TMLAB RESEARCH PROJECTS
Our research interests are broadly in bioorganic and natural products chemistry; biosynthesis of microbial secondary metabolites; and the interface of molecular genetics, enzymology, and chemistry to create and develop novel pharmaceutically active leads. Currently, a number of research projects are being pursued in our laboratory:
I. BIOSYNTHETIC STUDIES AND ENGINEERED PRODUCTION OF BIOACTIVE NATURAL PRODUCTS
Natural products continue to play an important role in drug discovery. About two-third of recently approved pharmaceuticals are natural product-derived or natural-inspired chemical entities. The rich biological activities and structural diversity accentuate their role as essential components of our therapeutic arsenal. Our laboratory employs a multidisciplinary approach that utilizes cutting-edge technologies in molecular genetics, enzymology, and chemistry to access, study, utilize, and manipulate genes and enzymes involved in natural product biosynthesis. Currently, we focus our study on the biosynthesis of microbial derived natural products, particularly pseudosugars (aminocyclitols) and polyketides. We carry out in-depth studies on their modes of formation in Nature and harness their biosynthetic machineries to generate analogs of bioactive compounds.
I.a. Biosynthesis of Aminocyclitols (Pseudooligosaccharides, Pseudoglycosides, Sugar Mimetics)
Our group has been working on deciphering the biosynthesis of aminocyclitols, such as pseudooligosaccharides, pseudoglycosides, and other sugar mimetics. We have made many contributions to the field, such as establishing aminocyclitol biosynthetic pathways, characterizing sugar-phosphate cyclases, cyclitol epimerases, cyclitol kinases, cyclitol nucleotidyltransferases, and pseudoglycosyltransferases.
• Sugar phosphate Cyclases (SPCs)
Sugar Phosphate Cyclases (SPCs) are a group of enzymes that catalyze the cyclization of sugar phosphates to produce a variety of cyclitol intermediates that serve as the building blocks of many primary metabolites, e.g., aromatic amino acids, and clinically relevant secondary metabolites, e.g., aminocyclitol/aminoglycoside and ansamycin antibiotics. More information about SPCs can be found in the following publications: ChemBioChem 2007, 8, 239-248; JACS 2012, 134, 12219-12229 and Biochemistry 2014, 53, 4250-4260.
• Pseudoglycosyltransferases (PsGTs)
Recently, we discovered that a number of putative glycosyltransferase (GT) enzymes can catalyze nonglycosidic C-N couplings in the biosynthesis of pseudosugar-containing natural products (e.g., aminocyclitols). These ‘pseudoglycosyltransferases’ (PsGTs) utilize nucleotidyl diphosphate (NDP)-valienol as the donor and sugars/nonsugars as acceptors to give pseudoglycosides (JACS 2011, 133, 12124-12135 and PLoS ONE 2012, 7(9):e44934). Despite their unique catalytic activity and important role in natural products biosynthesis, little is known about the molecular basis governing their substrate specificity and catalysis. Our comparative biochemical and kinetic studies using recombinant OtsA (a GT), VldE (a PsGT), and their chimeric proteins with a variety of sugar and pseudosugar substrates revealed their distinct substrate specificities and catalytic activities (Chemistry & Biology 2015, 22, 724-733). We found that the chimeric enzymes could produce hybrid pseudo-(amino)disaccharides and an amino group in the acceptor is necessary to facilitate a coupling reaction with a pseudosugar donor. We also found that the N-terminal domains of the enzymes not only play a major role in selecting the acceptors, but also control the type of nucleotidyl diphosphate moiety of the donors.
• Biosynthesis and development of ribomimetics
Sugar mimetics have long been known for their important biological activities. Many of them are currently used in clinics as antimicrobials (e.g., streptomycin, gentamycin, neomycin), antivirals (e.g., oseltamivir, peramivir, entecavir), and anti-diabetics (e.g., acarbose, voglibose). Among naturally-occurring sugar mimetics are the aminocyclopentitols, which contain a five-membered cyclitol unit resembling ribose (ribomimetics). However, due to their broad-spectrum toxicity and/or low production yield, none has yet been developed for clinical use. Therefore, addressing these limitations may provide new paths to the exploitation of their full potential as new drug leads. The long-term goals of this project are to understand the biosynthesis of ribomimetic natural products and to develop new ribomimetic-based drugs to combat infectious diseases. We focus effort on interrogating the biosynthesis of the ribomimetic-containing antibiotic pactamycin and developing new pactamycin analogs as drug leads against bacteria, viruses, and malarial parasites. Our study revealed the formation of the pactamycin core structure involves highly unusual discrete polyketide synthases, a broad-spectrum glycosyltransferase, and a radical SAM enzyme (Nat. Chem. Biol. 2019).
We also found that the tailoring pathway to pactamycin is exceptionally perplexing, due to the activity of numerous promiscuous tailoring enzymes. Furthermore, we have developed genetic, synthetic, and chemo-enzymatic strategies (involving a broad-spectrum ketoacyl-ACP synthase (KAS) III-like protein) to produce new pactamycin analogs and other ribomimetic compounds, some of which have improved biological properties (ACS Chem. Biol. 2017; MedChemComm 2019).
