Research

The MacMillan lab philosophy on natural products is predicated on the concept of using natural products as a tool for biological discovery – whether that be the development of new pharmaceuticals or as probes for biochemical pathways. The only way to accomplish this goal is by integration of cutting edge methods in identifying new chemical entities with highly innovative biology. Our researchplatform is based on the use of marine-derived microorganisms as a source of secondary metabolites, development of high-throughput methods of generating fraction libraries for biological evaluation and new NMR based techniques for solving complex structures.

Microbial Library: Terrestrial actinomycetes have been the predominate source of fermentation-derived natural products. It has been speculated that less than 1% of actinomycete produced natural products have been discovered, but new methods and resources are needed to tap into chemical diversity. In this regard, we have focused on developing techniques to unlock the diversity of marine-derived actinomycetes. The marine environment in general has emerged over the past 30 years as a frontier in natural products discovery, with a focus on sponges, tunicates and algae. More recently the vast microbial diversity of the oceans has been reported through large metagenomic analyses. With this as a starting point, we have collected marine sediments from a large variety of locations around the world, including the Bahamas, Tonga, Ecuador and in the Gulf of Mexico (our backyard!).

Mangroves Hypersaline lakes Andean Cloud Forests*

Utilizing a combination of existing methods and new strategies we have built a continually expanding collection of unique marine bacteria for our discovery efforts. One particular area where we have had success is in the use of biochemical and environmental factors to enrich for novel actinomycete diversity.  Our approach aims to take advantage of the importance of bacterial small-molecule communication, such as quorum sensing, as a way to stimulate growth of specific types of bacteria.

Integration with High-Throughput Screening: We have integrated our library of natural products into a format that is amenable to a variety of high-throughput screening platforms. As such, our collection has been screened in a variety of unique biological assays against cancer, opportunistic pathogens, neurological disorders, etc.  The assay formats combine a variety of target-based approaches and high-content phenotypic screens. These efforts rely on collaboration with a variety of researchers at UTSW and other academic institutions (UCSF, Texas A&M). Examples of the types of biological targets we are interested in include a number of novel targets in the apoptotic and autophagy pathways, selective agents for Gram-negative pathogens and targeting of cancer stem cells. We are able to utilize the biological expertise of world-class biologists at UTSW to utilize natural products as a tool to help unravel complex biological processes. For more information about the types of biological assays that we are currently utilizing contact Prof. MacMillan.

Structural Elucidation and Techniques for Rapid Deconvolution using NMR:
Identification of a biologically active compound is the starting point in the development of natural products as drug leads or tools for biological study. We utilize a combination of MS, NMR and X-ray crystallographic techniques to fully assign the planar structure of complex natural products. Structure determination is always an interesting challenge, as every class of molecule has its own difficulties and curiosities. Some structures, such as the macrolide mangrolide A can readily be solved by interpretation of 2D-NMR data, while complex alkaloids like ammosamide D require the use of X-ray crystallography. To solve structures by NMR, we utilize a Varian System 600 MHz NMR and/or a Varian 800 MHz NMR with a cryoprobe (when necessary). Stereochemistry is the other major challenge associated with new natural products and requires the use of analytical techniques in conjunction with organic synthesis. The MacMillan lab focuses on tackling difficult stereochemical challenges through the use of NMR and CD based techniques. Examples of the types of chemical structures that we have focused on are listed below.

In addition to the use of NMR to solve structures of biologically active molecules, we utilize hyphenated NMR techniques, specifically LC-SPE-NMR, to dereplicate complex natural product mixtures and obtain structural information in a rapid, high-throughput manner. In particular the system we utilize is our Varian 600 MHz NMR, fitted with a 60 µL flow probe that is connected to an HPLC system and Leap System X-Y-Z robot. The schematic of the system below shows conceptually how the system works. To fully utilize the system, there are a number of analytical challenges that need to be managed including the HPLC conditions, SPE trapping and elution and sample introduction. This methodology is powerful when attempting to analyze a large number of active hits.

New NMR Methods – MDEC: Advances in NMR instrumentation have pushed the limits of small molecule NMR, with higher fields allowing better signal dispersion and modern probe design permitting analysis and quantification on the nanomolar level. One of the remaining limitations of small molecule NMR is the ability to obtain relative configuration of small molecules. Since the 1959 report by Karplus the use of coupling constants to determine relative configuration in conformationally rigid small molecules has become routine. More recently, J-based configuration analysis has been applied to determine the relative stereochemistry of complex acyclic and macrocylic small molecules and natural products. The utility of J-based methods relies on the ability to measure discrete coupling constants between protons (JHH), which can be difficult in molecules with complex multiplets and significant signa overlap. The existing methods, such as E.COSY and 2D J-resolved suffer from lack of sensitivity and complex data analysis. Alternatively, classic homonuclear decoupling can be used to simplify a complex multiplet, but is limited to irradiating a single proton.  Due to the challenges of measuring coupling constants, many reports of natural products and synthetic small molecules do not report vital coupling information – rather they only assign signals as multiplets. We have developed a method that allows for the simultaneous decoupling of multiple signals, greatly simplifying signal overlap (Espindola et al., JACS, 2009). This pulse can be integrated with other 1D experiments, such as 1D-TOCSY to isolate individual signals in a complex spectra. Below is an example of an MDEC experiment to isolate individual coupling constants in menthol.

For questions about any of the above projects, please contact John MacMillan at