A High Through-put Platform for Recombinant Antibodies to Folded Proteins.

Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade.

The Minderoo-Monaco Commission on Plastics and Human Health.

Background: Plastics have conveyed great benefits to humanity and made possible some of the most significant advances of modern civilization in fields as diverse as medicine, electronics, aerospace, construction, food packaging, and sports. It is now clear, however, that plastics are also responsible for significant harms to human health, the economy, and the earth's environment. These harms occur at every stage of the plastic life cycle, from extraction of the coal, oil, and gas that are its main feedstocks through to ultimate disposal into the environment. The extent of these harms not been systematically assessed, their magnitude not fully quantified, and their economic costs not comprehensively counted. Goals: The goals of this Minderoo-Monaco Commission on Plastics and Human Health are to comprehensively examine plastics' impacts across their life cycle on: (1) human health and well-being; (2) the global environment, especially the ocean; (3) the economy; and (4) vulnerable populations-the poor, minorities, and the world's children. On the basis of this examination, the Commission offers science-based recommendations designed to support development of a Global Plastics Treaty, protect human health, and save lives. Report Structure: This Commission report contains seven Sections. Following an Introduction, Section 2 presents a narrative review of the processes involved in plastic production, use, and disposal and notes the hazards to human health and the environment associated with each of these stages. Section 3 describes plastics' impacts on the ocean and notes the potential for plastic in the ocean to enter the marine food web and result in human exposure. Section 4 details plastics' impacts on human health. Section 5 presents a first-order estimate of plastics' health-related economic costs. Section 6 examines the intersection between plastic, social inequity, and environmental injustice. Section 7 presents the Commission's findings and recommendations. Plastics: Plastics are complex, highly heterogeneous, synthetic chemical materials. Over 98% of plastics are produced from fossil carbon- coal, oil and gas. Plastics are comprised of a carbon-based polymer backbone and thousands of additional chemicals that are incorporated into polymers to convey specific properties such as color, flexibility, stability, water repellence, flame retardation, and ultraviolet resistance. Many of these added chemicals are highly toxic. They include carcinogens, neurotoxicants and endocrine disruptors such as phthalates, bisphenols, per- and poly-fluoroalkyl substances (PFAS), brominated flame retardants, and organophosphate flame retardants. They are integral components of plastic and are responsible for many of plastics' harms to human health and the environment.Global plastic production has increased almost exponentially since World War II, and in this time more than 8,300 megatons (Mt) of plastic have been manufactured. Annual production volume has grown from under 2 Mt in 1950 to 460 Mt in 2019, a 230-fold increase, and is on track to triple by 2060. More than half of all plastic ever made has been produced since 2002. Single-use plastics account for 35-40% of current plastic production and represent the most rapidly growing segment of plastic manufacture.Explosive recent growth in plastics production reflects a deliberate pivot by the integrated multinational fossil-carbon corporations that produce coal, oil and gas and that also manufacture plastics. These corporations are reducing their production of fossil fuels and increasing plastics manufacture. The two principal factors responsible for this pivot are decreasing global demand for carbon-based fuels due to increases in 'green' energy, and massive expansion of oil and gas production due to fracking.Plastic manufacture is energy-intensive and contributes significantly to climate change. At present, plastic production is responsible for an estimated 3.7% of global greenhouse gas emissions, more than the contribution of Brazil. This fraction is projected to increase to 4.5% by 2060 if current trends continue unchecked. Plastic Life Cycle: The plastic life cycle has three phases: production, use, and disposal. In production, carbon feedstocks-coal, gas, and oil-are transformed through energy-intensive, catalytic processes into a vast array of products. Plastic use occurs in every aspect of modern life and results in widespread human exposure to the chemicals contained in plastic. Single-use plastics constitute the largest portion of current use, followed by synthetic fibers and construction.Plastic disposal is highly inefficient, with recovery and recycling rates below 10% globally. The result is that an estimated 22 Mt of plastic waste enters the environment each year, much of it single-use plastic and are added to the more than 6 gigatons of plastic waste that have accumulated since 1950. Strategies for disposal of plastic waste include controlled and uncontrolled landfilling, open burning, thermal conversion, and export. Vast quantities of plastic waste are exported each year from high-income to low-income countries, where it accumulates in landfills, pollutes air and water, degrades vital ecosystems, befouls beaches and estuaries, and harms human health-environmental injustice on a global scale. Plastic-laden e-waste is particularly problematic. Environmental Findings: Plastics and plastic-associated chemicals are responsible for widespread pollution. They contaminate aquatic (marine and freshwater), terrestrial, and atmospheric environments globally. The ocean is the ultimate destination for much plastic, and plastics are found throughout the ocean, including coastal regions, the sea surface, the deep sea, and polar sea ice. Many plastics