An exon skipping screen identifies antitumor drugs that are potent modulators of pre-mRNA splicing, suggesting new therapeutic applications.

Agents that modulate pre-mRNA splicing are of interest in multiple therapeutic areas, including cancer. We report our recent screening results with the application of a cell-based Triple Exon Skipping Luciferase Reporter (TESLR) using a library that is composed of FDA approved drugs, clinical compounds, and mechanistically characterized tool compounds. Confirmatory assays showed that three clinical antitumor therapeutic candidates (milciclib, PF-3758309 and PF-562271) are potent splicing modulators and that these drugs are, in fact, nanomolar inhibitors of multiple kinases involved in the regulation the spliceosome. We also report the identification of new SF3B1 antagonists (sudemycinol C and E) and show that these antagonists can be used to develop a displacement assay for SF3B1 small molecule ligands. These results further support the broad potential for the development of agents that target the spliceosome for the treatment of cancer and other diseases, as well as new avenues for the discovery of new chemotherapeutic agents for a range of diseases.

Splicing Factor 3B Subunit 1 Interacts with HIV Tat and Plays a Role in Viral Transcription and Reactivation from Latency.

The main obstacle to an HIV cure is the transcriptionally inert proviruses that persist in resting CD4 T cells and other reservoirs. None of the current approaches has significantly reduced the size of the viral reservoir. Hence, alternative approaches, such as permanent blocking of viral transcription, to achieve a sustained remission, need urgent attention. To identify cellular factors that may be important for this approach, we sought for host targets that when altered could block HIV transcription and reactivation. Here, we identified splicing factor 3B subunit 1 (SF3B1) as a critical HIV dependency factor required for viral replication. SF3B1 is a splicing factor involved in directing chromatin and nascent gene transcripts to appropriate splice sites. Inhibitors of SF3B1 are currently in development for cancer and have been found to be nontoxic to normal cells compared to malignant cells. Knockdown of SF3B1 abrogated HIV replication in all cell types tested. SF3B1 interacted with viral protein Tat in vitro and in vivo Genetic or pharmacologic inhibition of SF3B1 prevented Tat-mediated HIV transcription and RNA polymerase II association with the HIV promoter. In addition, an inhibitor of SF3B1 prevented HIV reactivation from latency irrespective of the latency-reversing agent used. The data show that SF3B1 is involved in viral transcription and reactivation from latency and may serve as a therapeutic target in the HIV cure efforts.IMPORTANCE The reason why HIV cannot be cured by current therapy is because of viral persistence in resting T cells. One approach to permanent HIV remission that has received less attention is the so-called "block and lock" approach. The idea behind this approach is that the virus could be permanently disabled in patients if viral genome or surrounding chromatin could be altered to silence the virus, thus enabling patients to stop therapy. In this work, we have identified splicing factor 3B subunit 1 (SF3B1) as a potential target for this approach. SF3B1 interacts with the viral protein Tat, which is critical for viral transcription. Inhibition of SF3B1 prevents HIV transcription and reactivation from latency. Since there are preclinical inhibitors for this protein, our findings could pave the way to silence HIV transcription, potentially leading to prolonged or permanent remission.

Protein-Structure Assisted Optimization of 4,5-Dihydroxypyrimidine-6-Carboxamide Inhibitors of Influenza Virus Endonuclease.

Influenza is a serious hazard to human health that causes hundreds of thousands of deaths annually. Though vaccines and current therapeutics can blunt some of the perilous impact of this viral infection, new treatments are needed due to the constantly evolving nature of this virus. Recently, our growing understanding of an essential influenza viral protein, PA, has led to the development of focused libraries of new small molecules that specifically target the active site of the PA influenza endonuclease, which we report here. Our overarching approach has been to proactively develop lead inhibitors that are less likely to rapidly develop clinical resistance by optimizing inhibitors that retain activity against induced resistant mutants. Here, we report details behind the discovery of new potent inhibitors of wild type and resistant mutant endonucleases along with their high-resolution co-crystal structure-activity relationships. These results add to our understanding of nuclease protein targets and potentially serve as starting points for a new therapeutic approach to the treatment of influenza.

A triple exon-skipping luciferase reporter assay identifies a new CLK inhibitor pharmacophore.

