Identification of a small molecule splicing inhibitor targeting UHM domains

Abstract

Splicing factor mutations are frequent in myeloid neoplasms, blood cancers, and solid tumors. Cancer cells harboring these mutations present a particular vulnerability to drugs that target splicing factors such as SF3b155 or CAPERα. Still, the arsenal of chemical probes that target the spliceosome is very limited. U2AF homology motifs (UHMs) are common protein interaction domains among splicing factors. They present a hydrophobic pocket ideally suited to anchor small molecules with the aim to inhibit protein-protein interaction. Here, we combined a virtual screening of a small molecules database and an in vitro competition assay and identified a small molecule, we named UHMCP1 that prevents the SF3b155/U2AF 65 interaction. NMR analyses and molecular dynamics simulations confirmed the binding of this molecule in the hydrophobic pocket of the U2AF 65 UHM domain. We further provide evidence that UHMCP1 impacts RNA splicing and cell viability and is therefore an interesting novel compound targeting an UHM domain with potential anticancer properties.

Somatic Mutations in Core Spliceosome Components Promote Tumorigenesis and Generate an Exploitable Vulnerability in Human Cancer

“…Recently, a high throughput screening identified a small molecule, UHMCP1, which prevents the SF3B1/U2AF65 interaction. UHMCP1 interacts with the hydrophobic pocket of the U2AF65 UHM domain, thus strongly impacting RNA splicing and cell viability of cancer cells [ 80 ]. Another study identified phenothiazine derivatives, such as 7,8-dihydroxyperphenazine and 7,8-dimethoxyperphenazine, as molecules targeting the early assembly of the spliceosome [ 81 ].…”

Profiling the Binding Activities of Peptides and Inhibitors to the U2 Auxiliary Factor Homology Motif (UHM) Domains

“…UHMCP1 ( 1 ) and analogs ( 2 – 7 ) were prepared via a reaction sequence of reductive amination and subsequent deprotection of Boc group using HCl or hydrolysis of an ester group as shown in Scheme . Note that UHMCP1 ( 1 ) and 4 have four stereoisomers according to the report, whereas 2 , 3 , and 5 – 7 have two stereoisomers, respectively.…”

“…The binding affinity of UHMCP1 to other three UHM domains was much weaker. Based on the microplate assay, UHMCP1 had an IC 50 value of 30 μM to U2AF2-UHM . When the amine group was positioned in the fused diazabicyclo-octane ring, the IC 50 values of 2 to SPF45-UHM, RBM39-UHM and PUF60-UHM were not markedly changed but were ∼5-fold lower with U2AF1-UHM.…”

“…In addition to the ULMs from splicing factors, we included a recently discovered ULM in Rev HIV-1 and a less well-characterized ULM in ATXN1. Small-molecule inhibitors targeting the UHM domains have recently been reported. − We synthesized seven analogous compounds based on the tryptophan scaffold and characterized the selectivity of these inhibitors against different UHM domains.…”

“…Another study by Hanzawa et al identified a PUF60-UHM inhibitor, DS89092425, and reported that a terminal amine group in DS89092425 contributed importantly to the binding affinity. Recently, Kobayashi et al combined virtual screening and in vitro assays to discover an indole containing small-molecule inhibitor (UHMCP1, Figure ) of the U2AF2-UHM domain (IC 50 ∼ 30 μM) . UHMCP1 and cmp9 were reported to induce splicing pattern changes in selective genes by inhibiting the UHM domains in HEK293 cells and a reconstituted minigene assay respectively.…”