Light Activation of <i>Staphylococcus aureus</i> Toxin YoeB_<i>Sa</i>1 Reveals Guanosine-Specific Endoribonuclease Activity

The <i>Staphylococcus aureus</i> chromosome harbors two homologues of the YefM-YoeB toxin–antitoxin (TA) system. The toxins YoeB_<i>Sa</i>1 and YoeB_<i>Sa</i>2 possess ribosome-dependent ribonuclease (RNase) activity in <i>Escherichia coli</i>. This activity is similar to that of the <i>E. coli</i> toxin YoeB_<i>Ec</i>, an enzyme that, in addition to ribosome-dependent RNase activity, possesses ribosome-independent RNase activity <i>in vitro</i>. To investigate whether YoeB_<i>Sa</i>1 is also a ribosome-independent RNase, we expressed YoeB_<i>Sa</i>1 using a novel strategy and characterized its <i>in vitro</i> RNase activity, sequence specificity, and kinetics. Y88 of YoeB_<i>Sa</i>1 was critical for <i>in vitro</i> activity and cell culture toxicity. This residue was mutated to <i>o</i>-nitrobenzyl tyrosine (ONBY) via unnatural amino acid mutagenesis. YoeB_<i>Sa</i>1-Y88ONBY could be expressed in the absence of the antitoxin YefM_<i>Sa</i>1 in <i>E. coli</i>. Photocaged YoeB_<i>Sa</i>1-Y88ONBY displayed UV light-dependent RNase activity toward free mRNA <i>in vitro</i>. The <i>in vitro</i> ribosome-independent RNase activity of YoeB_<i>Sa</i>1-Y88ONBY, YoeB_<i>Sa</i>1-Y88F, and YoeB_<i>Sa</i>1-Y88TAG was significantly reduced or abolished. In contrast to YoeB_<i>Ec</i>, which cleaves RNA at both adenosine and guanosine with a preference for adenosine, YoeB_<i>Sa</i>1 cleaved mRNA specifically at guanosine. Using this information, a fluorometric assay was developed and used to determine the kinetic parameters for ribosome-independent RNA cleavage by YoeB_<i>Sa</i>1

Access to a Structurally Complex Compound Collection via Ring Distortion of the Alkaloid Sinomenine

Many compound collections used in high-throughput screening are composed of members whose structural complexity is considerably lower than that of natural products. We previously reported a strategy for the synthesis of complex and diverse small molecules from natural products using ring-distortion reactions, called complexity-to-diversity (CtD), and herein, CtD is applied in the synthesis of 16 diverse scaffolds and 65 total compounds from the alkaloid natural product sinomenine. Chemoinformatic analysis shows that these compounds possess complex ring systems and marked three-dimensionality

Total Synthesis, Stereochemical Assignment, and Biological Activity of All Known (−)-Trigonoliimines

A full account of our concise and enantioselective total syntheses of all known (−)-trigonoliimine alkaloids is described. Our retrobiosynthetic analysis of these natural products enabled identification of a single bistryptamine precursor as a precursor to all known trigonoliimines through a sequence of transformations involving asymmetric oxidation and reorganization. Our enantioselective syntheses of these alkaloids enabled the revision of the absolute stereochemistry of (−)-trigonoliimines A, B, and C. We report that trigonoliimines A, B, C and structurally related compounds showed weak anticancer activities against HeLa and U-937 cells

Efficient NQO1 Substrates are Potent and Selective Anticancer Agents

A major goal of personalized medicine in oncology is the identification of drugs with predictable efficacy based on a specific trait of the cancer cell, as has been demonstrated with gleevec (presence of Bcr-Abl protein), herceptin (Her2 overexpression), and iressa (presence of a specific EGFR mutation). This is a challenging task, as it requires identifying a cellular component that is altered in cancer, but not normal cells, and discovering a compound that specifically interacts with it. The enzyme NQO1 is a potential target for personalized medicine, as it is overexpressed in many solid tumors. In normal cells NQO1 is inducibly expressed, and its major role is to detoxify quinones via bioreduction; however, certain quinones become more toxic after reduction by NQO1, and these compounds have potential as selective anticancer agents. Several quinones of this type have been reported, including mitomycin C, RH1, EO9, streptonigrin, β-lapachone, and deoxynyboquinone (DNQ). However, no unified picture has emerged from these studies, and the key question regarding the relationship between NQO1 processing and anticancer activity remains unanswered. Here, we directly compare these quinones as substrates for NQO1 <i>in vitro</i>, and for their ability to kill cancer cells in culture in an NQO1-dependent manner. We show that DNQ is a superior NQO1 substrate, and we use computationally guided design to create DNQ analogues that have a spectrum of activities with NQO1. Assessment of these compounds definitively establishes a strong relationship between <i>in vitro</i> NQO1 processing and induction of cancer cell death and suggests these compounds are outstanding candidates for selective anticancer therapy