The splicing of pre-mRNA is a critical process in normal cells and is deregulated in cancer. Compounds that modulate this process have recently been shown to target a specific vulnerability in tumors. We have developed a novel cell-based assay that specifically activates luciferase in cells exposed to SF3B1 targeted compounds, such as sudemycin D6. This assay was used to screen a combined collection of approved drugs and bioactive compounds. This screening approach identified several active hits, the most potent of which were CGP-74514A and aminopurvalanol A, both have been reported to be cyclin-dependent kinases (CDKs) inhibitors. We found that these compounds, and their analogs, show significant cdc2-like kinase (CLK) inhibition and clear structure-activity relationships (SAR) at CLKs. We prepared a set of analogs and were able to 'dial out' the CDK activity and simultaneously developed CLK inhibitors with low nanomolar activity. Thus, we have demonstrated the utility of our exon-skipping assay and identified new molecules that exhibit potency and selectivity for CLK, as well as some structurally related dual CLK/CDK inhibitors.

Mutant U2AF1-expressing cells are sensitive to pharmacological modulation of the spliceosome.

Somatic mutations in spliceosome genes are detectable in ∼50% of patients with myelodysplastic syndromes (MDS). We hypothesize that cells harbouring spliceosome gene mutations have increased sensitivity to pharmacological perturbation of the spliceosome. We focus on mutant U2AF1 and utilize sudemycin compounds that modulate pre-mRNA splicing. We find that haematopoietic cells expressing mutant U2AF1(S34F), including primary patient cells, have an increased sensitivity to in vitro sudemycin treatment relative to controls. In vivo sudemycin treatment of U2AF1(S34F) transgenic mice alters splicing and reverts haematopoietic progenitor cell expansion induced by mutant U2AF1 expression. The splicing effects of sudemycin and U2AF1(S34F) can be cumulative in cells exposed to both perturbations-drug and mutation-compared with cells exposed to either alone. These cumulative effects may result in downstream phenotypic consequences in sudemycin-treated mutant cells. Taken together, these data suggest a potential for treating haematological cancers harbouring U2AF1 mutations with pre-mRNA splicing modulators like sudemycins.

The spliceosome is a therapeutic vulnerability in MYC-driven cancer.

MYC (also known as c-MYC) overexpression or hyperactivation is one of the most common drivers of human cancer. Despite intensive study, the MYC oncogene remains recalcitrant to therapeutic inhibition. MYC is a transcription factor, and many of its pro-tumorigenic functions have been attributed to its ability to regulate gene expression programs. Notably, oncogenic MYC activation has also been shown to increase total RNA and protein production in many tissue and disease contexts. While such increases in RNA and protein production may endow cancer cells with pro-tumour hallmarks, this increase in synthesis may also generate new or heightened burden on MYC-driven cancer cells to process these macromolecules properly. Here we discover that the spliceosome is a new target of oncogenic stress in MYC-driven cancers. We identify BUD31 as a MYC-synthetic lethal gene in human mammary epithelial cells, and demonstrate that BUD31 is a component of the core spliceosome required for its assembly and catalytic activity. Core spliceosomal factors (such as SF3B1 and U2AF1) associated with BUD31 are also required to tolerate oncogenic MYC. Notably, MYC hyperactivation induces an increase in total precursor messenger RNA synthesis, suggesting an increased burden on the core spliceosome to process pre-mRNA. In contrast to normal cells, partial inhibition of the spliceosome in MYC-hyperactivated cells leads to global intron retention, widespread defects in pre-mRNA maturation, and deregulation of many essential cell processes. Notably, genetic or pharmacological inhibition of the spliceosome in vivo impairs survival, tumorigenicity and metastatic proclivity of MYC-dependent breast cancers. Collectively, these data suggest that oncogenic MYC confers a collateral stress on splicing, and that components of the spliceosome may be therapeutic entry points for aggressive MYC-driven cancers.

Pharmacodynamic assays to facilitate preclinical and clinical development of pre-mRNA splicing modulatory drug candidates.

The spliceosome has recently emerged as a new target for cancer chemotherapy and novel antitumor spliceosome targeted agents are under development. Here, we describe two types of novel pharmacodynamic assays that facilitate drug discovery and development of this intriguing class of innovative therapeutics; the first assay is useful for preclinical optimization of small-molecule agents that target the SF3B1 spliceosomal protein in animals, the second assay is an ex vivo validated, gel-based assay for the measurement of drug exposure in human leukocytes. The first assay utilizes a highly specific bioluminescent splicing reporter, based on the skipping of exons 4-11 of a Luc-MDM2 construct, which specifically yields active luciferase when treated with small-molecule spliceosome modulators. We demonstrate that this reporter can be used to monitor alternative splicing in whole cells in vitro. We describe here that cell lines carrying the reporter can be used in vivo for the efficient pharmacodynamic analysis of agents during drug optimization and development. We also demonstrate dose- and time-dependent on-target activity of sudemycin D6 (SD6), which leads to dramatic tumor regression. The second assay relies on the treatment of freshly drawn human blood with SD6 ex vivo treatment. Changes in alternative splicing are determined by RT-PCR using genes previously identified in in vitro experiments. The Luc-MDM2 alternative splicing bioluminescent reporter and the splicing changes observed in human leukocytes should allow for the more facile translation of novel splicing modulators into clinical application.

