Our laboratory uses small organic molecules to identify and define novel targets for the treatment of a variety of intractable biomedical problems. We use the tools of synthetic organic chemistry, biochemistry, combinatorial chemistry, high-throughput screening, and cell biology to explore disease states that have for a variety of reasons resisted the standard paradigm of drug discovery and development. In the course of this work we often obtain clinical samples from patients in an effort to both define the levels of a target in the patient population and to test the efficacy of our compounds in these clinical isolates. We are actively using small molecules to define novel biological targets for the treatment of cancer, neurodegeneration, and drug-resistant bacteria. Thus the products of this work are both novel compounds and novel protein or RNA targets.

Using small molecules to define novel anti-cancer targets

          Programmed cell death (apoptosis) is an extremely important biological cascade that is continuously at work in higher multi-cellular organisms. Through this process, damaged or otherwise unwanted cells are removed from the organism in a systematic fashion, without provoking the inflammatory response. Unfortunately, when apoptosis goes awry there are disastrous consequences for the organism. For example, too little apoptosis is linked to a variety of cancers, and too much apoptosis has been implicated in disorders of premature cell death such as Alzheimer’s disease, Parkinson’s disease, ALS, stroke, myocardial infarction, and many others.

          We are synthesizing a variety of natural products, designed compounds, and combinatorial libraries in an effort to identify novel and selective small molecule inducers of programmed cell death, with a keen eye towards untreatable cancers such as late stage malignant melanoma. In this regard we first discovered a compound (13-D) that selectively induces apoptosis in cancer cells (J. Am. Chem. Soc. 2003, 125, 14672-14673, ChemBioChem 2006, 7, 1916-1922).

          In more recent studies, we have explicitly targeted melanoma. Late-stage malignant melanoma is essentially untreatable with chemotherapeutics; patients receiving this diagnosis have an average survival time of six months. Even in cell culture, many standard anti-cancer drugs are not effective at killing melanoma. By creating a combinatorial library and looking for small molecules that arrest cells in the G1 phase of the cell cycle, we discovered a class of compounds (triphenylmethyl amides; TPMAs) that potently induce apoptosis in melanoma cells (J. Am. Chem. Soc. 2005, 127, 8686-8696).  Systematic and detailed analysis of these TPMAs (and of phosphorous-containing derivatives that we created (TPMPs)) led to the surprising discovery that, although the triphenylmethyl group is present in several anti-cancer compounds, these compounds can exert their death-inducing effect through a variety of different mechanisms.  For example, the methoxy-TPMPs (TPMP-III-2, below) affect cell death by inhibiting tubulin polymerization, while other triphenylmethyl-containing compounds inhibit Eg5, or cause depletion of cellular Ca²⁺ (J. Am. Chem. Soc. 2008, 130, 10274-10281; Bioorg. Med. Chem. Lett. 2008, in press).  Efforts are underway to identify the precise molecular target of a fourth class of these compounds, and a subset of these compounds have been licensed to LinkCore Phrama for development toward a Phase I clinical trial.

Some anti-cancer compounds discovered in the Hergenrother laboratory.

          A growing area of interest in our laboratories is the development of "personalized" strategies for combating cancer, strategies in which we exploit defined molecular defects in cancer to specifically kill these cells.  In an exciting result, we have identified a small molecule that specifically turns "on" a protein that is elevated in many cancer types, and by turning this protein on we are able to kill the cancer cell.  Activation of the major apoptotic pathways ultimately results in the conversion of procaspase-3 to caspase-3.  Caspase-3 is a cysteine protease that has a multitude of substrates in the cell; once the active caspase-3 enzyme is generated, these substrates are rapidly consumed and the cell dies.  A hallmark of cancer is its resistance to apoptosis.  This resistance typically occurs through mutations in or aberrant expression levels of proteins in the apoptotic cascade.  The net result is that the "circuit" connecting the upstream pro-apoptotic signal to the activation of procaspase-3 is broken.  We have shown that the compound PAC-1 directly activates procaspase-3 to caspase-3 both in vitro and in cell culture (Nature Chem. Biol. 2006, 2, 543).  We surveyed the colon tumors from 23 colon cancer patients, and the results showed that the cancerous cells had elevated levels of procaspase-3 relative to the adjacent non-cancerous tissue.  The efficacy of PAC-1 in inducing apoptotic death is proportional to the concentration of procaspase-3 in these cells.  PAC-1 was also shown to be quite effective in three animal models of cancer.  In addition, we have recently determined exactly how PAC-1 activates procaspase-3 in vitro (manuscript submitted). 

