Synthesis and evaluation of carbocyanine dyes as PRMT inhibitors and imaging agents

Abstract

Summary Protein arginine methylation regulates multiple biological processes. Deregulation of protein arginine methyltransferase (PRMT) activities has been observed in many disease phenotypes. Small molecule probes that target PRMTs with strong affinity and selectivity can be used as valuable tools to dissect biological mechanisms of arginine methylation and establish the role of PRMT proteins in a disease process. In this work, we report synthesis and evaluation of a class of carbocyanine compounds containing indolium, benz[e]indolium or benz[c,d]indolium heterocyclic moieties that bind to the predominant arginine methyltransferase PRMT1 and inhibit its methyltransferase activity at low micromolar potencies. In particular, the developed molecules have long wavelength colorimetric and fluorometric photoactivities, which can be used for optical and near-infrared fluorescence imaging in cells or biological tissues. Together, these new chemical probes have potential application in PRMT studies both as enzyme inhibitors and as fluorescent dyes for microscope imaging.

Small Molecule Inhibitors of Protein Arginine Methyltransferases

“…In a general procedure, the radioisotope-labeled methyl group from [ 3 H]-SAM or [ 14 C]-SAM is first enzymatically transferred into a peptide or protein substrate of PRMTs. Prior to quantitation by autoradiography or liquid scintillation counting, the methylated substrates have to be separated from unreacted SAM by using different approaches such as polyacrylamide gel electrophoresis (radiometric gel assay, RGA) [70–76], pipette chromatography (ZipTip assay) [77], and filtering on glass fiber or phosphocellulose paper discs (radiometric filter assay, RFA) [37, 73, 78–90]. To eliminate the washing step required for the above described radiometric assays, scintillation proximity assay (SPA) has been implemented [80, 91–101], in which the scintillation signals depend on the micrometer proximity between biotinylated substrates and streptavidin-coated scintillants (either FlashPlates or streptavidin-coated microscopic beads).…”

“…We recently screened a serial carbocyanine compounds against PRMT1 using the RFA assay [78], among which compound 11 was discovered with promising potency (IC 50 = 4.1 μM) compared to AMI-1 and compound 13 ( stilbamidine) (137 μM and 105 μM, respectively, determined under the same assay condition [89]). This compound also inhibits PRMT3, -5 and -6 with slightly weaker potency but is totally inactive to CARM1.…”

“…Consistently, the global cellular methylation declines with 24h-treatment of 12 and the effect is significant at 100 nM of the compound for Meg01 and MOLM13 cells. One unique advantage of carbocyanine inhibitors is their strong fluorescent property, which can be utilized for subcellular tracking using fluorescence imaging microscopy [78]. …”

Histone Methyltransferase Inhibitors for Cancer Therapy

“…Later, Purandare et al identified a series of pyrazole amide containing compounds as potential inhibitors through high-throughput screening; preliminary optimization of these compounds led to Methylgene compound 7a ( [95,96]. It is also worth mentioning the carbocyanine inhibitors developed by Sinha et al, which are not only selective PRMT1 inhibitors (Table 17.1, entry 41, IC50 5 4.1 6 0.1) but also a photoactive chemical probe for the further study and characterization of PRMT inhibitors [98]. It is also worth mentioning the carbocyanine inhibitors developed by Sinha et al, which are not only selective PRMT1 inhibitors (Table 17.1, entry 41, IC50 5 4.1 6 0.1) but also a photoactive chemical probe for the further study and characterization of PRMT inhibitors [98].…”

2-{(E)-2-[(3E)-2-Chloro-3-{(2E)-2-[1,1-dimethyl-3-(3-phenylpropyl)-1,3-dihydro-2H-benzo[e]indol-2-ylidene]-ethylidene}cyclohex-1-en-1-yl]ethenyl}-1,1-dimethyl-3-(3-phenylpropyl)-1H-benzo[e]indolium Iodide

“…High-resolution accurate mass spectra (HRMS) were obtained either at the Georgia State University Mass Spectrometry Facility using a Waters Q-TOF micro (ESI-Q-TOF) mass spectrometer or utilizing a Waters Micromass LCT TOF ES+ Premier Mass Spectrometer (Waters Corporation, Milford, MA, USA). The synthetic procedures for the preparation of 1, 2, and 3 have been previously reported by our lab [15,16].…”