Synthesis and Anticancer Activity of All Known (−)-Agelastatin Alkaloids

The full details of our enantioselective total syntheses of (−)-agelastatins A–F (<b>1</b>–<b>6</b>), the evolution of a new methodology for synthesis of substituted azaheterocycles, and the first side-by-side evaluation of all known (−)-agelastatin alkaloids against nine human cancer cell lines are described. Our concise synthesis of these alkaloids exploits the intrinsic chemistry of plausible biosynthetic precursors and capitalizes on a late-stage synthesis of the C-ring. The critical copper-mediated cross-coupling reaction was expanded to include guanidine-based systems, offering a versatile preparation of substituted imidazoles. The direct comparison of the anticancer activity of all naturally occurring (−)-agelastatins in addition to eight advanced synthetic intermediates enabled a systematic analysis of the structure–activity relationship within the natural series. Significantly, (−)-agelastatin A (<b>1</b>) is highly potent against six blood cancer cell lines (20–190 nM) without affecting normal red blood cells (>333 μM). (−)-Agelastatin A (<b>1</b>) and (−)-agelastatin D (<b>4</b>), the two most potent members of this family, induce dose-dependent apoptosis and arrest cells in the G2/M-phase of the cell cycle; however, using confocal microscopy, we have determined that neither alkaloid affects tubulin dynamics within cells

Synthesis and Anticancer Activity of All Known (−)-Agelastatin Alkaloids

The full details of our enantioselective total syntheses of (−)-agelastatins A–F (<b>1</b>–<b>6</b>), the evolution of a new methodology for synthesis of substituted azaheterocycles, and the first side-by-side evaluation of all known (−)-agelastatin alkaloids against nine human cancer cell lines are described. Our concise synthesis of these alkaloids exploits the intrinsic chemistry of plausible biosynthetic precursors and capitalizes on a late-stage synthesis of the C-ring. The critical copper-mediated cross-coupling reaction was expanded to include guanidine-based systems, offering a versatile preparation of substituted imidazoles. The direct comparison of the anticancer activity of all naturally occurring (−)-agelastatins in addition to eight advanced synthetic intermediates enabled a systematic analysis of the structure–activity relationship within the natural series. Significantly, (−)-agelastatin A (<b>1</b>) is highly potent against six blood cancer cell lines (20–190 nM) without affecting normal red blood cells (>333 μM). (−)-Agelastatin A (<b>1</b>) and (−)-agelastatin D (<b>4</b>), the two most potent members of this family, induce dose-dependent apoptosis and arrest cells in the G2/M-phase of the cell cycle; however, using confocal microscopy, we have determined that neither alkaloid affects tubulin dynamics within cells

Synthesis of Dimeric ADP-Ribose and Its Structure with Human Poly(ADP-ribose) Glycohydrolase

Poly­(ADP-ribosyl)­ation is a common post-translational modification that mediates a wide variety of cellular processes including DNA damage repair, chromatin regulation, transcription, and apoptosis. The difficulty associated with accessing poly­(ADP-ribose) (PAR) in a homogeneous form has been an impediment to understanding the interactions of PAR with poly­(ADP-ribose) glycohydrolase (PARG) and other binding proteins. Here we describe the chemical synthesis of the ADP-ribose dimer, and we use this compound to obtain the first human PARG substrate-enzyme cocrystal structure. Chemical synthesis of PAR is an attractive alternative to traditional enzymatic synthesis and fractionation, allowing access to products such as dimeric ADP-ribose, which has been detected but never isolated from natural sources. Additionally, we describe the synthesis of an alkynylated dimer and demonstrate that this compound can be used to synthesize PAR probes including biotin and fluorophore-labeled compounds. The fluorescently labeled ADP-ribose dimer was then utilized in a general fluorescence polarization-based PAR–protein binding assay. Finally, we use intermediates of our synthesis to access various PAR fragments, and evaluation of these compounds as substrates for PARG reveals the minimal features for substrate recognition and enzymatic cleavage. Homogeneous PAR oligomers and unnatural variants produced from chemical synthesis will allow for further detailed structural and biochemical studies on the interaction of PAR with its many protein binding partners

