Identifying the molecular targets for the beneficial or detrimental effects of small-molecule drugs is an important and currently unmet challenge. We have developed a method, drug affinity responsive ...target stability (DARTS), which takes advantage of a reduction in the protease susceptibility of the target protein upon drug binding. DARTS is universally applicable because it requires no modification of the drug and is independent of the mechanism of drug action. We demonstrate use of DARTS to identify known small-molecule-protein interactions and to reveal the eukaryotic translation initiation machinery as a molecular target for the longevity-enhancing plant natural product resveratrol. We envisage that DARTS will also be useful in global mapping of protein-metabolite interaction networks and in label-free screening of unlimited varieties of compounds for development as molecular imaging agents.
The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyperactivation of the mammalian TOR (mTOR) pathway in human cancers, strategies to enhance TOR ...pathway inhibition are needed. We used a yeast-based screen to identify small-molecule enhancers of rapamycin (SMERs) and discovered an inhibitor (SMER3) of the Skp1-Cullin-F-box (SCF)Met30 ubiquitin ligase, a member of the SCF E3-ligase family, which regulates diverse cellular processes including transcription, cell-cycle control and immune response. We show here that SMER3 inhibits SCFMet30 in vivo and in vitro, but not the closely related SCFCdc4. Furthermore, we demonstrate that SMER3 diminishes binding of the F-box subunit Met30 to the SCF core complex in vivo and show evidence for SMER3 directly binding to Met30. Our results show that there is no fundamental barrier to obtaining specific inhibitors to modulate function of individual SCF complexes.
•DS-5670a, an mRNA vaccine, targets the RBD of the SARS-CoV-2 spike protein.•Immunologically-naïve adults received 2 injections of DS-5670a, 4 weeks apart.•Safety, immunogenicity and pharmacokinetics ...were evaluated in two dose groups.•DS-5670a was well tolerated and most TEAEs were mild or moderate.•60 µg of mRNA provided greater humoral immunity and will be investigated further.
DS-5670a is a vaccine candidate for coronavirus disease 2019 (COVID-19) harnessing a novel modality composed of messenger ribonucleic acid (mRNA) encoding the receptor-binding domain (RBD) from the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encapsulated in lipid nanoparticles. Here, we report the safety, immunogenicity, and pharmacokinetic profile of DS-5670a from a phase 2 clinical trial in healthy adults who were immunologically naïve to SARS-CoV-2.
The study consisted of an open-label, uncontrolled, dose-escalation part and a double-blind, randomized, uncontrolled, 2-arm, parallel-group part. A total of 80 Japanese participants were assigned to receive intramuscular DS-5670a, containing either 30 or 60 µg of mRNA, as two injections administered 4 weeks apart. Safety was assessed by characterization of treatment-emergent adverse events (TEAEs). Immunogenicity was assessed by neutralization titers against SARS-CoV-2, anti-RBD immunoglobulin (Ig)G levels, and SARS-CoV-2 spike-specific T cell responses. Plasma pharmacokinetic parameters of DS-5670a were also evaluated.
Most solicited TEAEs were mild or moderate with both the 30 and 60 µg mRNA doses. Four participants (10%) in the 60 µg mRNA group developed severe redness at the injection site, but all cases resolved without treatment. There were no serious TEAEs and no TEAEs leading to discontinuation. Humoral immune responses in both dose groups were greater than those observed in human convalescent serum; the 60 µg mRNA dose produced better responses. Neutralization titers were found to be correlated with anti-RBD IgG levels (specifically IgG1). DS-5670a elicited antigen-specific T helper 1-polarized cellular immune responses.
The novel mRNA-based vaccine candidate DS-5670a provided favorable immune responses against SARS-CoV-2 with a clinically acceptable safety profile. Confirmatory trials are currently ongoing to evaluate the safety and immunogenicity of DS-5670a as the primary vaccine and to assess the immunogenicity when administered as a heterologous or homologous booster.
Trial registry: https://jrct.niph.go.jp/latest-detail/jRCT2071210086.
The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control
1
. With prevalent hyper-activation of the mTOR pathway in human cancers
2
, novel strategies to enhance TOR ...pathway inhibition are highly desirable. We used a yeast-based platform to identify small-molecule enhancers of rapamycin (SMERs) and discovered an inhibitor of the SCF
Met30
ubiquitin ligase (SMER3). The large SCF (Skp1-Cullin-F-box) family of ubiquitin ligases performs important functions in diverse cellular processes including transcription, cell-cycle control, and immune response
3
. Accordingly, there would be great value in developing SCF ligase inhibitors that act by a defined mechanism to specifically inactivate ligase activity. We show here that SMER3 selectively inhibits SCF
Met30
in vivo
and
in vitro
, but not the closely related SCF
Cdc4
. Our results demonstrate that there is no fundamental barrier to obtaining specific inhibitors to modulate function of individual SCF complexes, and suggest new strategies for combination therapy with rapamycin.
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