nm of SAR302503, which showed growth of innumerable colonies (Supplementary Figure 1b).
Cellular RNA was isolated using RNA-Bee (Tel-Test, Friendswood, TX, USA) according to the manufacturer's instructions and transcribed into cDNA using the ImProm-II Reverse Transcription System (Promega, Madison, WI, USA).
Cellular mRNA isolation was performed using RNA-Bee (TEL-TEST, Friendswood, TX, USA) according to the manufacturer's instructions and was transcribed into cDNA using Superscript II (Invitrogen, Carlsbad, CA, USA).
Total cellular RNA was extracted by RNA-Bee™ RNA isolation kit (TEL-TEST, Friendswood, TX) in accordance with the manufacturer's instructions.
For the preparation of polyA mRNA cellular RNA, resting or stimulated Jurkat cells were lysed in STAT60™ (TEL-TEST, INC., Friendswood, TX) as described [ 6].
Buffy coats were obtained by Ficoll density gradient centrifugation, and total cellular RNA was isolated using a guanidinium thiocyanate-phenol-chloroform solution (RNA STAT-60™; TEL-TEST, Friendswood, TX).
Total cellular RNA was isolated from 107 cells with RNA-Bee as recommended by the manufacturer (Tel-Test), followed by extraction with an equal volume of phenol-chloroform and then chloroform prior to ethanol precipitation.