          In July of 2007 PAC-1 was licensed by BioLineRx for development as a human anti-cancer drug (http://www.biolinerx.com/NewsPr.asp?i=65).  In addition, the veterinary medicine school at UIUC has initiated a Phase I trial with PAC-1 in dogs with cancer; parties interested in enrolling their pet should see this link for information:

UIUC Vet Med Clinical Trial

          Further screening for activators of other enzymes led to the surprising discovery that some compounds in high-throughput screening collections are general enzyme activators (J. Med. Chem. 2008, 51, 2346-2349).  In other work, the compound 8H/343 was identified as a potent inducer of cell death in cancer cells as well as an inhibitor of the CYP enzymes and CYP enzyme expression (J. Am. Chem. Soc. 2005, 127, 8686-8696; Carcinogenesis 2007, 28, 1052-1057).  The synthesis and evaluation of several other classes of anti-cancer compounds are on going in our laboratory, including natural products, and compounds identified through high-throughput screens.

For reviews on DNA as a target for anti-cancer compounds, compound collections for high-throughput screening, target identification using affinity chromatography, and isosteric replacements of the phosphate group in inhibitor design, please see:

Palchaudhuri, R.; Hergenrother, P. J. “DNA as a Target for Anti-Cancer Compounds: Methods to Determine the Mode of Binding and the Mechanism of Action” Curr. Opin. Biotechnol. 2007, 18, 497-503.

Hergenrother, P. J. “Obtaining and screening compound collections: A user’s guide and a call to chemists” Curr. Opin. Chem. Biol. 2006, 10, 213-218.

Leslie, B. J.; Hergenrother, P. J. “Identification of the cellular targets of bioactive small organic molecules using affinity reagents” Chem. Soc. Rev. 2008, 37, 1347-1360                             

Nottbohm, A. C.; Hergenrother, P. J. “Replacing the Irreplaceable: Cyclic compounds as Novel Phosphate Mimics” The Encyclopedia of Chemical Biology, 2008, in press.

Using small molecules to define novel targets for the treatment of neurodegenerative disorders

          In the anti-apoptotic area, we are synthesizing certain natural products that appear to have cytoprotective properties, and then using these compounds to identify novel biological targets.  In addition, we have targeted the inhibition of certain proteins involved in poly(ADP-ribose) synthesis and catabolism: PARP, PARG, and Apoptosis Inducing Factor (AIF).

          A bottleneck in this field is the lack of facile assays that can be used for high-throughput screening.  We have thus developed several of these assays in our laboratory. For example we have recently reported high-throughput assays for the assessment of PARP (Anal. Biochem. 2004, 326, 78-86) and PARG (Anal. Biochem. 2004, 333, 256-264) activity. We have also developed a simple chromogenic substrate and have used it to evaluate several of the PARP isozymes (Angew. Chem. Int. Ed. 2007, 46, 2066-2069).  Our PARP assay can also be applied to differentiate apoptotic cell death from necrotic cell death in both whole cells and animal tissue (ChemBioChem 2005, 6, 53-55, Resuscitation 2007, 75, 173-183).

Some enzyme assays and high-throughput screens developed in the Hergenrother lab.