The splicing modulator sudemycin induces a specific antitumor response and cooperates with ibrutinib in chronic lymphocytic leukemia.

Mutations or deregulated expression of the components of the spliceosome can influence the splicing pattern of several genes and contribute to the development of tumors. In this context, we report that the spliceosome modulator sudemycin induces selective cytotoxicity in primary chronic lymphocytic leukemia (CLL) cells when compared with healthy lymphocytes and tumor cells from other B-lymphoid malignancies, with a slight bias for CLL cases with mutations in spliceosome-RNA processing machinery. Consistently, sudemycin exhibits considerable antitumor activity in NOD/SCID/IL2Rγ-/- (NSG) mice engrafted with primary cells from CLL patients. The antileukemic effect of sudemycin involves the splicing modulation of several target genes important for tumor survival, both in SF3B1-mutated and -unmutated cases. Thus, the apoptosis induced by this compound is related to the alternative splicing switch of MCL1 toward its proapoptotic isoform. Sudemycin also functionally disturbs NF-κB pathway in parallel with the induction of a spliced RELA variant that loses its DNA binding domain. Importantly, we show an enhanced antitumor effect of sudemycin in combination with ibrutinib that might be related to the modulation of the alternative splicing of the inhibitor of Btk (IBTK). In conclusion, we provide first evidence that the spliceosome is a relevant therapeutic target in CLL, supporting the use of splicing modulators alone or in combination with ibrutinib as a promising approach for the treatment of CLL patients.

Sudemycin E influences alternative splicing and changes chromatin modifications.

Sudemycin E is an analog of the pre-messenger RNA splicing modulator FR901464 and its derivative spliceostatin A. Sudemycin E causes the death of cancer cells through an unknown mechanism. We found that similar to spliceostatin A, sudemycin E binds to the U2 small nuclear ribonucleoprotein (snRNP) component SF3B1. Native chromatin immunoprecipitations showed that U2 snRNPs physically interact with nucleosomes. Sudemycin E induces a dissociation of the U2 snRNPs and decreases their interaction with nucleosomes. To determine the effect on gene expression, we performed genome-wide array analysis. Sudemycin E first causes a rapid change in alternative pre-messenger RNA splicing, which is later followed by changes in overall gene expression and arrest in the G2 phase of the cell cycle. The changes in alternative exon usage correlate with a loss of the H3K36me3 modification in chromatin encoding these exons. We propose that sudemycin E interferes with the ability of U2 snRNP to maintain an H3K36me3 modification in actively transcribed genes. Thus, in addition to the reversible changes in alternative splicing, sudemycin E causes changes in chromatin modifications that result in chromatin condensation, which is a likely contributing factor to cancer cell death.

Pre-mRNA splicing-modulatory pharmacophores: the total synthesis of herboxidiene, a pladienolide-herboxidiene hybrid analog and related derivatives.

Herboxidiene is a natural product that has previously been shown to exhibit antitumor activity by targeting the spliceosome. This activity makes herboxidiene a valuable starting point for the development of anticancer drugs. Here, we report an improved enantioselective synthesis of herboxidiene and the first report of its biologically active totally synthetic analog: 6-norherboxidiene. The synthesis of the tetrahydropyran moiety utilizes the novel application of inverse electron-demand Diels-Alder chemistry and the Ferrier-type rearrangement as key steps. We report, for the first time, cytotoxicity IC50s for synthetic herboxidiene and analogs in human tumor cell lines. We have also demonstrated that synthetic herboxidiene and its analogs can potently modulate the alternate splicing of MDM-2 pre-mRNA.

Optimization of antitumor modulators of pre-mRNA splicing.

The spliceosome regulates pre-mRNA splicing, which is a critical process in normal mammalian cells. Recently, recurrent mutations in numerous spliceosomal proteins have been associated with a number of cancers. Previously, natural product antitumor agents have been shown to interact with one of the proteins that is subject to recurrent mutations (SF3B1). We report the optimization of a class of tumor-selective spliceosome modulators that demonstrate significant in vivo antitumor activity. This optimization culminated in the discovery of sudemycin D6, which shows potent cytotoxic activity in the melanoma line SK-MEL-2 (IC50 = 39 nM) and other tumor cell lines, including JeKo-1 (IC50 = 22 nM), HeLa (IC50 = 50 nM), and SK-N-AS (IC50 = 81 nM). We also report improved processes for the synthesis of these compounds. Our work supports the idea that sudemycin D6 is worthy of further investigation as a novel preclinical anticancer agent with application in the treatment of numerous human cancers.

