Selected approaches for rational drug design and high throughput screening to identify anti-cancer molecules

Abstract

Structure-based modeling combined with rational drug design, and high throughput screening approaches offer significant potential for identifying and developing lead compounds with therapeutic potential. The present review focuses on these two approaches using explicit examples based on specific derivatives of Gossypol generated through rational design and applications of a cancer-specificpromoter derived from Progression Elevated Gene-3. The Gossypol derivative Sabutoclax (BI-97C1) displays potent anti-tumor activity against a diverse spectrum of human tumors. The model of the docked structure of Gossypol bound to Bcl-XL provided a virtual structure-activity-relationship where appropriate modifications were predicted on a rational basis. These structure-based studies led to the isolation of Sabutoclax, an optically pure isomer of Apogossypol displaying superior efficacy and reduced toxicity. These studies illustrate the power of combining structure-based modeling with rational design to predict appropriate derivatives of lead compounds to be empirically tested and evaluated for bioactivity. Another approach to cancer drug discovery utilizes a cancer-specific promoter as readouts of the transformed state. The promoter region of Progression Elevated Gene-3 is such a promoter with cancer-specific activity. The specificity of this promoter has been exploited as a means of constructing cancer terminator viruses that selectively kill cancer cells and as a systemic imaging modality that specifically visualizes in vivo cancer growth with no background from normal tissues. Screening of small molecule inhibitors that suppress the Progression Elevated Gene-3-promoter may provide relevant lead compounds for cancer therapy that can be combined with further structure-based approaches leading to the development of novel compounds for cancer therapy.

Fig. 1

(A) Structure of Gossypol, Apogossypol and BI97D10. (B) Structure of compound 8r. (C) Structure of Sabutoclax (BI-97C1). Adapted from Wei et al. (24), J. Med. Chem., 2009.

Fig. 2

Photograph comparing tumor growth of E11 and its derivatives in nude mice. Pictures were taken 20 days postinjection with the indicated cell type. Cell lines: E11, H5ts125-transformed primary rat embryo cell line showing the non-progressed localized tumor phenotype; E11-NMT, nude mouse tumor-derived E11 clone, showing the progressed aggressive transformed phenotype; E11/PEG-3 S, a clone of E11 cells stably overexpressing a transduced PEG-3 gene in the sense orientation (S), displaying an aggressive progressed transformed phenotype, gain-of-function phenotype; E11-NMT/PEG-3 AS, a clone of E11-NMT cells stably expressing an antisense construct (AS) targeting the PEG-3 gene, displaying a non-progressed transformed phenotype, loss-of-function phenotype. Taken from Su et al. (29), Proc. Natl. Acad. Sci. USA, 1999.

Fig. 3

PEG-3-promoter drives the expression of green fluorescence protein (GFP) only in cancer cells but not in normal cells. The indicated cells were infected with either Ad.CMV-GFP or Ad.PEG-GFP at a moi of 100 pfu per cell, and GFP expression was analyzed by an immunofluorescence microscope at 2 d postinfection. Cell types: HuPEC, early passage primary human prostate epithelial cells; Du-145, PC-3 and LNCaP: human prostate carcinoma cell lines; P69, SV40 T-antigen immortalized normal human prostate epithelial cell line; M2182, is a P69-derived clone that is tumorigenic but not metastatic in animal models; M12, is a P69-derived clone displaying both tumorigenic and metastatic properties in animal models; HMEC, early passage primary human mammary epithelial cells; MCF7, T47D, MDA-MB-157, MDA-MB-231 and MDA-MB-453, human breast carcinoma cell lines; PHFA, early passage normal human primary fetal astrocytes; U251MG, T98G and U87MG, human glioblastoma multiforme cell lines. Taken from Su et al. (32), Proc. Natl. Acad. Sci. USA, 2005.

Fig. 4

Cancer-specific PEG-3-promoter activity shown by bioluminescence imaging in experimental metastasis models of human melanoma (Mel) and breast cancer (BCa). (a) Bioluminescence imaging of a representative healthy control mouse (Ctrl-2). (b) Bioluminescence imaging showing firefly luciferase expression observed in a representative melanoma model (Mel-3). Each mouse was imaged from four directions (V, ventral; L, left side; R, right side; D, dorsal views) to cover the entire body. Pseudocolor images from the two groups were adjusted to the same threshold. (c) Quantification of bioluminescence imaging signal intensity in the control group (Ctrl) and melanoma group at 24 and 48 h after injection of pPEG-Luc–PEI polyplex. Quantified values are shown in total flux. ***P < 0.0001. (d,e) CT scans and gross anatomical views of lung from one representative mouse from the control group (d) and the melanoma group (e). (f,g) Bioluminescence imaging of one representative mouse from the control group (f, Ctrl-3) and the experimental breast cancer metastasis group (g, BCa-1). The pseudocolor images were adjusted to the same threshold. (h) Quantification of bioluminescent signal intensity in the Ctrl and breast cancer groups at 24 and 48 h after injection of pPEG-Luc–PEI polyplex. **P = 0.0066. (i) A CT image and a macroscopic view of lung from a representative breast cancer mouse. Displayed bioluminescent images (a,b,f,g) were obtained at 48 h after the systemic delivery of pPEG-Luc–PEI polyplex. Black arrows (e,i) indicate metastatic nodules observed in the lung. Scale bars, 5 mm. From Bhang et al. (37), Nature Medicine, 2011.

