In designing a search for novel prototype antibiotics for AIDS-related opportunistic infec- tions, it seems reasonable to assume that if new agents are to be found that have different structures with different or supplemental activities from the ones in current use or devel- opment, then a source other than the more traditional microorganisms must also be inves- tigated. In particular, the higher plants are a logical choice, chiefly because of their seemingly infinite variety of secondary metabolites. Antifungal agents appear to be widely distributed among the higher plants,19 but very few have been evaluated for their activity
against human pathogenic fungi and essentially none of these has been evaluated in animal models of disseminated mycoses.
For many years our group has pursued a program to detect, isolate, and structurally characterize novel antibiotics, especially antifungal antibiotics, from higher plants.20-24 The
fundamental components of our program are similar to those of any natural product drug discovery program: sourcing and sample acquisition, biological evaluation, isolation and structure elucidation (including dereplication), and selection and optimization of lead compounds to identify pharmaceutical candidates for further development (Figure 7.2). Within the discipline, much debate surrounds the specifics of how best to accomplish each of these, and much attention and resource are devoted to developing more efficient meth- ods to meet the many challenges associated with these activities. A brief overview of our general approach, as well as some of the challenges that must be met and key decisions that must be made in any natural product drug discovery program, follows.
7.3.1 Sourcing and Sample Acquisition and Preparation
The goal of sourcing and sample acquisition is to obtain the maximum chemodiversity and therapeutically useful biological activity within the minimum number of collected samples.
FIGURE 7.2
Overall schematic of drug discovery from natural sources. ©2000 by CRC Press LLC
A number of strategies may be developed to select and acquire sources of natural products that are most likely to yield desirable compounds. In our program, these range from utiliz- ing ethnobotanical and folklore information on medicinal plant use to strategies to explore the relationship of genetic biodiversity and environmental factors with chemodiversity and specific biological activities. Consideration should be given to the fact that the greatest biodiversity occurs in the tropical regions, and many times in developing countries. Col- lections should be done as collaborative efforts with attention to issues of ownership of intellectual property, laws governing access to biodiversity, and political, social, and eco- nomic factors.
Once samples are acquired, voucher specimens must be maintained according to standard accepted methods and the collected specimens must be extracted or otherwise processed to prepare samples for biological evaluation. The goal of sample handling and preparation is to select for positives (remove nuisance compounds), prepare the samples to be compatible with existing (and future) bioassays, and store both the collected unprocessed material and the processed samples in a manner that is easily retrievable and maximizes stability.
The key decisions in sourcing and sampling preparation are what strategies to employ, including what sources on which to focus (plant, marine, microbial, insect, etc.); what quantity to collect; and how to store and process the collected samples. There remains much to be done in this area, especially in the validation of various sourcing strategies and in designing and validating protocols for storage and preparation of collected specimens and processed samples to maximize reproducible biological activities and stable chemical composition.
7.3.2 Biological Evaluation
The core foundation of every drug discovery program is the biological assays. The goal here is to identify compounds with selective and specific biological effects on contempo- rary and relevant disease targets, and to predict effectively in vivo efficacy, toxicity, and pharmacokinetics. Typically, a tier of assays may be established, beginning with a primary assay that has a relative high throughput capacity and is designed to detect samples with the most promise for yielding interesting compounds, i.e, screening out the vast majority of samples with low to moderate activity or with nonselective activity. Prior to major advances in molecular biology and genomics, most assays for the discovery of new antibi- otics were based on identifying compounds that inhibited the growth of the target patho- gens. This remains an important component of our approach, and we utilize a combination of mechanism-specific, targeted subcellular assays and mechanism-blind, whole cell assays. In this way, agents acting by a novel mechanism are less likely to be missed in the primary screening. Secondary assays are usually designed to corroborate and quantitate the activity observed in the primary assay, to establish the spectrum of activity, to provide insight into the mode of action, and to predict in vivo pharmaceutical properties. An impor- tant criterion for determining the relative importance of a lead compound is evidence to suggest it acts by a novel mechanism of action. For antifungal agents, this may be accom- plished initially by relatively simple methods such as determining if the compound is active against strains resistant to other known agents, if the compound inhibits known anti- fungal targets (e.g., ergosterol biosynthesis), or how it affects general biochemical path- ways (e.g., protein or nucleic acid synthesis). Results that suggest a novel mechanism of action should be followed up with more extensive studies to determine the molecular site of action, which may include studies such as determining where the compound is localized subcellularly (this usually requires availability of labeled compound) and comparative protein analysis of treated vs. untreated cells.
©2000 by CRC Press LLC
7.3.3 Isolation and Structure Elucidation
One of the major challenges in natural product drug discovery is determining which of a number of approximately equally active samples to pursue for further study, usually for isolation and structure elucidation of the active constituents. This is best accomplished through a system of prioritization coupled with the process known as dereplication. Since isolation and structure elucidation can be among the most laborious, time-consuming, and expensive steps in natural product drug discovery, much attention is given to developing reliable methods of dereplication. The goal of dereplication is to select, for followup isola- tion and structure elucidation, only extracts that are likely to yield novel chemotypes, i.e., to “deselect” compounds with known activity profiles and/or structures. One of the best ways to achieve dereplication is to access unique sources, so that there is a greater assur- ance of obtaining novel chemotypes. Also, advances in coupling spectroscopic and chro- matographic techniques can provide rapid and reliable structural information on very small quantities of material, thus facilitating dereplication.25 It is also important to use the
literature — chemotaxonomy forms the cornerstone of dereplication.
It is also important to prioritize samples for further study. Priority assignment may be based on a combination of factors, including biological activity profile, the results of liter- ature searches that indicate minimal previous work on a specimen, and the availability of sufficient biomass for larger-scale fractionation. Once a priority position is assigned to a given sample, bioassay-directed fractionation is carried out to isolate pure active constitu- ents. Structural characterization of isolated active compounds is accomplished most often using state-of-the-art spectroscopic techniques such as high-field NMR, mass spectroscopy, and various methods to determine absolute and relative stereochemistry.
7.3.4 Lead Selection and Optimization
Structurally novel compounds (different from existing antifungal compounds) showing potent in vitro fungicidal activity, coupled with evidence of in vivo efficacy in an animal model of disseminated mycoses and evidence of a novel mechanism of action, are consid- ered lead compounds and are subject to further studies aimed at developing a promising pharmaceutical candidate for preclinical studies and clinical trials. These studies involve medicinal chemistry to accomplish total synthesis, analog synthesis for SAR studies, molecular modeling, and computer-aided drug design, as well as analytical and physical chemistry and studies to establish the pharmaceutical properties of the compound.26 These
studies are aimed at identifying the most suitable candidate for preclinical and clinical development.