Publications by authors named "Paul J Hergenrother"

137 Publications

Facilitating Compound Entry as a Means to Discover Antibiotics for Gram-Negative Bacteria.

Acc Chem Res 2021 03 26;54(6):1322-1333. Epub 2021 Feb 26.

Department of Chemistry Institute for Genomic Biology, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801, United States.

It has been over half a century since the last class of antibiotics active against the most problematic Gram-negative bacteria was approved by the Food and Drug Administration (FDA). The major challenge with developing antibiotics to treat these infections is not drug-target engagement but rather the inability of most small molecules to traverse the Gram-negative membranes, be retained, and accumulate within the cell. Despite an abundance of lead compounds, limited understanding of the physicochemical properties needed for compound accumulation (or avoidance of efflux) in Gram-negative bacteria has precluded a generalizable approach for developing Gram-negative antibiotics. Indeed, in many instances, despite years of intensive derivatization efforts and the synthesis of hundreds of compounds aimed at building in Gram-negative activity, little or no progress has been made in expanding the spectrum of activity for many Gram-positive-only antibiotics. In this Account, we describe the discovery and successful applications of a promising strategy for enhancing the accumulation of Gram-positive-only antibiotics as a means of imbuing compounds with broad-spectrum activity.Utilizing a prospective approach examining the accumulation in for more than 180 diverse compounds, we found that small molecules have an increased likelihood to accumulate in when they contain an ionizable itrogen, have low hree-dimensionality, and are igid. Implementing these guidelines, codified as the "eNTRy rules" and assisted by web application www.entry-way.org, we have facilitated compound entry and systematically built Gram-negative activity into Gram-positive-only antibiotics. Though each antibiotic will have case-specific considerations, we describe a set of important criteria to consider when selecting candidate Gram-positive-only antibiotics for conversion to Gram-negative-active versions via the eNTRy rules. As detailed herein, using this blueprint the spectrum of activity was expanded for three antibiotic classes that engage three different biological targets: DNA gyrase inhibitor 6DNM, FabI inhibitor Debio-1452, and FMN riboswitch inhibitor Ribocil C. In each scenario, the eNTRy rules guided the synthesis of key analogues predisposed to accumulate in Gram-negative bacteria leading to compounds that display antibiotic activity (minimum inhibitory concentrations (MIC) ≤8 μg mL) against and other Gram-negative ESKAPE pathogens. While the eNTRy rules will continue to be refined and enhanced as more accumulation data is gathered, on the basis of these collective results and on other examples not covered herein it is clear that the eNTRy rules are actionable for the development of novel broad-spectrum antibiotics from Gram-positive-only compounds. By enabling the prediction of compound accumulation, the eNTRy rules should facilitate the process of discovering and developing novel antibiotics active against Gram-negative bacteria.
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http://dx.doi.org/10.1021/acs.accounts.0c00895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7969460PMC
March 2021

A Phosphonate Natural Product Made by Pantoea ananatis is Necessary and Sufficient for the Hallmark Lesions of Onion Center Rot.

mBio 2021 02 2;12(1). Epub 2021 Feb 2.

Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

is the primary cause of onion center rot. Genetic data suggest that a phosphonic acid natural product is required for pathogenesis; however, the nature of the molecule is unknown. Here, we show that produces at least three phosphonates, two of which were purified and structurally characterized. The first, designated pantaphos, was shown to be 2-(hydroxy[phosphono]methyl)maleate; the second, a probable biosynthetic precursor, was shown to be 2-(phosphonomethyl)maleate. Purified pantaphos is both necessary and sufficient for the hallmark lesions of onion center rot. Moreover, when tested against mustard seedlings, the phytotoxic activity of pantaphos was comparable to the widely used herbicides glyphosate and phosphinothricin. Pantaphos was also active against a variety of human cell lines but was significantly more toxic to glioblastoma cells. Pantaphos showed little activity when tested against a variety of bacteria and fungi. is a significant plant pathogen that targets a number of important crops, a problem that is compounded by the absence of effective treatments to prevent its spread. Our identification of pantaphos as the key virulence factor in onion center rot suggests a variety of approaches that could be employed to address this significant plant disease. Moreover, the general phytotoxicity of the molecule suggests that it could be developed into an effective herbicide to counter the alarming rise in herbicide-resistant weeds.
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http://dx.doi.org/10.1128/mBio.03402-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7858074PMC
February 2021

Synthesis of Fusidic Acid Derivatives Yields a Potent Antibiotic with an Improved Resistance Profile.

ACS Infect Dis 2021 02 1;7(2):493-505. Epub 2021 Feb 1.

Department of Chemistry, University of Illinois at Urbana-Champaign, Roger Adams Laboratory, 600 South Mathews Avenue, Urbana, Illinois 61801, United States.

