Synthesis and Conformational Analysis of FR901464-Based RNA Splicing Modulators and Their Synergism in Drug-Resistant Cancers

Abstract

FR901464 is a cytotoxic natural product that binds splicing factor 3B subunit 1 (SF3B1) and PHD finger protein 5A (PHF5A), the components of the human spliceosome. The amide-containing tetrahydropyran ring binds SF3B1, and it remains unclear how the substituents on the ring contribute to the binding. Here, we synthesized meayamycin D, an analogue of FR901464, and three additional analogues to probe the conformation through methyl scanning. We discovered that the amide-containing tetrahydropyran ring assumes only one of the two possible chair conformations and that methylation of the nitrogen distorts the chair form, dramatically reducing cytotoxicity. Meayamycin D induced alternative splicing of MCL-1, showed strong synergism with venetoclax in drug-resistant lung cancer cells, and was cancer-specific over normal cells. Meayamycin D incorporates an alkyl ether and shows a long half-life in mouse plasma. The characteristics of meayamycin D may provide an approach to designing other bioactive L-shaped molecules.

Structure-activity relationship study of splicing modulators on Hsh155/SF3B1 through chemical synthesis and yeast genetics

“…46 Next, we investigated the stability of 2'-Me meayamycin D and 3'-Me meayamycin D in mouse CD1 plasma (Figure S4). 3'-Me meayamycin D has comparable stability in plasma compared to meayamycin D (t1/2 = 13 h) 38 with a halflife of 16 h. Meanwhile, 2'-Me meayamycin D has a higher half-life of 30 h, which may be attributed to steric shielding of the amide bond. To better understand the binding pocket for meayamycin D on SF3B1, we analyzed the crystal structure 28 and identified four residues that are near the C2' and C3' methyl group: L1066, R1074, T1077, and V1078 (Figure 4).…”

“…The synthesis of 2'-Me meayamycin D began with 1, which was prepared in a single step from commercially available ethyl-(S)-lactate (Scheme 1). 38 Sequentially, lactate 1 was reduced with diisobutylaluminum hydride (DIBALH), and the in-situ-generated aldehyde was submitted to an Ando-Horner-Wadsworth-Emmons reaction 40 with phosphonate 9 41 to yield the a-methylated enoate 2 in 52% yield, with an E/Z ratio of 6:94. Enoate 2 was hydrolyzed to acid 3 in quantitative yield.…”

“…The data for meayamycin A and meayamycin D are the same as previously reported since these controls were tested in the same experiment as the previous report. 38 We evaluated the ability of 2'-Me meayamycin D and 3'-Me meayamycin D to decrease the abundance of proteins whose expression is dependent on splicing of their respective pre-mRNAs (Figure 3). 3'-Me meayamycin D (GI50 = 5 nM) showed a comparable decrease in myeloid cell leukemia 1 (MCL-1) protein abundance to meayamycin D (GI50 = 2 nM), while 2'-Me meayamycin D showed only small changes in protein levels at concentrations up to 1 µM.…”

“…18,[25][26][27][29][30][31][32][33][34][35][36][37] In the cryo-EM structure the SSA-SF3B1 complex, the enamide occupied the narrow neck region of the protein pocket (Figure 2). To gain additional insights, we decided to study the SAR around the C2' and C3' positions of FR901464 with our more metabolically stable analog, meayamycin D. 38 The region between L1066 and V1078 residues of human SF3B1 appear to interact with the C2' and C3' positions of SSA. We previously compared the Zenamide (naturally occurring) to the E-enamide and the C2'-C3' saturated equivalent.…”