appear to resist breakdown in the ocean and could persist in the global environment for decades. Macro- and micro-plastic particles have been identified in hundreds of marine species in all major taxa, including species consumed by humans. Trophic transfer of microplastic particles and the chemicals within them has been demonstrated. Although microplastic particles themselves (>10 µm) appear not to undergo biomagnification, hydrophobic plastic-associated chemicals bioaccumulate in marine animals and biomagnify in marine food webs. The amounts and fates of smaller microplastic and nanoplastic particles (MNPs <10 µm) in aquatic environments are poorly understood, but the potential for harm is worrying given their mobility in biological systems. Adverse environmental impacts of plastic pollution occur at multiple levels from molecular and biochemical to population and ecosystem. MNP contamination of seafood results in direct, though not well quantified, human exposure to plastics and plastic-associated chemicals. Marine plastic pollution endangers the ocean ecosystems upon which all humanity depends for food, oxygen, livelihood, and well-being. Human Health Findings: Coal miners, oil workers and gas field workers who extract fossil carbon feedstocks for plastic production suffer increased mortality from traumatic injury, coal workers' pneumoconiosis, silicosis, cardiovascular disease, chronic obstructive pulmonary disease, and lung cancer. Plastic production workers are at increased risk of leukemia, lymphoma, hepatic angiosarcoma, brain cancer, breast cancer, mesothelioma, neurotoxic injury, and decreased fertility. Workers producing plastic textiles die of bladder cancer, lung cancer, mesothelioma, and interstitial lung disease at increased rates. Plastic recycling workers have increased rates of cardiovascular disease, toxic metal poisoning, neuropathy, and lung cancer. Residents of "fenceline" communities adjacent to plastic production and waste disposal sites experience increased risks of premature birth, low birth weight, asthma, childhood leukemia, cardiovascular disease, chronic obstructive pulmonary disease, and lung cancer.During use and also in disposal, plastics release toxic chemicals including additives and residual monomers into the environment and into people. National biomonitoring surveys in the USA document population-wide exposures to these chemicals. Plastic additives disrupt endocrine function and increase risk for premature births, neurodevelopmental disorders, male reproductive birth defects, infertility, obesity, cardiovascular disease, renal disease, and cancers. Chemical-laden MNPs formed through the environmental degradation of plastic waste can enter living organisms, including humans. Emerging, albeit still incomplete evidence indicates that MNPs may cause toxicity due to their physical and toxicological effects as well as by acting as vectors that transport toxic chemicals and bacterial pathogens into tissues and cells.Infants in the womb and young children are two populations at particularly high risk of plastic-related health effects. Because of the exquisite sensitivity of early development to hazardous chemicals and children's unique patterns of exposure, plastic-associated exposures are linked to increased risks of prematurity, stillbirth, low birth weight, birth defects of the reproductive organs, neurodevelopmental impairment, impaired lung growth, and childhood cancer. Early-life exposures to plastic-associated chemicals also increase the risk of multiple non-communicable diseases later in life. Economic Findings: Plastic's harms to human health result in significant economic costs. We estimate that in 2015 the health-related costs of plastic production exceeded $250 billion (2015 Int$) globally, and that in the USA alone the health costs of disease and disability caused by the plastic-associated chemicals PBDE, BPA and DEHP exceeded $920 billion (2015 Int$). Plastic production results in greenhouse gas (GHG) emissions equivalent to 1.96 gigatons of carbon dioxide (CO2e) annually. Using the US Environmental Protection Agency's (EPA) social cost of carbonmetric, we estimate the annual costs of these GHG emissions to be $341 billion (2015 Int$).These costs, large as they are, almost certainly underestimate the full economic losses resulting from plastics' negative impacts on human health and the global environment. All of plastics' economic costs-and also its social costs-are externalized by the petrochemical and plastic manufacturing industry and are borne by citizens, taxpayers, and governments in countries around the world without compensation. Social Justice Findings: The adverse effects of plastics and plastic pollution on human health, the economy and the environment are not evenly distributed. They disproportionately affect poor, disempowered, and marginalized populations such as workers, racial and ethnic minorities, "fenceline" communities, Indigenous groups, women, and children, all of whom had little to do with creating the current plastics crisis and lack the political influence or the resources to address it. Plastics' harmful impacts across its life cycle are most keenly felt in the Global South, in small island states, and in disenfranchised areas in the Global North. Social and environmental justice (SEJ) principles require reversal of these inequitable burdens to ensure that no group bears a disproportionate share of plastics' negative impacts and that those who benefit economically from plastic bear their fair share of its currently externalized costs. Conclusions: It is now clear that current patterns of plastic production, use, and disposal are not sustainable and are responsible for significant harms to human health, the environment, and the economy as well as for deep societal injustices.The main driver of these worsening harms is an almost exponential and still accelerating increase in global plastic production. Plastics' harms