Fusidic acid (FA) is a potent steroidal antibiotic that has been used in Europe for more than 60 years to treat a variety of infections caused by Gram-positive pathogens. Despite its clinical success, FA requires significantly elevated dosing (3 g on the first day, 1.2 g on subsequent days) to minimize resistance, as FA displays a high resistance frequency, and a large shift in minimum inhibitory concentration is observed for resistant bacteria. Despite efforts to improve on these aspects, all previously constructed derivatives of FA have worse antibacterial activity against Gram-positive bacteria than the parent natural product. Here, we report the creation of a novel FA analogue that has equivalent potency against clinical isolates of (s) and () as well as an improved resistance profile when compared to FA. Importantly, this new compound displays efficacy against an FA-resistant strain of in a soft-tissue murine infection model. This work delineates the structural features of FA necessary for potent antibiotic activity and demonstrates that the resistance profile can be improved for this scaffold and target.
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http://dx.doi.org/10.1021/acsinfecdis.0c00869DOI Listing
February 2021

Synthetic Studies on Selective, Proapoptotic Isomalabaricane Triterpenoids Aided by Computational Techniques.

J Am Chem Soc 2021 Feb 19;143(4):2138-2155. Epub 2021 Jan 19.

Roger Adams Laboratory, Department of Chemistry, University of Illinois, Urbana, Illinois 61801, United States.

The isomalabaricanes comprise a large family of marine triterpenoids with fascinating structures that have been shown to be selective and potent apoptosis inducers in certain cancer cell lines. In this article, we describe the successful total syntheses of the isomalabaricanes stelletin A, stelletin E, and rhabdastrellic acid A, as well as the development of a general strategy to access other natural products within this unique family. High-throughput experimentation and computational chemistry methods were used in this endeavor. A preliminary structure-activity relationship study of stelletin A revealed the core motif of the isomalabaricanes to be critical for their cytotoxic activity.
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http://dx.doi.org/10.1021/jacs.0c12569DOI Listing
February 2021

Compound Uptake into Can Be Facilitated by -Alkyl Guanidiniums and Pyridiniums.

ACS Infect Dis 2021 01 23;7(1):162-173. Epub 2020 Nov 23.

Roger Adams Laboratory, Department of Chemistry, University of Illinois, Urbana, Illinois 61801, United States.

Multidrug-resistant Gram-negative bacterial infections are on the rise, and with no FDA approvals for new classes of broad-spectrum antibiotics in over 50 years, these infections constitute a major threat to human health. A significant challenge is the inability of most compounds to accumulate in Gram-negative bacteria. Recently developed predictive guidelines show that appending a primary amine to an appropriately shaped compound can enhance Gram-negative accumulation. Here, we report that other positively charged nitrogen functional groups, namely, -alkyl guanidiniums and pyridiniums, can also facilitate compound uptake into Gram-negative bacteria. The accumulation of a set of 60 nonantibiotic compounds, consisting of 20 primary amines and their corresponding guanidiniums and pyridiniums, was assessed in . We also installed these alternate functional groups onto antibiotic scaffolds and assessed their accumulation and antibacterial activity in Gram-negative bacteria. The results suggest that other positively-charged, nitrogen-containing functional groups should be considered when designing antibiotics with Gram-negative activity.
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http://dx.doi.org/10.1021/acsinfecdis.0c00715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7796962PMC
January 2021

Re-engineering natural products to engage new biological targets.

Nat Prod Rep 2020 11;37(11):1395-1403

Department of Chemistry, Institute for Genomic Biology, Cancer Center at Illinois, University of Illinois, Urbana-Champaign, USA.

Covering: up to 2020 Natural products have a long history in drug discovery, with their inherent biological activity often tailored by medicinal chemists to arrive at the final drug product. This process is illustrated by numerous examples, including the conversion of epothilone to ixabepilone, erythromycin to azithromycin, and lovastatin to simvastatin. However, natural products are also fruitful starting points for the creation of complex and diverse compounds, especially those that are markedly different from the parent natural product and accordingly do not retain the biological activity of the parent. The resulting products have physiochemical properties that differ considerably when compared to traditional screening collections, thus affording an opportunity to discover novel biological activity. The synthesis of new structural frameworks from natural products thus yields value-added compounds, as demonstrated in the last several years with multiple biological discoveries emerging from these collections. This Highlight details a handful of these studies, describing new compounds derived from natural products that have biological activity and cellular targets different from those evoked/engaged by the parent. Such re-engineering of natural products offers the potential for discovering compounds with interesting and unexpected biological activity.
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http://dx.doi.org/10.1039/d0np00059kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7720426PMC
November 2020

Gram-Negative Antibiotic Active Through Inhibition of an Essential Riboswitch.

J Am Chem Soc 2020 06 8;142(24):10856-10862. Epub 2020 Jun 8.

Department of Pathobiology, University of Illinois, Urbana, Illinois 61802, United States.

