Vitaliy M. Sviripa

Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii.

Botryococcene biosynthesis is thought to resemble that of squalene, a metabolite essential for sterol metabolism in all eukaryotes. Squalene arises from an initial condensation of two molecules of farnesyl diphosphate (FPP) to form presqualene diphosphate (PSPP), which then undergoes a reductive rearrangement to form squalene. In principle, botryococcene could arise from an alternative rearrangement of the presqualene intermediate. Because of these proposed similarities, we predicted that a botryococcene synthase would resemble squalene synthase and hence isolated squalene synthase-like genes from Botryococcus braunii race B. While B. braunii does harbor at least one typical squalene synthase, none of the other three squalene synthase-like (SSL) genes encodes for botryococcene biosynthesis directly. SSL-1 catalyzes the biosynthesis of PSPP and SSL-2 the biosynthesis of bisfarnesyl ether, while SSL-3 does not appear able to directly utilize FPP as a substrate. However, when combinations of the synthase-like enzymes were mixed together, in vivo and in vitro, robust botryococcene (SSL-1+SSL-3) or squalene biosynthesis (SSL1+SSL-2) was observed. These findings were unexpected because squalene synthase, an ancient and likely progenitor to the other Botryococcus triterpene synthases, catalyzes a two-step reaction within a single enzyme unit without intermediate release, yet in B. braunii, these activities appear to have separated and evolved interdependently for specialized triterpene oil production greater than 500 MYA. Coexpression of the SSL-1 and SSL-3 genes in different configurations, as independent genes, as gene fusions, or targeted to intracellular membranes, also demonstrate the potential for engineering even greater efficiencies of botryococcene biosynthesis.

Fluorinated N,N-dialkylaminostilbenes repress colon cancer by targeting methionine S-adenosyltransferase 2A.

Methionine S-adenosyltransferase 2A (MAT2A) is the catalytic subunit for synthesis of S-adenosylmethionine (SAM), the principal methyl donor in many biological processes. MAT2A is up-regulated in many cancers, including liver cancer and colorectal cancer (CRC) and is a potentially important drug target. We developed a family of fluorinated N,N-dialkylaminostilbene agents, called FIDAS agents, that inhibit the proliferation of CRC cells in vitro and in vivo. Using a biotinylated FIDAS analogue, we identified the catalytic subunit of MAT2A as the direct and exclusive binding target of these FIDAS agents. MAT2B, an associated regulatory subunit of MAT2A, binds indirectly to FIDAS agents through its association with MAT2A. FIDAS agents inhibited MAT2A activity in SAM synthesis, and depletion of MAT2A by shRNAs inhibited CRC cell growth. A novel FIDAS agent delivered orally repressed CRC xenografts in athymic nude mice. These findings suggest that FIDAS analogues targeting MAT2A represent a family of novel and potentially useful agents for cancer treatment.

Fluorinated N,N-Dialkylaminostilbenes for Wnt Pathway Inhibition and Colon Cancer Repression

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality in the United States. CRC is initiated by mutations of the tumor suppressor gene, adenomatous polyposis coli (APC), or β-catenin gene. These mutations stabilize β-catenin and constitutively activate Wnt/β-catenin target genes, such as c-Myc and cyclin D1, ultimately leading to cancer. Naturally occurring stilbene derivatives, resveratrol and pterostilbene, inhibit Wnt signaling and repress CRC cell proliferation but are ineffective at concentrations less than 10 μM. To understand the structure--activity relationship within these stilbene derivatives and to develop more efficacious Wnt inhibitors than these natural products, we synthesized and evaluated a panel of fluorinated N,N-dialkylaminostilbenes. Among this panel, (E)-4-(2,6-difluorostyryl)-N,N-dimethylaniline (4r) inhibits Wnt signaling at nanomolar levels and inhibits the growth of human CRC cell xenografts in athymic nude mice at a dosage of 20 mg/kg. These fluorinated N,N-dialkylaminostilbenes appear to inhibit Wnt signaling downstream of β-catenin, probably at the transcriptional level.

Synthesis and characterization of pseudocantharidins, novel phosphatase modulators that promote the inclusion of exon 7 into the SMN (survival of motoneuron) pre-mRNA.

Alternative pre-mRNA splicing is a central element of eukaryotic gene expression. Its deregulation can lead to disease, and methods to change splice site selection are developed as potential therapies. Spinal muscular atrophy is caused by the loss of the SMN1 (survival of motoneuron 1) gene. A therapeutic avenue for spinal muscular atrophy treatment is to promote exon 7 inclusion of the almost identical SMN2 (survival of motoneuron 2) gene. The splicing factor tra2-beta1 promotes inclusion of this exon and is antagonized by protein phosphatase (PP) 1. To identify new compounds that promote exon 7 inclusion, we synthesized analogs of cantharidin, an inhibitor of PP1, and PP2A. Three classes of compounds emerged from these studies. The first class blocks PP1 and PP2A activity, blocks constitutive splicing in vitro, and promotes exon 7 inclusion in vivo. The second class has no measurable effect on PP1 activity but activates PP2A. This class represents the first compounds described with these properties. These compounds cause a dephosphorylation of Thr-33 of tra2-beta1, which promotes exon 7 inclusion. The third class had no detectable effect on phosphatase activity and could promote exon 7 via allosteric effects. Our data show that subtle changes in similar compounds can turn a phosphatase inhibitor into an activator. These chemically related compounds influence alternative splicing by distinct mechanisms.

2',6'-Dihalostyrylanilines, pyridines, and pyrimidines for the inhibition of the catalytic subunit of methionine S-adenosyltransferase-2.