are further magnified by low rates of recovery and recycling and by the long persistence of plastic waste in the environment.The thousands of chemicals in plastics-monomers, additives, processing agents, and non-intentionally added substances-include amongst their number known human carcinogens, endocrine disruptors, neurotoxicants, and persistent organic pollutants. These chemicals are responsible for many of plastics' known harms to human and planetary health. The chemicals leach out of plastics, enter the environment, cause pollution, and result in human exposure and disease. All efforts to reduce plastics' hazards must address the hazards of plastic-associated chemicals. Recommendations: To protect human and planetary health, especially the health of vulnerable and at-risk populations, and put the world on track to end plastic pollution by 2040, this Commission supports urgent adoption by the world's nations of a strong and comprehensive Global Plastics Treaty in accord with the mandate set forth in the March 2022 resolution of the United Nations Environment Assembly (UNEA).International measures such as a Global Plastics Treaty are needed to curb plastic production and pollution, because the harms to human health and the environment caused by plastics, plastic-associated chemicals and plastic waste transcend national boundaries, are planetary in their scale, and have disproportionate impacts on the health and well-being of people in the world's poorest nations. Effective implementation of the Global Plastics Treaty will require that international action be coordinated and complemented by interventions at the national, regional, and local levels.This Commission urges that a cap on global plastic production with targets, timetables, and national contributions be a central provision of the Global Plastics Treaty. We recommend inclusion of the following additional provisions:The Treaty needs to extend beyond microplastics and marine litter to include all of the many thousands of chemicals incorporated into plastics.The Treaty needs to include a provision banning or severely restricting manufacture and use of unnecessary, avoidable, and problematic plastic items, especially single-use items such as manufactured plastic microbeads.The Treaty needs to include requirements on extended producer responsibility (EPR) that make fossil carbon producers, plastic producers, and the manufacturers of plastic products legally and financially responsible for the safety and end-of-life management of all the materials they produce and sell.The Treaty needs to mandate reductions in the chemical complexity of plastic products; health-protective standards for plastics and plastic additives; a requirement for use of sustainable non-toxic materials; full disclosure of all components; and traceability of components. International cooperation will be essential to implementing and enforcing these standards.The Treaty needs to include SEJ remedies at each stage of the plastic life cycle designed to fill gaps in community knowledge and advance both distributional and procedural equity.This Commission encourages inclusion in the Global Plastic Treaty of a provision calling for exploration of listing at least some plastic polymers as persistent organic pollutants (POPs) under the Stockholm Convention.This Commission encourages a strong interface between the Global Plastics Treaty and the Basel and London Conventions to enhance management of hazardous plastic waste and slow current massive exports of plastic waste into the world's least-developed countries.This Commission recommends the creation of a Permanent Science Policy Advisory Body to guide the Treaty's implementation. The main priorities of this Body would be to guide Member States and other stakeholders in evaluating which solutions are most effective in reducing plastic consumption, enhancing plastic waste recovery and recycling, and curbing the generation of plastic waste. This Body could also assess trade-offs among these solutions and evaluate safer alternatives to current plastics. It could monitor the transnational export of plastic waste. It could coordinate robust oceanic-, land-, and air-based MNP monitoring programs.This Commission recommends urgent investment by national governments in research into solutions to the global plastic crisis. This research will need to determine which solutions are most effective and cost-effective in the context of particular countries and assess the risks and benefits of proposed solutions. Oceanographic and environmental research is needed to better measure concentrations and impacts of plastics <10 µm and understand their distribution and fate in the global environment. Biomedical research is needed to elucidate the human health impacts of plastics, especially MNPs. Summary: This Commission finds that plastics are both a boon to humanity and a stealth threat to human and planetary health. Plastics convey enormous benefits, but current linear patterns of plastic production, use, and disposal that pay little attention to sustainable design or safe materials and a near absence of recovery, reuse, and recycling are responsible for grave harms to health, widespread environmental damage, great economic costs, and deep societal injustices. These harms are rapidly worsening.While there remain gaps in knowledge about plastics' harms and uncertainties about their full magnitude, the evidence available today demonstrates unequivocally that these impacts are great and that they will increase in severity in the absence of urgent and effective intervention at global scale. Manufacture and use of essential plastics may continue. However, reckless increases in plastic production, and especially increases in the manufacture of an ever-increasing array of unnecessary single-use plastic products, need to be curbed.Global intervention against the plastic crisis is needed now because the costs of failure to act will be immense.