Multidrug-resistant Gram-negative (GN) infections for which there are few available treatment options are increasingly common. The development of new antibiotics for these pathogens is challenging because of the inability of most small molecules to accumulate inside GN bacteria. Using recently developed predictive guidelines for compound accumulation in , we have converted the antibiotic Ribocil C, which targets the flavin mononucleotide (FMN) riboswitch, from a compound lacking whole-cell activity against wild-type GN pathogens into a compound that accumulates to a high level in , is effective against Gram-negative clinical isolates, and has efficacy in mouse models of GN infections. This compound allows for the first assessment of the translational potential of FMN riboswitch binders against wild-type Gram-negative bacteria.
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http://dx.doi.org/10.1021/jacs.0c04427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405991PMC
June 2020

Imidazotetrazines as Weighable Diazomethane Surrogates for Esterifications and Cyclopropanations.

Angew Chem Int Ed Engl 2020 01 13;59(5):1857-1862. Epub 2019 Dec 13.

Department of Chemistry and Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.

Diazomethane is one of the most versatile reagents in organic synthesis, but its utility is limited by its hazardous nature. Although alternative methods exist to perform the unique chemistry of diazomethane, these suffer from diminished reactivity and/or correspondingly harsher conditions. Herein, we describe the repurposing of imidazotetrazines (such as temozolomide, TMZ, the standard of care for glioblastoma) for use as synthetic precursors of alkyl diazonium reagents. TMZ was employed to conduct esterifications and metal-catalyzed cyclopropanations, and results show that methyl ester formation from a wide variety of substrates is especially efficient and operationally simple. TMZ is a commercially available solid that is non-explosive and non-toxic, and should find broad utility as a replacement for diazomethane.
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http://dx.doi.org/10.1002/anie.201911896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6982548PMC
January 2020

Implementation of permeation rules leads to a FabI inhibitor with activity against Gram-negative pathogens.

Nat Microbiol 2020 01 18;5(1):67-75. Epub 2019 Nov 18.

Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

Gram-negative bacterial infections are a significant public health concern, and the lack of new drug classes for these pathogens is linked to the inability of most drug leads to accumulate inside Gram-negative bacteria. Here, we report the development of a web application-eNTRyway-that predicts compound accumulation (in Escherichia coli) from its structure. In conjunction with structure-activity relationships and X-ray data, eNTRyway was utilized to re-design Debio-1452-a Gram-positive-only antibiotic-into versions that accumulate in E. coli and possess antibacterial activity against high-priority Gram-negative pathogens. The lead compound Debio-1452-NH3 operates as an antibiotic via the same mechanism as Debio-1452, namely potent inhibition of the enoyl-acyl carrier protein reductase FabI, as validated by in vitro enzyme assays and the generation of bacterial isolates with spontaneous target mutations. Debio-1452-NH3 is well tolerated in vivo, reduces bacterial burden in mice and rescues mice from lethal infections with clinical isolates of Acinetobacter baumannii, Klebsiella pneumoniae and E. coli. This work provides tools for the facile discovery and development of high-accumulating compounds in E. coli, and a general blueprint for the conversion of Gram-positive-only compounds into broad-spectrum antibiotics.
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http://dx.doi.org/10.1038/s41564-019-0604-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953607PMC
January 2020

Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program.

Acta Neuropathol 2019 12 19;138(6):1053-1074. Epub 2019 Aug 19.

Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet, Barcelona, Catalonia, Spain.

Tumors have aberrant proteomes that often do not match their corresponding transcriptome profiles. One possible cause of this discrepancy is the existence of aberrant RNA modification landscapes in the so-called epitranscriptome. Here, we report that human glioma cells undergo DNA methylation-associated epigenetic silencing of NSUN5, a candidate RNA methyltransferase for 5-methylcytosine. In this setting, NSUN5 exhibits tumor-suppressor characteristics in vivo glioma models. We also found that NSUN5 loss generates an unmethylated status at the C3782 position of 28S rRNA that drives an overall depletion of protein synthesis, and leads to the emergence of an adaptive translational program for survival under conditions of cellular stress. Interestingly, NSUN5 epigenetic inactivation also renders these gliomas sensitive to bioactivatable substrates of the stress-related enzyme NQO1. Most importantly, NSUN5 epigenetic inactivation is a hallmark of glioma patients with long-term survival for this otherwise devastating disease.
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http://dx.doi.org/10.1007/s00401-019-02062-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851045PMC
December 2019

Procaspase-3 Overexpression in Cancer: A Paradoxical Observation with Therapeutic Potential.

ACS Chem Biol 2019 11 16;14(11):2335-2348. Epub 2019 Jul 16.

Department of Chemistry and Institute for Genomic Biology , University of Illinois at Urbana-Champaign , Urbana , Illinois , United States.