Another major target we are pursing is the inhibition of Apoptosis Inducing Factor (AIF).  After insult to the cell, AIF is released from the mitochondria and translocates to the nucleus, where it binds DNA and induces chromatin condensation. There is much evidence implicating AIF in neurodegenerative disorders such as Parkinson’s disease.  In exciting recent work, we have identified the first small molecule inhibitor of AIF.  To do this we developed a new tool for Chemical Biology: A general method to rapidly detect protein—DNA interactions through the use of photonic crystal biosensors (ACS Chem. Biol. 2008, 3, 437-448).  This was developed in collaboration with the laboratory of Professor Brian Cunningham’s laboratory in the Department of Electrical Engineering at UIUC.  A small scale screen using this method revealed the compound ATA as the first small molecule inhibitor of the AIF—DNA interaction.

Once identified, these and other compounds are being used to biologically explore and potentially treat various degenerative-type conditions, including Parkinson’s disease, stroke, and ALS. During the course of this work, methodology for organic synthesis often needs to be developed, such as efficient ways to create chiral amino alcohols (Org. Lett. 2003, 5, 281-284) and b-amino acids (Org. Lett. 2003, 5, 2107-2109). We have also used structural information to assist us in the design and synthesis of potent inhibitors of caspase-3 and caspase-7 (Org. Lett. 2005, 7, 3529-3532).

Some methodologies for organic synthesis developed in the Hergenrother lab.

For reviews on PARG inhibition and the role of poly(ADP-ribose) in cell death, please see:

Heeres, J. T.; Hergenrother, P. J. “Poly(ADP-Ribose) Makes a Date with Death” Curr. Opin. Chem. Biol. 2007, 11, 644-653.

Nottbohm, A. C.; Hergenrother, P. J. “The Promises and Pitfalls of Small-Molecule Inhibition of Poly(ADP-Ribose) Glycohydrolase (PARG)” Book Chapter in: Drug Discovery Research: New Frontiers in the Post-Genomic Era; Wiley & Sons, 2007, 163-185.

Using small molecules to define novel targets to combat drug-resistant bacteria and a general paradigm for small molecule-RNA binding

          Bacteria that are resistant to multiple antibiotics are increasingly commone. In the hospital setting, such bacteria are especially prevalent; it has recently been estimated that over one-third of enterococci in intensive care units are resistant to vancomycin, generally regarded as the antibiotic of last resort. Although modern science has successfully kept bacteria largely at bay over the last 50 years with a bevy of antibiotics, these drugs generally have only three targets: the bacterial ribosome, the cell wall, or DNA gyrase. It is imperative that new antibacterial compounds and compounds with a novel mechanism of action are discovered and developed. In reality, the problem is not that we lack drugs to kill bacteria (for instance enterococci and Staphylococcus aureus are quite sensitive to many common antibiotics), but rather that bacteria have acquired foreign pieces of DNA that encode resistance-mediating proteins. Bacteria that harbor this laterally transferred DNA, which often reside on a plasmid, are now resistant to multiple antibiotics. We are thus developing a general strategy to eliminate plasmids from bacterial cells and thus sensitize bacteria to antibiotics. As our first foray into this area, we have elected to mimic a natural mechanism of plasmid elimination, known as plasmid incompatibility. Thus, we have targeted small stretches of RNA with small molecules in an effort to vanquish plasmids from bacterial cells.

 In an exciting result, we disclosed that the compound apramycin binds tightly to a RNA incompatibility determinant, causes plasmid loss in a dose dependant manner, and re-sensitizes bacteria to antibiotics to which they were previously resistant (J. Am. Chem. Soc. 2004, 126, 15402-15404). We have also reported on the ability of other structurally related molecules to mimic incompatibility and induce plasmid elimination (Biochemistry 2005, 44, 6800-6808).