Correction: The Minderoo-Monaco Commission on Plastics and Human Health.

[This corrects the article DOI: 10.5334/aogh.4056.].

Design, Synthesis, and Characterization of 4-Aminoquinazolines as Potent Inhibitors of the G Protein-Coupled Receptor Kinase 6 (GRK6) for the Treatment of Multiple Myeloma.

Both previous and additional genetic knockdown studies reported herein implicate G protein-coupled receptor kinase 6 (GRK6) as a critical kinase required for the survival of multiple myeloma (MM) cells. Therefore, we sought to develop a small molecule GRK6 inhibitor as an MM therapeutic. From a focused library of known kinase inhibitors, we identified two hits with moderate biochemical potencies against GRK6. From these hits, we developed potent (IC50 < 10 nM) analogues with selectivity against off-target kinases. Further optimization led to the discovery of an analogue (18) with an IC50 value of 6 nM against GRK6 and selectivity against a panel of 85 kinases. Compound 18 has potent cellular target engagement and antiproliferative activity against MM cells and is synergistic with bortezomib. In summary, we demonstrate that targeting GRK6 with small molecule inhibitors represents a promising approach for MM and identify 18 as a novel, potent, and selective GRK6 inhibitor.

Pancratistatin induces apoptosis in clinical leukemia samples with minimal effect on non-cancerous peripheral blood mononuclear cells.

BACKGROUND: Pancratistatin, a natural compound extracted from Hymenocallis littoralis, can selectively induce apoptosis in several cancer cell lines. In this ex vivo study, we evaluated the effect of pancratistatin on peripheral blood mononuclear cells obtained from 15 leukemia patients prior to clinical intervention of newly diagnosed patients, as well as others of different ages in relapse and at various disease progression states. RESULTS: Mononuclear cells from healthy volunteers and leukemia patients were exposed to 1 microM pancratistatin for up to 48 h. Irrespective of leukemia type, pancratistatin induced apoptosis in the leukemic samples, with minimal effects on non-cancerous peripheral blood mononuclear control cells. CONCLUSION: Our results show that pancratistatin is an effective and selective anti-cancer agent with potential for advancement to clinical trials.

Chemoenzymatic synthesis of Amaryllidaceae constituents and biological evaluation of their C-1 analogues. The next generation synthesis of 7-deoxypancratistatin and trans-dihydrolycoricidine.

An efficient synthesis of C-1 derivatives of 7-deoxypancratistatin is reported. The key steps include the following: selective opening of an epoxide with aluminum acetylide in the presence of an aziridine; solid-state silica-gel-catalyzed opening of an aziridine; and oxidative cleavage of a phenanthrene core and its recyclization to phenanthridone to provide the key C-1 aldehyde 22. The conversion of this aldehyde to C-1 acetoxymethyl and C-1 hydroxymethyl derivatives is described along with the evaluation of their biological activity against several cancer cell lines and in an apoptosis study. The C-1 acetoxymethyl derivative has shown promising activity comparable to that of the natural product. In addition, a total synthesis of trans-dihydrolycoricidine and a formal total synthesis of 7-deoxypancratistatin are reported from aldehyde 22. Detailed experimental and spectral data are provided for all new compounds.

Selective cytotoxicity of pancratistatin-related natural Amaryllidaceae alkaloids: evaluation of the activity of two new compounds.

