Alex H. F. Lee

Itraconazole and Arsenic Trioxide Inhibit Hedgehog Pathway Activation and Tumor Growth Associated with Acquired Resistance to Smoothened Antagonists

Recognition of the multiple roles of Hedgehog signaling in cancer has prompted intensive efforts to develop targeted pathway inhibitors. Leading inhibitors in clinical development act by binding to a common site within Smoothened, a critical pathway component. Acquired Smoothened mutations, including SMO(D477G), confer resistance to these inhibitors. Here, we report that itraconazole and arsenic trioxide, two agents in clinical use that inhibit Hedgehog signaling by mechanisms distinct from that of current Smoothened antagonists, retain inhibitory activity in vitro in the context of all reported resistance-conferring Smoothened mutants and GLI2 overexpression. Itraconazole and arsenic trioxide, alone or in combination, inhibit the growth of medulloblastoma and basal cell carcinoma in vivo, and prolong survival of mice with intracranial drug-resistant SMO(D477G) medulloblastoma.

Polymerase Amplification, Cloning, and Gene Expression of Benzo-Homologous yDNA Base Pairs

A widened DNA base‐pair architecture is studied in an effort to explore the possibility of whether new genetic system designs might possess some of the functions of natural DNA. In the “yDNA” system, pairs are homologated by addition of a benzene ring, which yields (in the present study) benzopyrimidines that are correctly paired with purines. Here we report initial tests of ability of the benzopyrimidines yT and yC to store and transfer biochemical and biological information in vitro and in bacterial cells. In vitro primer extension studies with two polymerases showed that the enzymes could insert the correct nucleotides opposite these yDNA bases, but with low selectivity. PCR amplifications with a thermostable polymerase resulted in correct pairings in 15–20 % of the cases, and more successfully when yT or yC were situated within the primers. Segments of DNA containing one or two yDNA bases were then ligated into a plasmid and tested for their ability to successfully lead the expression of an active protein in vivo. Although active at only a fraction of the activity of fully natural DNA, the unnatural bases encoded the correct codon bases in the majority of cases when singly substituted, and yielded functioning green fluorescent protein. Although the activities with native polymerases are modest with these large base pairs, this is the first example of encoding protein in vivo by an unnatural DNA base pair architecture.

Synthesis of 5-(7-hydroxyhept-3-enyl)-1,2-dithiolan-3-one 1-oxide, a core functionality of antibiotic leinamycin

5-(7-Hydroxyhept-3-enyl)-1,2-dithiolan-3-one 1-oxide 1 possessing both the 1,2-dithiolan-3-one 1-oxide five-membered ring and the double bond at the gamma position of the heterocycle, characteristic of the antibiotic leinamycin, was synthesized. In addition, the activated ester form of 1 was prepared that may be useful for coupling 1 to certain DNA-binding agents.

Structure and Replication of yDNA: A Novel Genetic Set Widened by Benzo-homologation

In a functioning genetic system, the information‐encoding molecule must form a regular self‐complementary complex (for example, the base‐paired double helix of DNA) and it must be able to encode information and pass it on to new generations. Here we study a benzo‐widened DNA‐like molecule (yDNA) as a candidate for an alternative genetic set, and we explicitly test these two structural and functional requirements. The solution structure of a 10 bp yDNA duplex is measured by using 2D‐NMR methods for a simple sequence composed of T–yA/yA–T pairs. The data confirm an antiparallel, right‐handed, hydrogen‐bonded helix resembling B‐DNA but with a wider diameter and enlarged base‐pair size. In addition to this, the abilities of two different polymerase enzymes (Klenow fragment of DNA pol I (Kf) and the repair enzyme Dpo4) to synthesize and extend the yDNA pairs T–yA, A–yT, and G–yC are measured by steady‐state kinetics studies. Not surprisingly, insertion of complementary bases opposite yDNA bases is inefficient due to the larger base‐pair size. We find that correct pairing occurs in several cases by both enzymes, but that common and relatively efficient mispairing involving T–yT and T–yC pairs interferes with fully correct formation and extension of pairs by these polymerases. Interestingly, the data show that extension of the large pairs is considerably more efficient with the flexible repair enzyme (Dpo4) than with the more rigid Kf enzyme. The results shed light on the properties of yDNA as a candidate for an alternative genetic information‐encoding molecule and as a tool for application in basic science and biomedicine.

G-quadruplex as a new class of structural entities for directing the formation of circular oligodeoxyribonucleotides

It has been demonstrated for the first time that G-quadruplex is capable of acting as a template for directing the sequence-specific formation of certain circular oligodeoxyribonucleotides with high efficiency.

Pilot study on the bioactivity of vitamin d in the skin after oral supplementation.

Laboratory studies suggest that vitamin D (VD) supplementation inhibits skin carcinogenesis. However, epidemiologic studies report mixed findings in the association between circulating VD levels and skin cancer risk. We conducted a clinical study to determine whether oral cholecalciferol supplementation would exert direct bioactivity in human skin through modulation of the VD receptor (VDR). We enrolled 25 individuals with serum 25-hydroxyvitamin-D levels <30 ng/mL and with skin photodamage to take 50,000 IU of cholecalciferol biweekly for 8 to 9 weeks. Then, we obtained baseline and end-of-study skin biopsies from photodamaged (PD) and photoprotected (PP) skin, and from benign nevi (BN) and tested for mRNA expression of VDR and cytochrome P450-24 (CYP24), and markers of keratinocytic differentiation. High-dose cholecalciferol supplementation significantly elevated circulating levels of 25-hydroxyvitamin-D (P < 0.0001) and 1,25-dihydroxyvitamin-D (P < 0.0001). VDR expression in PD- and PP-skin showed minimum changes after supplementation. CYP24 expression in PD- and PP-skin was increased after supplementation by 186%, P = 0.08, and 134%, P = 0.07, respectively. In BNs from 11 participants, a trend for higher VDR and CYP24 expression was observed (average of 20%, P = 0.08, and 544%, P = 0.09, respectively). Caspase-14 expression at the basal layer in PD skin samples was the only epidermal differentiation marker that was significantly increased (49%, P < 0.0001). High-dose cholecalciferol supplementation raised serum VD metabolite levels concurrently with CYP24 mRNA and caspase-14 levels in the skin. Our findings of significant variability in the range of VDR and CYP24 expression across study samples represent an important consideration in studies evaluating the role of VD as a skin cancer chemopreventive agent. Cancer Prev Res; 8(6); 563–9. ©2015 AACR.

Synthesis of 5-(7-Hydroxyheptyl)-1,2-dithiolan-3-one 1-Oxide, a Core Functionality of Antibiotic Leinamycin

5-(7-Hydroxyheptyl)-1,2-dithiolan-3-one 1-oxide was designed and synthesized in our laboratories that contain the heterocycle of 1,2-dithiolan-3-one 1-oxide, a reactive core of antibiotic leinamycin. In addition, the activated ester of 5-(7-hydroxyheptyl)-1,2-dithiolan-3-one 1-oxide was prepared, which presumably is useful for coupling this DNA-cleaving functionality to certain DNA-binding agents.

Benzotrithiole 2-oxide: a new family of thiol-activated DNA-cleaving functionalities.

It was demonstrated in our studies that benzotrithiole 2-oxide was capable of causing efficient DNA cleavage in the presence of 2-mercaptoethanol or glutathione and exhibited potent cytotoxic properties against certain cancer cell lines.