Inhibition of the catalytic subunit of the heterodimeric methionine S-adenosyl transferase-2 (MAT2A) with fluorinated N,N-dialkylaminostilbenes (FIDAS agents) offers a potential avenue for the treatment of liver and colorectal cancers where upregulation of this enzyme occurs. A study of structure–activity relationships led to the identification of the most active compounds as those with (1) either a 2,6-difluorostyryl or 2-chloro-6-fluorostyryl subunit, (2) either an N-methylamino or N,N-dimethylamino group attached in a para orientation relative to the 2,6-dihalostyryl subunit, and (3) either an N-methylaniline or a 2-(N,N-dimethylamino)pyridine ring. These modifications led to FIDAS agents that were active in the low nanomolar range, that formed water-soluble hydrochloride salts, and that possessed the desired property of not inhibiting the human hERG potassium ion channel at concentrations at which the FIDAS agents inhibit MAT2A. The active FIDAS agents may inhibit cancer cells through alterations of methylation reactions essential for cancer cell survival and growth.

Arylquins target vimentin to trigger Par-4 secretion for tumor cell apoptosis

The tumor suppressor protein Par-4, which is secreted by normal cells, selectively induces apoptosis in cancer cells. We identified a 3-arylquinoline derivative, designated Arylquin 1, as a potent Par-4 secretagogue in cell cultures and mice. Mechanistically, Arylquin 1 binds to vimentin, displaces Par-4 from vimentin for secretion and triggers the efficient paracrine apoptosis of diverse cancer cells. Thus, targeting vimentin with Par-4 secretagogues efficiently induces paracrine apoptosis of tumor cells.

Antineoplastic Isoflavonoids Derived from Intermediate ortho-Quinone Methides Generated from Mannich Bases.

The regioselective condensations of various 7‐hydroxyisoflavonoids with bis(N,N‐dimethylamino)methane in a Mannich reaction provided C‐8 N,N‐dimethylaminomethyl‐substituted isoflavonoids in good yield. Similar condensations of 7‐hydroxy‐8‐methylisoflavonoids led to the C‐6‐substituted analogs. Thermal eliminations of dimethylamine from these C‐6 or C‐8 N,N‐dimethylaminomethyl‐substituted isoflavonoids generated ortho‐quinone methide intermediates within isoflavonoid frameworks for the first time. Despite other potential competing outcomes, these ortho‐quinone methide intermediates trapped dienophiles including 2,3‐dihydrofuran, 3,4‐dihydro‐2H‐pyran, 3‐(N,N‐dimethylamino)‐5,5‐dimethyl‐2‐cyclohexen‐1‐one, 1‐morpholinocyclopentene, and 1‐morpholinocyclohexene to give various inverse electron‐demand Diels–Alder adducts. Several adducts derived from 8‐N,N‐dimethylaminomethyl‐substituted isoflavonoids displayed good activity in the 1–10 μm concentration range in an in vitro proliferation assay using the PC‐3 prostate cancer cell line.

Halogenated diarylacetylenes repress c-myc expression in cancer cells.

Halogenated diarylacetylenes that possess fluorine or chlorine substituents in one aryl ring and N-methylamino or N,N-dimethylamino in the other aryl ring inhibit the proliferation of LS174T colon cancer cells through the repression of c-myc expression and induction of the cyclin-dependent kinase inhibitor-1 (i.e., p21(Wif1/Cip1)) and represent potentially useful antineoplastic agents.

Fluorinated N,N'-Diarylureas As Novel Therapeutic Agents Against Cancer Stem Cells

Colorectal cancer is the second-leading cause of cancer-related mortality in the United States. More than 50% of patients with colorectal cancer will develop local recurrence or distant organ metastasis. Cancer stem cells play a major role in the survival and metastasis of cancer cells. In this study, we examined the effects of novel AMP-activated protein kinase (AMPK) activating compounds on colorectal cancer metastatic and stem cell lines as potential candidates for chemotherapy. We found that activation of AMPK by all fluorinated N,N-diarylureas (FND) compounds at micromolar levels significantly inhibited the cell-cycle progression and subsequent cellular proliferation. In addition, we demonstrated that select FNDs significantly increased apoptosis in colorectal cancer metastatic and cancer stem cells. Therefore, FNDs hold considerable promise in the treatment of metastatic colorectal cancer, through elimination of both regular cancer cells and cancer stem cells. Mol Cancer Ther; 16(5); 831–7. ©2017 AACR.

N-Aryl benzenesulfonamide inhibitors of [3H]-thymidine incorporation and β-catenin signaling in human hepatocyte-derived Huh-7 carcinoma cells.

Structure-activity relationships (SAR) in 2,5-dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535) were examined as part of a program to identify agents that inhibit the Wnt/β-catenin signaling pathway that is frequently upregulated in hepatocellular carcinoma (HCC). FH535 was reported as an inhibitor of both β-catenin in the Wnt signaling pathway and the peroxisome proliferator-activated receptor (PPAR). A β-catenin/T-cell factor (TCF)/Lymphoid-enhancer factor (LEF)-dependent assay (i.e., luciferase-based TOPFlash assay) as well as a [(3)H]-thymidine incorporation assay were used to explore SAR modifications of FH535. Although replacing the 2,5-dichlorophenylsulfonyl substituent in FH535 with a 2,6-dihalogenation pattern generally produced more biologically active analogs than FH535, other SAR modifications led only to FH535 analogs with comparable or slightly improved activity in these two assays. The absence of a clear SAR pattern in activity suggested a multiplicity of target effectors for N-aryl benzenesulfonamides.