Selective Small Molecule Inhibition of Poly(ADP-Ribose) Glycohydrolase (PARG)

The poly­(ADP-ribose) (PAR) post-translational modification is essential for diverse cellular functions, including regulation of transcription, response to DNA damage, and mitosis. Cellular PAR is predominantly synthesized by the enzyme poly­(ADP-ribose) polymerase-1 (PARP-1). PARP-1 is a critical node in the DNA damage response pathway, and multiple potent PARP-1 inhibitors have been described, some of which show considerable promise in the clinic for the treatment of certain cancers. Cellular PAR is efficiently degraded by poly­(ADP-ribose) glycohydrolase (PARG), an enzyme for which no potent, readily accessible, and specific inhibitors exist. Herein we report the discovery of small molecules that effectively inhibit PARG <i>in vitro</i> and in cellular lysates. These potent PARG inhibitors can be produced in two chemical steps from commercial starting materials and have complete specificity for PARG over the other known PAR glycohydrolase (ADP-ribosylhydrolase 3, ARH3) and over PARP-1 and thus will be useful tools for studying the biochemistry of PAR signaling

Dual Small-Molecule Targeting of Procaspase‑3 Dramatically Enhances Zymogen Activation and Anticancer Activity

Combination anticancer therapy typically consists of drugs that target different biochemical pathways or those that act on different targets in the same pathway. Here we demonstrate a new concept in combination therapy, that of enzyme activation with two compounds that hit the same biological target, but through different mechanisms. Combinations of procaspase-3 activators PAC-1 and 1541B show considerable synergy in activating procaspase-3 in vitro, stimulate rapid and dramatic maturation of procaspase-3 in multiple cancer cell lines, and powerfully induce caspase-dependent apoptotic death to a degree well exceeding the additive effect. In addition, the combination of PAC-1 and 1541B effectively reduces tumor burden in a murine lymphoma model at dosages for which the compounds alone have minimal or no effect. These data suggest the potential of PAC-1/1541B combinations for the treatment of cancer and, more broadly, demonstrate that differentially acting enzyme activators can potently synergize to give a significantly heightened biological effect

Small-Molecule Procaspase‑3 Activation Sensitizes Cancer to Treatment with Diverse Chemotherapeutics

Conventional chemotherapeutics remain essential treatments for most cancers, but their combination with other anticancer drugs (including targeted therapeutics) is often complicated by unpredictable synergies and multiplicative toxicities. As cytotoxic anticancer chemotherapeutics generally function through induction of apoptosis, we hypothesized that a molecularly targeted small molecule capable of facilitating a central and defining step in the apoptotic cascade, the activation of procaspase-3 to caspase-3, would broadly and predictably enhance activity of cytotoxic drugs. Here we show that procaspase-activating compound 1 (PAC-1) enhances cancer cell death induced by 15 different FDA-approved chemotherapeutics, across many cancer types and chemotherapeutic targets. In particular, the promising combination of PAC-1 and doxorubicin induces a synergistic reduction in tumor burden and enhances survival in murine tumor models of osteosarcoma and lymphoma. This PAC-1/doxorubicin combination was evaluated in 10 pet dogs with naturally occurring metastatic osteosarcoma or lymphoma, eliciting a biologic response in 3 of 6 osteosarcoma patients and 4 of 4 lymphoma patients. Importantly, in both mice and dogs, coadministration of PAC-1 with doxorubicin resulted in no additional toxicity. On the basis of the mode of action of PAC-1 and the high expression of procaspase-3 in many cancers, these results suggest the combination of PAC-1 with cytotoxic anticancer drugs as a potent and general strategy to enhance therapeutic response