Fig. 5

Detection and localization of metastatic masses by SPECT-CT imaging after the systemic administration of pPEG-HSV1tk. (a–j) Systemic metastatic sites were detected based on the whole body SPECT-CT images in three representative mice, Mel-2 (a,b), Mel-3 (c–h) and BCa-1 (i,j). (a,c,e,g) Transverse, coronal and sagittal views of co-registered SPECT-CT images of Mel-2 (a) and Mel-3 (c,e,g). All images were obtained at 24 h after [¹²⁵I]FIAU injection. (b,d,f,h) Gross anatomical details of the metastatic masses that were located on the basis of the SPECT-CT images (a,c,e,g). Multiple metastatic sites were detected by imaging in Mel-2 (a, dotted circle). Necropsy of the corresponding area revealed melanoma masses under the brown adipose tissue in the upper dorsal area (b, dotted circle). (c) Accumulated radioactivity was detected adjacent to the thoracic mid-spine (arrow), which corresponded to a tumor at this location (d, arrow). Additional metastatic sites demonstrated by SPECT-CT imaging (e,g, arrow and dotted circle) correlated with melanoma masses uncovered immediately above the diaphragm (f, dotted circle) and in the left inguinal lymph node (h, arrow), respectively. (i,j) Cross-comparison of PEG-3 promoter–mediated imaging and FDG-PET in a breast cancer metastasis model, BCa-1. Two nodules (Tu-1 and Tu-2) were detected by [¹²⁵I]FIAU-SPECT near the heart (i) and were confirmed by necropsy (j). Although Tu-1 was also detected by [¹⁸F]FDG-PET, Tu-2, a smaller nodule attached to the heart, was not obvious in the PET image. SPECT images were acquired 48 h after injection of [¹²⁵I]FIAU. The PET and SPECT images were acquired on the same day (i). (Tu, tumor; Ht, heart; Bf, brown fat.) Scale bars, 10 mm. From Bhang et al. (37), Nature Medicine, 2011.

Fig. 6

Conditionally replication competent adenovirus, CTV (Ad.PEG-E1A-mda-7), eradicates primary and distant tumors in athymic nude mice. Subcutaneous tumor xenografts from T47D cells were established in athymic nude mice in both right and left flanks, and only tumors on the left side were injected with PBS (control) or with the indicated Ad for 3 wk (total of seven injections). (A and C) Measurement of tumor volume. The data represent mean ± SD with a minimum of five mice per group. (B and D) Measurement of tumor weight at the end of the study. The data represent mean ± SD with at least five mice per group. Qualitatively similar results were obtained in an additional study. From Sarkar et al. (34) Proc. Natl. Acad. Sci. USA, 2005.

Fig. 7

Schematic representation of the screening strategy to recover selective inhibitors of the PEG-3-promoter. HeLa cells stably expressing the PEG-3-promoter will be utilized as the primary screen. A hit from the primary screen will be designated as a compound that inhibits the PEG-3-promoter but does not inhibit a non-specific promoter and is not toxic to untransformed CREF cells. Compounds that meet these criteria will be designated probe compounds for follow up studies. Cytotoxicity profiling of hits from the HTS campaign in human cancer and normal counterpart cells will facilitate lead selection. We will begin to dissect the mechanism of action of the PEG-3-promoter-inhibitors through cytotoxicity profiling in oncogenic CREF cells and by studying the activity of the compounds on the PEG-3-promoter mutated at the AP1 or PEA3 binding sites.

Fig. 8

Schematic representation of the PEG-3-promoter and the network of signaling pathways which it potentially monitors. ERK, p38, SAPK/JNK, Wnt, and Rho signaling activate transcription of AP-1 and PEA3, which are required for PEG-3-promoter activity. Small molecules that can inhibit PEG-3-promoter activity may be valuable anti-cancer therapeutics as well as probe compounds to study new pathways involved in catalyzing transformation.