Many anticancer strategies rely on the promotion of apoptosis in cancer cells as a means to shrink tumors. Crucial for apoptotic function are executioner caspases, most notably caspase-3, that proteolyze a variety of proteins, inducing cell death. Paradoxically, overexpression of procaspase-3 (PC-3), the low-activity zymogen precursor to caspase-3, has been reported in a variety of cancer types. Until recently, this counterintuitive overexpression of a pro-apoptotic protein in cancer has been puzzling. Recent studies suggest subapoptotic caspase-3 activity may promote oncogenic transformation, a possible explanation for the enigmatic overexpression of PC-3. Herein, the overexpression of PC-3 in cancer and its mechanistic basis is reviewed; collectively, the data suggest the potential for exploitation of PC-3 overexpression with PC-3 activators as a targeted anticancer strategy.
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http://dx.doi.org/10.1021/acschembio.9b00338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858495PMC
November 2019

Editorial overview: From the iceman to modern medicine.

Curr Opin Chem Biol 2019 06 6;50:A1-A2. Epub 2019 Jun 6.

Department of Chemistry, University of Illinois, Urbana, IL 61802, USA. Electronic address:

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http://dx.doi.org/10.1016/j.cbpa.2019.04.021DOI Listing
June 2019

Diverse compounds from pleuromutilin lead to a thioredoxin inhibitor and inducer of ferroptosis.

Nat Chem 2019 06 13;11(6):521-532. Epub 2019 May 13.

Department of Chemistry, Roger Adams Laboratory, University of Illinois, Urbana, IL, USA.

The chemical diversification of natural products provides a robust and general method for the creation of stereochemically rich and structurally diverse small molecules. The resulting compounds have physicochemical traits different from those in most screening collections, and as such are an excellent source for biological discovery. Herein, we subject the diterpene natural product pleuromutilin to reaction sequences focused on creating ring system diversity in few synthetic steps. This effort resulted in a collection of compounds with previously unreported ring systems, providing a novel set of structurally diverse and highly complex compounds suitable for screening in a variety of different settings. Biological evaluation identified the novel compound ferroptocide, a small molecule that rapidly and robustly induces ferroptotic death of cancer cells. Target identification efforts and CRISPR knockout studies reveal that ferroptocide is an inhibitor of thioredoxin, a key component of the antioxidant system in the cell. Ferroptocide positively modulates the immune system in a murine model of breast cancer and will be a useful tool to study the utility of pro-ferroptotic agents for treatment of cancer.
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http://dx.doi.org/10.1038/s41557-019-0261-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6639018PMC
June 2019

Structural analyses of NudT16-ADP-ribose complexes direct rational design of mutants with improved processing of poly(ADP-ribosyl)ated proteins.

Sci Rep 2019 04 11;9(1):5940. Epub 2019 Apr 11.

Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.

ADP-ribosylation is a post-translational modification that occurs on chemically diverse amino acids, including aspartate, glutamate, lysine, arginine, serine and cysteine on proteins and is mediated by ADP-ribosyltransferases, including a subset commonly known as poly(ADP-ribose) polymerases. ADP-ribose can be conjugated to proteins singly as a monomer or in polymeric chains as poly(ADP-ribose). While ADP-ribosylation can be reversed by ADP-ribosylhydrolases, this protein modification can also be processed to phosphoribosylation by enzymes possessing phosphodiesterase activity, such as snake venom phosphodiesterase, mammalian ectonucleotide pyrophosphatase/phosphodiesterase 1, Escherichia coli RppH, Legionella pneumophila Sde and Homo sapiens NudT16 (HsNudT16). Our studies here sought to utilize X-ray crystallographic structures of HsNudT16 in complex with monomeric and dimeric ADP-ribose in identifying the active site for binding and processing free and protein-conjugated ADP-ribose into phosphoribose forms. These structural data guide rational design of mutants that widen the active site to better accommodate protein-conjugated ADP-ribose. We identified that several HsNudT16 mutants (Δ17, F36A, and F61S) have reduced activity for free ADP-ribose, similar processing ability against protein-conjugated mono(ADP-ribose), but improved catalytic efficiency for protein-conjugated poly(ADP-ribose). These HsNudT16 variants may, therefore, provide a novel tool to investigate different forms of ADP-ribose.
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http://dx.doi.org/10.1038/s41598-019-39491-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459841PMC
April 2019

Immunohistochemical Characterization of Procaspase-3 Overexpression as a Druggable Target With PAC-1, a Procaspase-3 Activator, in Canine and Human Brain Cancers.

Front Oncol 2019 25;9:96. Epub 2019 Feb 25.

Department of Veterinary Clinical Medicine and Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, United States.

