Facile Access to Branched Multispecific Proteins.

Approaches that leverage orthogonal chemical reactions to generate protein-protein conjugates have expanded access to bespoke chimeras. Although the literature is replete with examples of the semisynthesis of bispecific proteins, few methods exist for the semisynthesis of protein conjugates of higher complexity (i.e., greater than two-protein fusions). The recent emergence of trispecific cell engagers for immune cell redirection therapies necessitates the development of chemical methods for the construction of trispecific proteins that would otherwise be inaccessible via natural protein synthesis. Here, we demonstrate that 3-bromo-5-methylene pyrrolone (3Br-5MP) can be used to effect the facile chemical synthesis of trispecific peptides and proteins with exquisite control over the addition of each monomer. The multimeric complexes maintain epitope functionality both in human cells and upon immobilization. We anticipate that facile access to trispecific proteins using this 3Br-5MP will have broad utility in basic science research and will quicken the pace of research to establish novel, multimeric immune cell redirection therapies.

In Vitro and In Vivo Characterization of the Anti-Zika Virus Activity of ProTides of 2'-C-ß-Methylguanosine.

The ProTide approach has emerged as a powerful tool to improve the intracellular delivery of nucleotide analogs with antiviral and anticancer activity. Here, we characterized the anti-ZIKV (ZIKV, Zika virus) activity of two ProTides of 2'-C-ß-methylguanosine. ProTide UMN-1001 is a 2'-C-ß-methylguanosine tryptamine phosphoramidate monoester, and ProTide UMN-1002 is a 2-(methylthio)-ethyl-2'-C-ß-methylguanosine tryptamine phosphoramidate diester. UMN-1002 undergoes stepwise intracellular activation to the corresponding nucleotide monophosphate followed by P-N bond cleavage by intracellular histidine triad nucleotide binding protein 1 (Hint1). UMN-1001 is activated by Hint1 but is less cell-permeable than UMN-1002. UMN-1001 and UMN-1002 were found to be more potent than 2'-C-ß-methylguanosine against ZIKV in human-derived microvascular endothelial and neuroblastoma cells and in reducing ZIKV RNA replication. Studies with a newborn mouse model of ZIKV infection demonstrated that, while treatment with 2'-C-ß-methylguanosine and UMN-1001 was lethal, treatment with UMN-1002 was nontoxic and significantly reduced ZIKV infection. Our data suggests that anchimeric activated ProTides of 2'-C-ß-methyl nucleosides should be further investigated for their potential as anti-ZIKV therapeutics.

Profiling of Small Ribosomal Subunits Reveals Modes and Regulation of Translation Initiation.

Translation initiation is often attributed as the rate-determining step of eukaryotic protein synthesis and key to gene expression control. Despite this centrality, the series of steps involved in this process is poorly understood. Here, we capture the transcriptome-wide occupancy of ribosomes across all stages of translation initiation, enabling us to characterize the transcriptome-wide dynamics of ribosome recruitment to mRNAs, scanning across 5' UTRs and stop codon recognition, in a higher eukaryote. We provide mechanistic evidence for ribosomes attaching to the mRNA by threading the mRNA through the small subunit. Moreover, we identify features that regulate the recruitment and processivity of scanning ribosomes and redefine optimal initiation contexts. Our approach enables deconvoluting translation initiation into separate stages and identifying regulators at each step.

Repression of oncogenic cap-mediated translation by 4Ei-10 diminishes proliferation, enhances chemosensitivity and alters expression of malignancy-related proteins in mesothelioma.

Activated cap-dependent translation promotes cancer by stimulating translation of mRNAs encoding malignancy-promoting proteins. The nucleoside monophosphate Protide, 4Ei-10, undergoes intracellular uptake and conversion by Hint1 to form 7-Cl-Ph-Ethyl-GMP. 7-Cl-Ph-Ethyl-GMP is an analog of cap and inhibits protein translation by binding and sequestering eIF4E thus blocking eIF4E from binding to the mRNA cap. The effects of inhibiting translation initiation by disruption of the eIF4F complex with 4Ei-10 were examined in malignant mesothelioma (MM). In a cell-free assay system, formation of the eIF4F complex was disabled in response to exposure to 4Ei-10. Treatment of MM with 4Ei-10 resulted in decreased cell proliferation, increased sensitivity to pemetrexed and altered expression of malignancy-related proteins. In light of these findings, suppression of translation initiation by small molecule inhibitors like 4Ei-10 alone or in combination with pemetrexed represents an encouraging strategy meriting further evaluation in the treatment of MM.