          More recently, we have begun an exciting effort to explicitly use plasmid-encoded traits against the drug-resistant bacteria.  Plasmids that harbor drug-resistant bacteria are generally large, and thus they require intricate mechanisms to maintain themselves in the bacterial population.  One way they do this is through the use of a plasmid encoded toxin, which is neutralized by a plasmid encoded antitoxin.  However, if the plasmid is lost during cell division, the labile antitoxin is rapidly degraded, and the toxin kills the cell.  Through a detailed epidemiological survey, we recently discovered that these toxin-antitoxin systems are ubiquitous and plasmid encoded in VRE (Proc. Natl. Acad. Sci. 2007, 104, 311-316).  The results of this survey suggest that toxin-antitoxin systems will make tractable antibacterial targets.  As a first step towards the identification of compounds that activate these toxins, we developed a high-throughput assay for assessment of MazF activity (Anal. Biochem. 2007, 371, 173-183).

Another morphology of bacteria that are very difficult to treat with conventional antibiotics are bacterial biofilms, or communities of bacteria that live together and share certain resources. We developed a novel high-throughput screen for anti-biofilm compounds and have applied it to discover an agent that both prevents the formation of bacterial biofilms and disrupts existing biofilms of Pseudomonas aeruginosa. P. aerginosa biofilms are a leading cause of mortality in Cystic Fibrosis (CF) patients. We have also shown that our compound is effective against P. aeruginosa isolated from the lungs of CF patients (Chem. Biol. 2005, 12, 789-796), and that nebulized versions of the compounds will distribute effectively in an in vitro model of delivery to the human lung (J. Applied Microbiol. 2008, 105, 380-388).

          These projects have spawned a more general effort in our laboratory to develop a paradigm for small molecule—RNA binding such that any RNA sequence can be targeted in vivo. This RNA-based approach for drug discovery has multiple advantages over the traditional protein-based approach, but development of compounds specific for one mRNA over all others has been hampered by the lack of a systematic strategy for small molecule—RNA binding. We are in the course of developing such a strategy based on the tethering together of compounds that are specific for a given RNA secondary structure. In this regard we recently described a novel class of molecules (deoxystreptamine dimers) that bind to RNA hairpin loops (J. Am. Chem. Soc. 2004, 126¸ 9196-9197). In subsequent work, we constructed a combinatorial library of 105 deoxystreptamine dimers and discovered compounds that were specific for RNA tetraloops, and still others that bind tightly and specifically to RNA octaloops (J. Am. Chem. Soc. 2005, 127, 12434-12435). We have found that these compounds appear to bind to their cognate RNA in an unusual, entropically-driven fashion (Biochemistry 2006, 45, 10928-10938).

Some small molecules that bind to RNA under investigation in the Hergenrother lab.

For reviews on plasmid-encoded resistance, combating multidrug-resistant bacteria through exploitation of plasmid-encoded resistance, anti-biofim compounds, and targeting RNA with small molecules, please see:

Williams, J. J.; Hergenrother, P. J. “Exposing Plasmids as the Achilles’ Heel of Drug-Resistant Bacteria” Curr. Opin. Chem. Biol. 2008, in press.

Thomas, J. R.; Hergenrother, P. J. “Targeting RNA with Small Molecules” Chem. Rev. 2008, 108, 1171-1224.

Moritz, E. M.; Hergenrother, P. J. “The Prevalence of Plasmids and Other Mobile Genetic Elements in Clinically Important Drug-Resistant Bacteria” Book Chapter in: Antimicrobial Resistance in Bacteria; Horizon Scientific Press, 2007, 25-53.

Musk, D. J.; Hergenrother, P. J. “Chemical Countermeasures for the Control of Bacterial Biofilms: Effective Compounds and Promising Targets” Curr. Med. Chem. 2006, 13, 2163-2177.       

DeNap, J. C. B.; Hergenrother, P. J. “Bacterial Death Comes Full Circle: Targeting Plasmid Replication in Drug-Resistant Bacteria”  Org. Biomol. Chem. 2005, 3, 959-966.