Gliomas and meningiomas are the most common brain neoplasms affecting both humans and canines, and identifying druggable targets conserved across multiple brain cancer histologies and comparative species could broadly improve treatment outcomes. While satisfactory cure rates for low grade, non-invasive brain cancers are achievable with conventional therapies including surgery and radiation, the management of non-resectable or recurrent brain tumors remains problematic and necessitates the discovery of novel therapies that could be accelerated through a comparative approach, such as the inclusion of pet dogs with naturally-occurring brain cancers. Evidence supports procaspase-3 as a druggable brain cancer target with PAC-1, a pro-apoptotic, small molecule activator of procaspase-3 that crosses the blood-brain barrier. Procaspase-3 is frequently overexpressed in malignantly transformed tissues and provides a preferential target for inducing cancer cell apoptosis. While preliminary evidence supports procaspase-3 as a viable target in preclinical models, with PAC-1 demonstrating activity in rodent models and dogs with spontaneous brain tumors, the broader applicability of procaspase-3 as a target in human brain cancers, as well as the comparability of procaspase-3 expressions between differing species, requires further investigation. As such, a large-scale validation of procaspase-3 as a druggable target was undertaken across 651 human and canine brain tumors. Relative to normal brain tissues, procaspase-3 was overexpressed in histologically diverse cancerous brain tissues, supporting procaspase-3 as a broad and conserved therapeutic target. Additionally, procaspase-3 expressing glioma and meningioma cell lines were sensitive to the apoptotic effects of PAC-1 at biologically relevant exposures achievable in cancer patients. Importantly, the clinical relevance of procaspase-3 as a potential prognostic variable was demonstrated in human astrocytomas of variable histologic grades and associated clinical outcomes, whereby tumoral procaspase-3 expression was negatively correlated with survival; findings which suggest that PAC-1 might provide the greatest benefit for patients with the most guarded prognoses.
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http://dx.doi.org/10.3389/fonc.2019.00096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397847PMC
February 2019

Enantioselective Synthesis of Isocarbostyril Alkaloids and Analogs Using Catalytic Dearomative Functionalization of Benzene.

J Am Chem Soc 2019 01 20;141(1):657-670. Epub 2018 Dec 20.

Roger Adams Laboratory, Department of Chemistry , University of Illinois , Urbana , Illinois 61801 , United States.

Enantioselective total syntheses of the anticancer isocarbostyril alkaloids (+)-7-deoxypancratistatin, (+)-pancratistatin, (+)-lycoricidine, and (+)-narciclasine are described. Our strategy for accessing this unique class of natural products is based on the development of a Ni-catalyzed dearomative trans-1,2-carboamination of benzene. The effectiveness of this dearomatization approach is notable, as only two additional olefin functionalizations are needed to construct the fully decorated aminocyclitol cores of these alkaloids. Installation of the lactam ring has been achieved through several pathways and a direct interconversion between natural products was established via a late-stage C-7 cupration. Using this synthetic blueprint, we were able to produce natural products on a gram scale and provide tailored analogs with improved activity, solubility, and metabolic stability.
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http://dx.doi.org/10.1021/jacs.8b12123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488038PMC
January 2019

(ADP-ribosyl)hydrolases: Structural Basis for Differential Substrate Recognition and Inhibition.

Cell Chem Biol 2018 12 21;25(12):1533-1546.e12. Epub 2018 Nov 21.

Sir William Dunn School of Pathology, Oxford University, South Parks Road, Oxford OX1 3RE, UK. Electronic address:

Protein ADP-ribosylation is a highly dynamic post-translational modification. The rapid turnover is achieved, among others, by ADP-(ribosyl)hydrolases (ARHs), an ancient family of enzymes that reverses this modification. Recently ARHs came into focus due to their role as regulators of cellular stresses and tumor suppressors. Here we present a comprehensive structural analysis of the enzymatically active family members ARH1 and ARH3. These two enzymes have very distinct substrate requirements. Our data show that binding of the adenosine ribose moiety is highly diverged between the two enzymes, whereas the active sites harboring the distal ribose closely resemble each other. Despite this apparent similarity, we elucidate the structural basis for the selective inhibition of ARH3 by the ADP-ribose analogues ADP-HPD and arginine-ADP-ribose. Together, our biochemical and structural work provides important insights into the mode of enzyme-ligand interaction, helps to understand differences in their catalytic behavior, and provides useful tools for targeted drug design.
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http://dx.doi.org/10.1016/j.chembiol.2018.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309922PMC
December 2018

Oxygen Starvation Unmasks a Killer.

Cell Chem Biol 2018 11;25(11):1313-1314

Department of Chemistry and Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. Electronic address:

In this issue of Cell Chemical Biology, Jacobsen et al. (2018) investigate the hypoxia selectivity of two cyclolipodepsipeptide natural products bearing a 4-amido-2,4-pentadienoate warhead. A switch in the cell death pathway under hypoxic conditions is observed, suggesting these electrophilic natural products have potential as a prodrug-free approach for treating hypoxic tumors.
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http://dx.doi.org/10.1016/j.chembiol.2018.10.028DOI Listing
November 2018

Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate.