Induction of the p53 Tumor Suppressor in Cancer Cells through Inhibition of Cap-Dependent Translation.

The p53 tumor suppressor plays a critical role in protecting normal cells from malignant transformation. Development of small molecules to reactivate p53 in cancer cells has been an area of intense research. We previously identified an internal ribosomal entry site (IRES) within the 5' untranslated region of p53 mRNA that mediates translation of the p53 mRNA independent of cap-dependent translation. Our results also show that in response to DNA damage, cells switch from cap-dependent translation to cap-independent translation of p53 mRNA. In the present study, we discovered a specific inhibitor of cap-dependent translation, 4EGI-1, that is capable of inducing the accumulation of p53 in cancer cells retaining wild-type p53. Our results show that 4EGI-1 causes an increase in p53 IRES activity, leading to increased translation of p53 mRNA. We also observed that 4EGI-1 induces cancer cell apoptosis in a p53-dependent manner. Furthermore, 4EGI-1 induces p53 in cancer cells without causing DNA double-strand breaks. In conclusion, we discovered a mechanistic link between inhibition of cap-dependent translation and enhanced p53 accumulation. This leads to apoptosis of cancer cells without causing collateral damage to normal cells, thus providing a novel and effective therapeutic strategy for cancer.

Anchimerically Activated ProTides as Inhibitors of Cap-Dependent Translation and Inducers of Chemosensitization in Mantle Cell Lymphoma.

The cellular delivery of nucleotides through various pronucleotide strategies has expanded the utility of nucleosides as a therapeutic class. Although highly successful, the highly popular ProTide system relies on a four-step enzymatic and chemical process to liberate the corresponding monophosphate. To broaden the scope and reduce the number of steps required for monophosphate release, we have developed a strategy that depends on initial chemical activation by a sulfur atom of a methylthioalkyl protecting group, followed by enzymatic hydrolysis of the resulting phosphoramidate monoester. We have employed this ProTide strategy for intracellular delivery of a nucleotide antagonist of eIF4E in mantle cell lymphoma (MCL) cells. Furthermore, we demonstrated that chemical inhibition of cap-dependent translation results in suppression of c-Myc expression, increased p27 expression, and enhanced chemosensitization to doxorubicin, dexamethasone, and ibrutinib. In addition, the new ProTide strategy was shown to enhance oral bioavailability of the corresponding monoester phosphoramidate.

Anchimerically Activatable Antiviral ProTides.

This work describes the synthesis and biological evaluation of an anchimerically activated proTide of 2'-C-ß-methylguanosine as an inhibitor of dengue virus 2 (DENV-2). The proTide incorporates a chemically cleavable 2-(methylthio)ethyl moiety and a HINT1 hydrolyzable tryptamine phosphoramidate. Inhibition of DENV-2 replication by proTide 6 was 5-fold greater than the parent nucleoside while displaying no apparent cytotoxicity. Furthermore, we demonstrate with a HINT1 inhibitor that the anti DENV-2 activity of the proTide correlates with the activity of HINT1. Taken together, these results demonstrate that a phosphoramidate based pronucleotide that undergoes an initial nonenzymatic activation step based on anchimeric assistance followed by P-N bond cleavage by HINT1 can be prepared.

Small-molecule inhibition of oncogenic eukaryotic protein translation in mesothelioma cells.

Deranged cap-mediated translation is implicated in the genesis, maintenance and progression of many human cancers including mesothelioma. In this study, disrupting the eIF4F complex by antagonizing the eIF4E-mRNA-cap interaction is assessed as a therapy for mesothelioma. Mesothelioma cells were treated with 4Ei-1, a membrane permeable prodrug that when converted to the active drug, 7-benzyl guanosine monophosphate (7Bn-GMP) displaces capped mRNAs from the eIF4F complex. Colony formation was measured in mesothelioma treated with 4Ei-1 alone or combined with pemetrexed. Proliferation was examined in cells treated with 4Ei-1. Binding to a synthetic cap-analogue was used to study the strength of eIF4F complex activation in lysates exposed to 4Ei-1. 4Ei-1 treatment resulted in a dose dependent decrease in colony formation and cell viability. Combination therapy of 4Ei-1 with pemetrexed further reduced colony number. Formation of eIF4F cap-complex decreased in response to 4Ei-1 exposure. 4Ei-1 is a novel prodrug that reduces proliferation, represses colony formation, diminishes association of eIF4F with the mRNA cap, and sensitizes mesothelioma cells to pemetrexed.