Dissecting paclitaxel-microtubule association: quantitative assessment of the 2'-OH group.

Paclitaxel (PTX) is a microtubule-stabilizing agent that is widely used in cancer chemotherapy. This structurally complex natural product acts by binding to ß-tubulin in assembled microtubules. The 2'-hydroxyl group in the flexible side chain of PTX is an absolute requirement for activity, but its precise role in the drug-receptor interaction has not been specifically investigated. The contribution of the 2'-OH group to the affinity and tubulin-assembly efficacy of PTX has been evaluated through quantitative analysis of PTX derivatives possessing side chain deletions: 2'-deoxy-PTX, N-debenzoyl-2'-deoxy-PTX, and baccatin III. The affinity of 2'-deoxy-PTX for stabilized microtubules was more than 100-fold lower than that of PTX and only ~3-fold greater than the microtubule affinity of baccatin III. No microtubule binding activity was detected for the analogue N-debenzoyl-2'-deoxy-PTX. The tubulin-assembly efficacy of each ligand was consistent with the microtubule binding affinity, as was the trend in cytotoxicities. Molecular dynamics simulations revealed that the 2'-OH group of PTX can form a persistent hydrogen bond with D26 within the microtubule binding site. The absence of this interaction between 2'-deoxy-PTX and the receptor can account for the difference in binding free energy. Computational analyses also provide a possible explanation for why N-debenzoyl-2'-deoxy-PTX is inactive, in spite of the fact that it is essentially a substituted baccatin III. We propose that the hydrogen bonding interaction between the 2'-OH group and D26 is the most important stabilizing interaction that PTX forms with tubulin in the region of the C-13 side chain. We further hypothesize that the substituents at the 3'-position function to orient the 2'-OH group for a productive hydrogen bonding interaction with the protein.

Radiosynthesis of antitumor spliceosome modulators.

A set of novel antitumor agents (the sudemycins) has recently been described that are analogs of the natural product FR901464. We report the radiosynthesis of two of these antitumor drug lead compounds, using a three step procedure: (1) ester hydrolysis, (2) Lindlar's catalyst/tritium gas to give a (S,Z)-4-acetoxypent-2-enoic acid derivative, and finally (3) amide bond formation. These labeled analogs are useful in developing a better understanding of the pharmacological properties of this new class of therapeutic lead compounds.

Sudemycins, novel small molecule analogues of FR901464, induce alternative gene splicing.

Two unrelated bacterial natural products, FR901464 and pladienolide B, have previously been shown to have significant antitumor activity in vivo. These compounds target the SF3b subunit of the spliceosome, with a derivative of pladienolide (E7107) entering clinical trials for cancer. However, due to the structural complexity of these molecules, their research and development has been significantly constrained. We have generated a set of novel analogues (Sudemycins) that possess the pharmacophore that is common to FR901464 and pladienolide, via a flexible enantioselective route, which allows for the production of gram quantities of drug. These compounds demonstrate cytotoxicity toward human tumor cell lines in culture and exhibit antitumor activity in a xenograft model. Here, we present evidence that Sudemycins are potent modulators of alternative splicing in human cells, both of endogenous genes and from minigene constructs. Furthermore, levels of alternative splicing are increased in tumor cells relative to normal cells, and these modifications can be observed in human tumor xenografts in vivo following exposure of animals to the drug. In addition, the change in the splicing pattern observed with the Sudemycins are similar to that observed with Spliceostatin A, a molecule known to interact with the SF3b subunit of the spliceosome. Hence, we conclude that Sudemycins can regulate the production of alternatively spliced RNA transcripts and these alterations are more prevalent in tumors, as compared to normal cells, following drug exposure. These studies suggest that modulation of alternative splicing may play a role in the antitumor activity of this class of agents.

Synthetic mRNA splicing modulator compounds with in vivo antitumor activity.

We report our progress on the development of new synthetic anticancer lead compounds that modulate the splicing of mRNA. We also report the synthesis and evaluation of new biologically active ester and carbamate analogues. Further, we describe initial animal studies demonstrating the antitumor efficacy of compound 5 in vivo. Additionally, we report the enantioselective and diastereospecific synthesis of a new 1,3-dioxane series of active analogues. We confirm that compound 5 inhibits the splicing of mRNA in cell-free nuclear extracts and in a cell-based dual-reporter mRNA splicing assay. In summary, we have developed totally synthetic novel spliceosome modulators as therapeutic lead compounds for a number of highly aggressive cancers. Future efforts will be directed toward the more complete optimization of these compounds as potential human therapeutics.