Cell Chem Biol 2018 12 11;25(12):1562-1570.e19. Epub 2018 Oct 11.

Department of Chemistry and Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 261 Roger Adams Lab Box 36-5, 600 S. Mathews Avenue, Urbana, IL 61801, USA. Electronic address:

The post-translational modification (PTM) and signaling molecule poly(ADP-ribose) (PAR) has an impact on diverse biological processes. This PTM is regulated by a series of ADP-ribosyl glycohydrolases (PARG enzymes) that cleave polymers and/or liberate monomers from their protein targets. Existing methods for monitoring these hydrolases rely on detection of the natural substrate, PAR, commonly achieved via radioisotopic labeling. Here we disclose a general substrate for monitoring PARG activity, TFMU-ADPr, which directly reports on total PAR hydrolase activity via release of a fluorophore; this substrate has excellent reactivity, generality (processed by the major PARG enzymes), stability, and usability. A second substrate, TFMU-IDPr, selectively reports on PARG activity only from the enzyme ARH3. Use of these probes in whole-cell lysate experiments has revealed a mechanism by which ARH3 is inhibited by cholera toxin. TFMU-ADPr and TFMU-IDPr are versatile tools for assessing small-molecule inhibitors in vitro and probing the regulation of ADP-ribosyl catabolic enzymes.
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http://dx.doi.org/10.1016/j.chembiol.2018.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309520PMC
December 2018

Tunable Stability of Imidazotetrazines Leads to a Potent Compound for Glioblastoma.

ACS Chem Biol 2018 11 8;13(11):3206-3216. Epub 2018 Nov 8.

Department of Neurosurgery , Johns Hopkins University School of Medicine , Baltimore , Maryland 21287 , United States.

Even in the era of personalized medicine and immunotherapy, temozolomide (TMZ), a small molecule DNA alkylating agent, remains the standard-of-care for glioblastoma (GBM). TMZ has an unusual mode-of-action, spontaneously converting to its active component via hydrolysis in vivo. While TMZ has been FDA approved for two decades, it provides little benefit to patients whose tumors express the resistance enzyme MGMT and gives rise to systemic toxicity through myelosuppression. TMZ was first synthesized in 1984, but certain key derivatives have been inaccessible due to the chemical sensitivity of TMZ, precluding broad exploration of the link between imidazotetrazine structure and biological activity. Here, we sought to discern the relationship between the hydrolytic stability and anticancer activity of imidazotetrazines, with the objectives of identifying optimal timing for prodrug activation and developing suitable compounds with enhanced efficacy via increased blood-brain barrier penetrance. This work necessitated the development of new synthetic methods to provide access to previously unexplored functionality (such as aliphatic, ketone, halogen, and aryl groups) at the C8 position of imidazotetrazines. Through synthesis and evaluation of a suite of compounds with a range of aqueous stabilities (from 0.5 to 40 h), we derive a predictive model for imidazotetrazine hydrolytic stability based on the Hammett constant of the C8 substituent. Promising compounds were identified that possess activity against a panel of GBM cell lines, appropriate hydrolytic and metabolic stability, and brain-to-serum ratios dramatically elevated relative to TMZ, leading to lower hematological toxicity profiles and superior activity to TMZ in a mouse model of GBM. This work points a clear path forward for the development of novel and effective anticancer imidazotetrazines.
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http://dx.doi.org/10.1021/acschembio.8b00864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6243397PMC
November 2018

Preparation of Structurally Diverse Compounds from the Natural Product Lycorine.

Org Lett 2018 09 11;20(18):5894-5898. Epub 2018 Sep 11.

Department of Chemistry, Roger Adams Laboratory , University of Illinois at Urbana-Champaign , 600 South Mathews Avenue , Urbana , Illinois 61801 , United States.

The synthesis of a 52-member compound collection from the natural product lycorine is reported, highlighted by divergent cross-coupling and substitution strategies and an unusual ring rearrangement induced by reaction with aryne intermediates.
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http://dx.doi.org/10.1021/acs.orglett.8b02562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6499378PMC
September 2018

Overcoming Resistance to Targeted Anticancer Therapies through Small-Molecule-Mediated MEK Degradation.

Cell Chem Biol 2018 08 14;25(8):996-1005.e4. Epub 2018 Jun 14.