II. DEVELOPMENT OF NATURAL SUNSCREENS
• De Novo Synthesis of a sunscreen compound in vertebrates (eLife 2015; 4:e05191)
In 1980, it was discovered that the roe of the Atlantic cod (Gadus morhua) had high levels of a novel compound that was given the name gadusol. Unknown were both the origin and the function of the compound, although it was speculated to be a sunscreen compound of dietary or symbiont origin because of its similarity to the core of the mycosporine-like amino acid (MAA) found in cyanobacteria, algae, and corals. MAAs and related gadusols have been proposed to fulfill a variety of functions such as sunscreen, antioxidant, stress response, intracellular nitrogen reservoir, and/or optical filter. These natural products are believed to play important ecologically role(s), such as for the survival of reef-building corals and other marine organisms exposed to high solar irradiance. In our recent work, we found that fish can actually synthesize gadusol de novo using a two-enzyme pathway involving a 2-epi-5-epi-valiolone synthase (EEVS) and a bifunctional methyltransferase-oxidoreductase (MT-Ox) protein. We further showed that in zebrafish, the two genes are induced during development and gadusol is produced in the embryos. Our genome mining showed that the analogous pathways are also present in amphibians, reptiles, and birds, suggesting that gadusol is important for the well being of these organisms. Furthermore, we demonstrated that engineered yeast containing the fish genes can produce and secrete gadusol, and yeast extracts containing gadusol can significantly increase the survival rate of yeast exposed with UVB.
III. NATURAL PRODUCTS AS CANCER CHEMOTHERAPY
• Investigation of Pactamycin Analogs as Anticancer Drugs
Pactamycin, a bacterial-derived natural product discovered by the Upjohn Company in the early 1960s, has been shown to have broad-spectrum growth inhibitory activity against bacteria, mammalian cells, viruses, and protozoa. This broad-spectrum activity is primarily due to its non-selective inhibition of protein synthesis. Through biosynthetic engineering approaches we produced novel analogues of pactamycin that inhibit proliferation and induce senescence of head and neck cancer cells (PLoS ONE 2015, 10(5): e0125322).
IV. INVESTIGATION OF NEW ANTI-TB AND ANTIMALARIAL DRUGS
Tuberculosis (TB) and malaria are the leading causes of death among infectious diseases. Mycobacterium tuberculosis, the causative agent of TB, infects one third of world population and kills approximately 2 million people each year. On the other hand, about 3.3 billion people (half of the world’s population) are at risk of malaria. Every year, malaria kills nearly one million people. Most of the victims were children under the age of 5. Every 30 seconds a child dies from malaria in Africa. This number is steadily increasing. Therefore, new drug discovery to combat TB and malaria is a matter of emergency. Our group is actively pursuing the discovery and development of new anti-TB and antimalarial drugs. We apply molecular genetics and synthetic approaches to generate novel derivatives of natural products and screen the compounds for their anti-TB and antimalarial activities (JBC, 2014, 289, 21142-21152; Chem. Biol. 2011, 18, 425-431; Org. Lett. 2013, 15, 1678-1681).
• Biosynthetic Engineering of Pactamycin Analogs
Using biosynthetic engineering approached our group has been able to produce a suite of pactamycin analogs, some of which exhibit excellent biological properties, presenting hope in their development as new sources of pharmaceuticals (ChemBioChem 2009, 10, 2253-2265; Chem. Biol. 2011, 18, 425-431; ChemBioChem 2016, 17, 1585-1588; Appl. Microbiol. Biotechnol. 2018 102, 10589-10601; Appl. Microbiol. Biotechnol. 2019, 103, 4337-4345)
V. NATURAL PRODUCTS IN PLANT PATHOGENS AND SYMBIOSIS
• UNBURDENING US NURSERIES: Integrating new technologies and innovative approaches to manage broad host range, gall-forming bacterial diseases
The United States is the world’s leading producer of floriculture products. Horticultural plants are high-value crops of significant worth because of their aesthetic qualities and uses in enhancing personal and community-shared environments. The crown gall and leafy gall diseases caused by Agrobacterium tumefaciens and phytopathogenic Rhodococcus, respectively, are a significant national problem. The symptoms are often grotesque and the abnormal growth phenotypes render diseased plants of no value, resulting in annual losses exceeding a million of dollars for some growers. As part of a research team at Oregon State University we are trying to address the needs of the nursery industry by rigorously test preventative products for control. Currently, we are developing compounds from plant extracts as green alternatives for control. To understand the epidemiology of these pathogens, the team will determine the genome sequences of isolates collected from infected plant tissues and environmental reservoirs from nurseries located across the US. The genomic information will be used to understand how pathogens establish within production sites, migrate between sites, and change, possibly as a consequence of practices (PLoS One 2014 Jul 10;9(7):e101996). The team will also use genome sequences to develop dipstick diagnostic tools that can be used on-site without specialized equipment or expertise. This will provide a more rapid diagnosis of pathogens and reduce response time for more effective control.
VI. DRUG DISCOVERY FROM INDONESIAN UNIQUE ECOSYSTEMS
Indonesia is known as a major center in the world for biodiversity, which represents a treasure trove of untapped biotechnological potential for pharmaceutical and agricultural uses. In collaboration with colleagues in the OSU College of Pharmacy and with Indonesian scientists, we embarked an investigation on bioactive natural products from microorganisms isolated from unique ecosystems in Indonesia. Bioactive natural products from microorganisms are screened and identified through a combination of genetic/biosynthetic screening approach and bioassay-guided isolation and purification. Novel natural products are subjected to a number of different screening assays that are available in-house and/or through the diverse bioactive screening programs of our collaborators, e.g., antibacterial, antifungal, antimalarial, and anticancer (Biochem. Pharmacol. 2015, 93, 251-265; Org. Lett. 2015, 17, 2526-2529).