Lead compounds bioassay

Bioassays have been likened to analytical machines insofar as pharmacologists use them to assign biological properties to compounds in the same way a chemist measures the physical-chemical properties of molecules. If the fundamental role of the medicinal chemist is to optimize the pharmaceutical properties of so-called lead compounds by structural modification, then the role of the pharmacologist in the drug discovery process is to select, develop, and apply bioassays to provide relevant robust data that inform the medicinal chemist of the impact of the modifications he makes. 

In order to identify novel lead compounds with antiviral effects, methanol and aqueous extracts of some medicinal plants in the Zingiberaceae family were screened for inhibition of proteases from human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV) and human cytomegalovirus (HCMV). By bioassay-guided fractionation, eight fiavones were isolated from the black rhizomes of Kaempferia parviflora Wall, ex Baker. The most effective inhibitors, 5-hydroxy-7-methoxyfiavone and 5,7-dimethoxyflavone, inhibited HIV-1 protease, with an inhibitory concentration 50 (IC50) values of 19 0,M. Moreover, 5-hydroxy-3,7-dimethoxyflavone inhibited HCV protease and HCMV protease, with IC50 values of 190 and 250 pM, respectively. 

Fig. 3.1 Drug discovery sources in context. Different types of chemicai compounds (top left) are tested against bioassays that are relevant to therapeutic targets, which are derived from several possible sources of information (right).The initial lead compounds discovered by the screening process are optimised by analogue synthesis and tested for appropriate pharmacokinetic properties.The candidate compounds then enter the development process involving regulatory toxicology studies and clinical trials.

In this chapter, a rationale of the structure-activity relationships of various series of bioactive secondary metabolites from Indo-Pacific marine invertebrates is reviewed. These include alkaloids, terpenes and polybrominated diphenyl ethers which were subjected to a series of bioassays in search for insecticidal, antibacterial, fungicidal, and cytotoxic lead compounds. From these various biotests, it was observed that the bioactivity of an analogue is not due to general toxicity but rather possesses a degree of specificity on a particular target biomolecule. The relationship between chemical structures and biological activity is related to the specific action of a compound. 

Following this experimental design, approximately 700 plant materials have been evaluated and this has resulted in over 6,500 bioassay results. A number of active principles have been obtained that are active with the in vitro test systems, and several of the isolates have retained activity by preventing formation of preneoplastic lesions in mammary organ culture. Thus far, three lead compounds have been shown to mediate considerable cancer chemopreventive activity in full-term tumorigenesis models (28,34,45,46) and are being studied in more advanced test systems. We remain hopeful that one or more discoveries resulting from this project will be deemed worthy of human intervention trials. 

After preliminary disclosure of promising antimalarial leads, there is an ongoing requirement for the evaluation of selectivity in more advanced phases of the development process. By monitoring the effects of natural and chemical derivatization of lead compounds upon potency and selectivity in an in vitro bioassay system such as that described here, the safest and most effective candidates can be promoted for further study in in vivo models. 

The SI value that is used as a cut-off parameter for prioritizing those samples that possess the most potent and selective antimalarial activity is somewhat subjective due to the nature of the work involved. In the preliminary stages of the work, a cutoff is established such that the volume of samples that are subjected to bioassay-directed fractionation and further analysis is limited to a practical number. Ideally, the SI cut-off should be as high as possible and in our laboratory it is presently around 100 however, we are limited by the activities of the samples at hand. It is also meaningful to consider that if the cut-off is set too high, many potentially important lead compounds may be discarded for the sake of pursuing the ideal drug. 

Recent advances in the discovery of novel anticancer agents from higher plants are reported. These compounds are uncovered based on bioassay-directed fractionation and tandem MS analysis to detect analogs of significant lead compounds. The evolution and current status of the screening program of the U.S. National Cancer Institute is reviewed. 

Pheromone elucidation often starts with the observation by a biologist that a pheromone may be involved in a specific behavior of an organism. This leads to bioassays being developed utilizing crude extracts of glands or secretions which if successful are then analyzed usually after further purification. Once the chemical structure of a potential pheromone(s) has been identified, purified extracts or synthetic compounds are then used in the bioassay in order to confirm the pheromonal activity of the specific compounds. 

Rossman TG, Molina M, Meyer L, Boon P, Klein CB, Wang Z, Li F, Lin WC, Kinney PL (1991) Performance of 133 compounds in the lambda induction endpoint of the Microscreen assay and a comparison with Salmonella mutagenicity and rodent carcinogenicity bioassays. Mutat Res 260 349-367 Roy NK, Rossman TG (1992) Mutagenesis and comutagenesis by lead compounds. Mutat Res 298 97-103... 

Traditional Chinese medicines (TCMs) serve as a major source of a variety of drug lead compounds. In the process of natural products development, bioassay-guided isolation is a rapid and validated method for isolation of compounds with bioactivities. This chapter describes bioassay-guided separation and purification of compounds from the crude extracts of TCMs. Two approaches including size-exclusion chromatography (SEC) and high performance liquid chromatography (HPLC) are described in detail. 

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