Department of Chemistry and Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 261 Roger Adams Lab Box 36-5, 600 S. Mathews Avenue, Urbana, IL 61801, USA. Electronic address:

The discovery of mutant or fusion kinases that drive oncogenesis, and the subsequent approval of specific inhibitors for these enzymes, has been instrumental in the management of some cancers. However, acquired resistance remains a significant problem in the clinic, limiting the long-term effectiveness of most of these drugs. Here we demonstrate a general strategy to overcome this resistance through drug-induced MEK cleavage (via direct procaspase-3 activation) combined with targeted kinase inhibition. This combination effect is shown to be general across diverse tumor histologies (melanoma, lung cancer, and leukemia) and driver mutations (mutant BRAF or EGFR, fusion kinases EML4-ALK and BCR-ABL). Caspase-3-mediated degradation of MEK kinases results in sustained pathway inhibition and substantially delayed or eliminated resistance in cancer cells in a manner far superior to combinations with MEK inhibitors. These data suggest the generality of drug-mediated MEK kinase cleavage as a therapeutic strategy to prevent resistance to targeted anticancer therapies.
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http://dx.doi.org/10.1016/j.chembiol.2018.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6097934PMC
August 2018

Strong and sustained activation of the anticipatory unfolded protein response induces necrotic cell death.

Cell Death Differ 2018 10 13;25(10):1796-1807. Epub 2018 Jun 13.

Department of Biochemistry, University of Illinois, Urbana, IL, 61801, USA.

The endoplasmic reticulum stress sensor, the unfolded protein response (UPR), regulates intracellular protein homeostasis. While transient activation of the reactive UPR by unfolded protein is protective, prolonged and sustained activation of the reactive UPR triggers CHOP-mediated apoptosis. In the recently characterized, evolutionarily conserved anticipatory UPR, mitogenic hormones and other effectors pre-activate the UPR; how strong and sustained activation of the anticipatory UPR induces cell death was unknown. To characterize this cell death pathway, we used BHPI, a small molecule that activates the anticipatory UPR through estrogen receptor α (ERα) and induces death of ERα cancer cells. We show that sustained activation of the anticipatory UPR by BHPI kills cells by inducing depletion of intracellular ATP, resulting in classical necrosis phenotypes, including plasma membrane disruption and leakage of intracellular contents. Unlike reactive UPR activation, BHPI-induced hyperactivation of the anticipatory UPR does not induce apoptosis or sustained autophagy. BHPI does not induce CHOP protein or PARP cleavage, and two pan-caspase inhibitors, or Bcl2 overexpression, have no effect on BHPI-induced cell death. Moreover, BHPI does not increase expression of autophagy markers, or work through recently identified programmed-necrosis pathways, such as necroptosis. Opening of endoplasmic reticulum IPR calcium channels stimulates cell swelling, cPLA2 activation, and arachidonic acid release. Notably, cPLA2 activation requires ATP depletion. Importantly, blocking rapid cell swelling or production of arachidonic acid does not prevent necrotic cell death. Rapid cell death is upstream of PERK activation and protein synthesis inhibition, and results from strong and sustained activation of early steps in the anticipatory UPR. Supporting a central role for ATP depletion, reversing ATP depletion blocks rapid cell death, and the onset of necrotic cell death is correlated with ATP depletion. Necrotic cell death initiated by strong and sustained activation of the anticipatory UPR is a newly discovered role of the UPR.
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http://dx.doi.org/10.1038/s41418-018-0143-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6180055PMC
October 2018

Going on offense against the gram-negative defense.

Proc Natl Acad Sci U S A 2018 06 25;115(26):6530-6532. Epub 2018 May 25.

Department of Chemistry, University of Illinois, Urbana, IL 61801;

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http://dx.doi.org/10.1073/pnas.1807278115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6042078PMC
June 2018

The challenge of converting Gram-positive-only compounds into broad-spectrum antibiotics.

Ann N Y Acad Sci 2019 01 15;1435(1):18-38. Epub 2018 Feb 15.

Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois.

Multidrug resistant Gram-negative bacterial infections are on the rise, and there is a lack of new classes of drugs to treat these pathogens. This drug shortage is largely due to the challenge of finding antibiotics that can permeate and persist inside Gram-negative species. Efforts to understand the molecular properties that enable certain compounds to accumulate in Gram-negative bacteria based on retrospective studies of known antibiotics have not been generally actionable in the development of new antibiotics. A recent assessment of the ability of >180 diverse small molecules to accumulate in Escherichia coli led to predictive guidelines for compound accumulation in E. coli. These "eNTRy rules" state that compounds are most likely to accumulate if they contain a nonsterically encumbered ionizable Nitrogen (primary amines are the best), have low Three-dimensionality (globularity ≤ 0.25), and are relatively Rigid (rotatable bonds ≤ 5). In this review, we look back through 50+ years of antibacterial research and 1000s of derivatives and assess this historical data set through the lens of these predictive guidelines. The results are consistent with the eNTRy rules, suggesting that the eNTRy rules may provide an actionable and general roadmap for the conversion of Gram-positive-only compounds into broad-spectrum antibiotics.
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http://dx.doi.org/10.1111/nyas.13598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6093809PMC
January 2019

How many human proteoforms are there?

Nat Chem Biol 2018 02;14(3):206-214

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA.