BACKGROUND: Pancratistatin (PST), a compound extracted from an Amaryllidaceae (AMD) family plant, has been shown to specifically induce apoptosis in cancer cells with no/minimal toxic effect on normal cells. A systematic synthetic approach has indicated that the minimum cytotoxic pharmacophore comprises the trans-fused b/c-ring system containing the 2, 3, 4-triol unit in the C-ring. To further explore the structure-activity relationship of this group of compounds we have investigated the anti-cancer efficacy and specificity of two PST-related natural compounds, AMD4 and AMD5. Both of these compounds lack the polyhydroxylated lycorane element of PST instead having a methoxy-substituted crinane skeleton. RESULTS: Our results indicate that AMD5 has efficacy and selectivity similar to PST, albeit at a 10-fold increased concentration. Interestingly AMD4 lacks apoptotic activity. CONCLUSION: Our results indicate that the phenanthridone skeleton in natural Amaryllidaceae alkaloids may be a significant common element for selectivity against cancer cells; furthermore, the configuration of the methoxy-side groups is responsible for higher binding affinity to the target protein/s thus making for a more efficient anti-cancer agent.

Selective apoptosis-inducing activity of crinum-type Amaryllidaceae alkaloids.

The selective apoptosis-inducing activity of Amaryllidaceae alkaloids belonging to the crinane-type is reported. A mini-library of natural and synthetic crinane alkaloids was assembled. Biological screening indicated crinamine 4 and haemanthamine 9 to be potent inducers of apoptosis in tumour cells at micromolar concentrations. Structure-activity relationships demonstrated the requirement for both an alpha-C2 bridge and a free hydroxyl at the C-11 position as pharmacophoric requirements for this activity.

Pancratistatin causes early activation of caspase-3 and the flipping of phosphatidyl serine followed by rapid apoptosis specifically in human lymphoma cells.

Recently a major research effort has been focused on the development of anticancer drugs by targeting the components of a biochemical pathway to induce apoptosis in cancerous cells. Some of the natural products (e.g. paclitaxel) have been proven to be useful in inducing apoptosis in cancer cells with limited specificity. Pancratistatin, a natural product isolated and characterized over a decade ago, has been shown to be cytostatic and antineoplastic. We investigated the specificity and biochemical mechanism of action of pancratistatin. Pancratistatin seemed to show more specificity than VP-16 or paclitaxel as an efficient inducer of apoptosis in human lymphoma (Jurkat) cells, with minimal effect on normal nucleated blood cells. Caspase-3 activation and exposure of phosphatidyl serine on the outer leaflet of the plasma membrane were earlier events than the generation of ROS and DNA fragmentation observed following pancratistatin treatment. This indicates a possible involvement of caspase-3 and plasma membrane proteins in the induction phase of apoptosis. Our results indicate that pancratistatin does not cause DNA double-strand breaks or DNA damage prior to the execution phase of apoptosis in cancer cells. Parallel experimentation with VP-16, a currently used medication for cancer treatment, indicated that VP-16 causes substantial DNA damage in normal non-cancerous blood cells, while pancratistatin does not cause any DNA double-strand breaks or DNA damage in non-cancerous cells. Taken together, our finding that pancratistatin induces apoptosis in cancer cells using non-genomic targets, and more importantly does not seem to have any affect non-cancerous cells, presents a significant platform to develop non-toxic anticancer therapies.

Potent Targeting of the STAT3 Protein in Brain Cancer Stem Cells: A Promising Route for Treating Glioblastoma.

The STAT3 gene is abnormally active in glioblastoma (GBM) and is a critically important mediator of tumor growth and therapeutic resistance in GBM. Thus, for poorly treated brain cancers such as gliomas, astrocytomas, and glioblastomas, which harbor constitutively activated STAT3, a STAT3-targeting therapeutic will be of significant importance. Herein, we report a most potent, small molecule, nonphosphorylated STAT3 inhibitor, 31 (SH-4-54) that strongly binds to STAT3 protein (K D = 300 nM). Inhibitor 31 potently kills glioblastoma brain cancer stem cells (BTSCs) and effectively suppresses STAT3 phosphorylation and its downstream transcriptional targets at low nM concentrations. Moreover, in vivo, 31 exhibited blood-brain barrier permeability, potently controlled glioma tumor growth, and inhibited pSTAT3 in vivo. This work, for the first time, demonstrates the power of STAT3 inhibitors for the treatment of BTSCs and validates the therapeutic efficacy of a STAT3 inhibitor for GBM clinical application.

An allosteric inhibitor of protein arginine methyltransferase 3.

