Phage display as a tool for the directed evolution of enzymes.

Since its introduction in 1985, phage display has had a tremendous impact on the discovery of peptides that bind to a variety of receptors, the generation of binding sites within predefined scaffolds, and the creation of high-affinity antibodies without immunization. Its application to enzymology has required the development of techniques that couple enzymatic activity to selection protocols based on affinity chromatography. Here, we describe both indirect methods, using transition-state analogues and suicide substrates, and direct methods, using the ability of active phage-enzymes to transform substrate into product. The methods have been applied to large libraries for mechanistic-based studies and to generate variants with new or improved properties. In addition, such techniques have been successfully used to select catalytic antibodies and improve their catalytic efficiency.

New Synthetic Strategies to Vitamin D Analogues Modified at the Side Chain and D Ring. Synthesis of 1alpha,25-Dihydroxy-16-ene-vitamin D(3) and C-20 Analogues(1).

Two efficient synthetic routes to 1alpha,25-dihydroxy-16-ene-vitamin D(3) (4a) and their C-20 analogues (3 and 4) have been developed. Key features common to both routes A and B are the introduction of side chains functionalized at C20 (17, 21, 19, and 25). In route A the CD side chain fragments 5 and 6 are prepared by S(N)2' syn displacement of allylic carbamates 8 and 9 (X = OCONHPh) by Li(2)Cu(3)R(5). The triene unit is then constructed by assembling the latter fragments with the A-ring fragment using the Wittig-Horner method (average yield of vitamin D analogue 35%, 11-13 steps from ketone 11). In route B, the S(N)2' syn displacement of the carbamate moiety by Li(2)Cu(3)R(5) is carried out on intermediates 12 and 13, both of which bear the vitamin D triene unit (average yield of vitamin D analogue 27%, 13-15 steps from ketone 11). The latter route is particularly attractive as an approach to diverse C-20 vitamin D analogues for biological screening.

Nuclear estrogen receptor targeted photodynamic therapy: selective uptake and killing of MCF-7 breast cancer cells by a C17alpha-alkynylestradiol-porphyrin conjugate.

We hypothesized that over-expression of estrogen receptor (ER) in hormone-sensitive breast cancer could be harnessed synergistically with the tumor-migrating effect of porphyrins to selectively deliver estrogen-porphyrin conjugates into breast tumor cells, and preferentially kill the tumor cells upon exposure to red light. In the present work we synthesized four (4) conjugates of C17-alpha-alkynylestradiol and chlorin e6-dimethyl ester with varying tether lengths, and showed that all these conjugates specifically bound to recombinant ER alpha. In a cellular uptake assay with ER-positive MCF-7 and ER-negative MDA-MB 231 human breast cancer cell-lines, we observed that one such conjugate (E17-POR, XIV) was selectively taken up in a dose-dependent and saturable manner by MCF-7 cells, but not by MDA-MB 231 cells. Furthermore, MCF-7 cells, but not MDA-MB 231 cells, were selectively and efficiently killed by exposure to red light after incubation with E17-POR. Therefore, the combination approach, including drug and process modalities has the potential to be applied clinically for hormone-sensitive cancers in organs where ER is significantly expressed. This could potentially be carried out either as monotherapy involving a photo-induced selective destruction of tumor cells and/or adjuvant therapy in post-surgical treatment for the destruction of residual cancer cells in tissues surrounding the tumor.

Photodynamic cell-kill analysis of breast tumor cells with a tamoxifen-pyropheophorbide conjugate.

We hypothesized that estrogen receptor (ER) in hormone-sensitive breast cancer cells could be targeted for selective photodynamic killing of tumor cell with antiestrogen-porphyrin conjugates by combining the over-expression of ER in hormone-sensitive breast cancer cells and tumor-retention property of porphyrin photosensitizers. In this study we describe that a tamoxifen (TAM)-pyropheophorbide conjugate that specifically binds to ER alpha, caused selective cell-kill in MCF-7 breast cancer cells upon light exposure. Therefore, it is a potential candidate for ER-targeted photodynamic therapy of cancers (PDT) of tissues and organs that respond to estrogens/antiestrogens.

Affinity labeling of the nuclear vitamin D receptor with nonsteroidal alkylating agents.

Synthesis of an affinity alkylating non steroidal mimic of 1alpha,25-dihydroxyvitamin D(3) and its radiolabeled counterpart is presented. We also report the affinity labeling of the VDR-ligand binding domain (VDR-LBD) with this analogue.