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.
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http://dx.doi.org/10.1038/nchembio.2576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5837046PMC
February 2018

Synergistic and targeted therapy with a procaspase-3 activator and temozolomide extends survival in glioma rodent models and is feasible for the treatment of canine malignant glioma patients.

Oncotarget 2017 Oct 7;8(46):80124-80138. Epub 2017 Jul 7.

Department of Neurosurgery, School of Medicine, Johns Hopkins University, Baltimore, MD, USA.

Purpose: Glioblastoma is a deadly brain cancer with a median survival time of ∼15 months. Ionizing radiation plus the DNA alkylator temozolomide (TMZ) is the current standard therapy. PAC-1, a procaspase-3 activating small molecule, is blood-brain barrier penetrant and has previously demonstrated ability to synergize with diverse pro-apoptotic chemotherapeutics. We studied if PAC-1 could enhance the activity of TMZ, and whether addition of PAC-1 to standard treatment would be feasible in spontaneous canine malignant gliomas.

Experimental Design: Using cell lines and online gene expression data, we identified procaspase-3 as a potential molecular target for most glioblastomas. We investigated PAC-1 as a single agent and in combination with TMZ against glioma cells in culture and in orthotopic rodent models of glioma. Three dogs with spontaneous gliomas were treated with an analogous human glioblastoma treatment protocol, with concurrent PAC-1.

Results: Procaspase-3 is expressed in gliomas, with higher gene expression correlating with increased tumor grade and decreased prognosis. PAC-1 is cytotoxic to glioma cells in culture and active in orthotopic rodent glioma models. PAC-1 added to TMZ treatments in cell culture increases apoptotic death, and the combination significantly increases survival in orthotopic glioma models. Addition of PAC-1 to TMZ and radiation was well-tolerated in 3 out of 3 pet dogs with spontaneous glioma, and partial to complete tumor reductions were observed.

Conclusions: Procaspase-3 is a clinically relevant target for treatment of glioblastoma. Synergistic activity of PAC-1/TMZ in rodent models and the demonstration of feasibility of the combined regime in canine patients suggest potential for PAC-1 in the treatment of glioblastoma.
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http://dx.doi.org/10.18632/oncotarget.19085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655184PMC
October 2017

Single-cell analysis of early antiviral gene expression reveals a determinant of stochastic IFNB1 expression.

Integr Biol (Camb) 2017 Nov;9(11):857-867

Center for Computational Biology and Biophysics, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA.

RIG-I-like receptors (RLRs) are cytoplasmic sensors of viral RNA that trigger the signaling cascade that leads to type I interferon (IFN) production. Transcriptional induction of RLRs by IFN is believed to play the role of positive feedback to further amplify viral sensing. We found that RLRs and several other IFN-stimulated genes (ISGs) are induced early in viral infection independent of IFN. Expression of these early ISGs requires IRF3/IRF7 and is highly correlated amongst them. Simultaneous detection of mRNA of IFNB1, viral replicase, and ISGs revealed distinct populations of IFNB1 expressing and non-expressing cells which are highly correlated with the levels of early ISGs but are uncorrelated with IFN-dependent ISGs and viral gene expression. Individual expression of RLRs made IFNB1 expression more robust and earlier, suggesting a causal relation between levels of RLR and induction of IFN.
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http://dx.doi.org/10.1039/c7ib00146kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6201300PMC
November 2017

Characterization of the Saffron Derivative Crocetin as an Inhibitor of Human Lactate Dehydrogenase 5 in the Antiglycolytic Approach against Cancer.

J Agric Food Chem 2017 Jul 7;65(28):5639-5649. Epub 2017 Jul 7.

Dipartimento di Farmacia, Università di Pisa , Via Bonanno 6 and 33, 56126 Pisa, Italy.

Inhibition of lactate dehydrogenase (LDH) represents an innovative approach to tackle cancer because this peculiar glycolytic metabolism is characteristic of most invasive tumor cells. An investigation into the biological properties of saffron extracts led to the discover of their LDH-inhibition properties. In particular, the most important saffron components, crocetin, was found to inhibit LDH (IC = 54.9 ± 4.7 μM). This carotenoid was independently produced by chemical synthesis, and its LDH-inhibition properties manifested via its antiproliferative activity against two glycolytic cancer cell lines (A549 and HeLa, IC = 114.0 ± 8.0 and 113.0 ± 11.1 μM, respectively). The results described in this article suggest that saffron may be a helpful alimentary component in the prevention of cancer that potentially contributes to the efficacy of approved cancer therapies.
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http://dx.doi.org/10.1021/acs.jafc.7b01668DOI Listing
July 2017

Natural products: Taming reactive benzynes.

Nat Chem 2017 May;9(6):504-506

Department of Chemistry at the University of Illinois at Urbana-Champaign, Urbana, Ilinois 61801, USA.

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http://dx.doi.org/10.1038/nchem.2786DOI Listing
May 2017