Antitumor compounds based on a natural product consensus pharmacophore.

We report the design and highly enantioselective synthesis of a potent analogue of the spliceosome inhibitor FR901464, based on a non-natural product scaffold. The design of this compound was facilitated by a pharmacophore hypothesis that assumed key interaction types that are common to FR901464 and an otherwise unrelated natural product (pladienolide). The synthesis allows for the preparation of numerous novel analogues. We present results on the in vitro activity for this compound against several tumor cell lines.

Specificity of the N-benzoyl transferase responsible for the last step of Taxol biosynthesis.

The last few steps in the biosynthesis of the anticancer drug Taxol in yew (Taxus) species are thought to involve the attachment of beta-phenylalanine to the C13-O-position of the advanced taxane diterpenoid intermediate baccatin III to yield N-debenzoyl-2'-deoxytaxol, followed by hydroxylation on the side chain at the C2'-position to afford N-debenzoyltaxol, and finally N-benzoylation to complete the pathway. A cDNA encoding the N-benzoyl transferase that catalyzes the terminal step of the reaction sequence was previously isolated from a family of transferase clones (derived from an induced Taxus cell cDNA library) by functional characterization of the corresponding recombinant enzyme using the available surrogate substrate N-debenzoyl-2'-deoxytaxol [K. Walker, R. Long, R. Croteau, Proc. Nat. Acad. Sci. USA 99 (2002) 9166-9171]. Semi-synthetic N-debenzoyltaxol was prepared by coupling of 7-triethylsilybaccatin III and (2R,3S)-beta-phenylisoserine protected as the N-Boc N,O-isopropylidene derivative by means of carbodiimide activation and formic acid deprotections. The selectivity of the recombinant N-transferase for N-debenzoyltaxol was evaluated, and the enzyme was shown to prefer, by a catalytic efficiency factor of two, N-debenzoyltaxol over N-debenzoyl-2'-deoxytaxol as the taxoid co-substrate in the benzoyl transfer reaction, consistent with the assembly sequence involving 2'-hydroxylation prior to N-benzoylation. Selectivity for the acyl/aroyl-CoA co-substrate was also examined, and the enzyme was shown to prefer benzoyl-CoA. Transfer from tigloyl-CoA to N-debenzoyltaxol to afford cephalomannine (Taxol B) was not observed, nor was transfer observed from hexanoyl-CoA or butanoyl-CoA to yield Taxol C or Taxol D, respectively. These results support the proposed sequence of reactions for C13-O-side chain assembly in Taxol biosynthesis, and suggest that other N-transferases are responsible for the formation of related, late pathway, N-acylated taxoids.

The dioxanone approach to (2S,3R)-2-C-methylerythritol 4-phosphate and 2,4-cyclodiphosphate, and various MEP analogues.

Efficient syntheses of the non-mevalonate pathway intermediates 2-C-methylerythritol 4-phosphate (MEP) and 2-C-methylerythritol 2,4-cyclodiphosphate (ME-2,4-cycloPP), as well as the parent tetrol 2-C-methylerythritol, in enantiopure form from (2S,4R)-cis-2-phenyl-4-tert-butyldimethylsilyloxy-1,3-dioxan-5-one are reported. The 2S configuration of the C-methyl group was installed by highly axial-face selective addition of CH3MgBr (20:1) to the chiral dioxanone carbonyl group. Primary selective mono-phosphorylation and 2,4-bis-phosphorylation, followed by desilation and hydrogenolysis to the free mono- and diphosphates, and, in the latter case, cyclization to form the eight-membered phosphoryl anhydride, afforded MEP and ME-2,4-cycloPP in good yields. The C2 epimeric analogues, 2-C-methylthreitol and its 4-phosphate, were accessed by LiAlH4 reduction of the cis,cis epoxide of (2S,4R)-4-tert-butyldimethylsilyloxymethyl-5-methylene-2-phenyl-1,3-dioxane, primary-selective phosphorylation, and cleavage of the silyl, benzylidene, and benzyl protecting groups. Regioselective cleavage of the acetal ring of 1,3-benzylidene 2-C-methylerythritol silyl ether by ozonolysis afforded a 1,2,3-triol 3-monobenzoate intermediate that was converted to the novel amino sugar, 1-amino-1-deoxy-2-C-methylerythritol.