PRMT3, a protein arginine methyltransferase, has been shown to influence ribosomal biosynthesis by catalyzing the dimethylation of the 40S ribosomal protein S2. Although PRMT3 has been reported to be a cytosolic protein, it has been shown to methylate histone H4 peptide (H4 1-24) in vitro. Here, we report the identification of a PRMT3 inhibitor (1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(2-cyclohexenylethyl)urea; compound 1) with IC50 value of 2.5 µM by screening a library of 16,000 compounds using H4 (1-24) peptide as a substrate. The crystal structure of PRMT3 in complex with compound 1 as well as kinetic analysis reveals an allosteric mechanism of inhibition. Mutating PRMT3 residues within the allosteric site or using compound 1 analogs that disrupt interactions with allosteric site residues both abrogated binding and inhibitory activity. These data demonstrate an allosteric mechanism for inhibition of protein arginine methyltransferases, an emerging class of therapeutic targets.

Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors.

Selective inhibition of protein methyltransferases is a promising new approach to drug discovery. An attractive strategy towards this goal is the development of compounds that selectively inhibit binding of the cofactor, S-adenosylmethionine, within specific protein methyltransferases. Here we report the three-dimensional structure of the protein methyltransferase DOT1L bound to EPZ004777, the first S-adenosylmethionine-competitive inhibitor of a protein methyltransferase with in vivo efficacy. This structure and those of four new analogues reveal remodelling of the catalytic site. EPZ004777 and a brominated analogue, SGC0946, inhibit DOT1L in vitro and selectively kill mixed lineage leukaemia cells, in which DOT1L is aberrantly localized via interaction with an oncogenic MLL fusion protein. These data provide important new insight into mechanisms of cell-active S-adenosylmethionine-competitive protein methyltransferase inhibitors, and establish a foundation for the further development of drug-like inhibitors of DOT1L for cancer therapy.

Pancratistatin induces apoptosis and autophagy in metastatic prostate cancer cells.

The Amaryllidaceae alkaloid pancratistatin has been proven to selectively induce apoptotic cell death in a variety of human cancer cells with an insignificant effect on non-cancerous cells. In this study we report, for the first time, the effects of pancratistatin (PST) on models of metastatic prostate cancer. The effects of pancratistatin on prostate cancer DU145 and LNCaP cell lines was assessed by microscopy, enzymatic activity assays and Western blotting. Apoptosis was determined by nuclear condensation and caspase activation, and autophagy was observed by MDC staining and LC3 expression levels. Human prostate xenografts were used to test the potential therapeutic efficacy of intra-tumor administration of pancratistatin in vivo. Pancratistatin treatment reduced cell viability and induced apoptosis in androgen-responsive (LNCaP) and androgen-refractory (DU145) prostate cancer cell lines in a dose- and time-dependent manner, but with an insignificant effect on normal human fibroblast (NHF) cells at the effective dose. Increased reactive oxygen species production and collapse of mitochondrial membrane potential resulted from treatment with pancratistatin in both cancer cell lines. This study presents the novel finding that pancratistatin treatment caused decreased migration capacity and increased autophagy levels in metastatic prostate cancer cells. Importantly, in this proof-of-concept study, pancratistatin reduced the volume of xenograft tumors compared to control-treated animals, and was well-tolerated. Our results highlight the potential of pancratistatin for clinical development as a selective therapeutic for treatment of metastatic prostate cancer.

Pancratistatin selectively targets cancer cell mitochondria and reduces growth of human colon tumor xenografts.

The naturally occurring Amaryllidaceae alkaloid pancratistatin exhibits potent apoptotic activity against a large panel of cancer cells lines and has an insignificant effect on noncancerous cell lines, although with an elusive cellular target. Many current chemotherapeutics induce apoptosis via genotoxic mechanisms and thus have low selectivity. The observed selectivity of pancratistatin for cancer cells promoted us to consider the hypothesis that this alkaloid targets cancer cell mitochondria rather than DNA or its replicative machinery. In this study, we report that pancratistatin decreased mitochondrial membrane potential and induced apoptotic nuclear morphology in p53-mutant (HT-29) and wild-type p53 (HCT116) colorectal carcinoma cell lines, but not in noncancerous colon fibroblast (CCD-18Co) cells. Interestingly, pancratistatin was found to be ineffective against mtDNA-depleted (ρ(0)) cancer cells. Moreover, pancratistatin induced cell death in a manner independent of Bax and caspase activation, and did not alter ß-tubulin polymerization rate nor cause double-stranded DNA breaks. For the first time we report the efficacy of pancratistatin in vivo against human colorectal adenocarcinoma xenografts. Intratumor administration of pancratistatin (3 mg/kg) caused significant reduction in the growth of subcutaneous HT-29 tumors in Nu/Nu mice (n = 6), with no apparent toxicity to the liver or kidneys as indicated by histopathologic analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Altogether, this work suggests that pancratistatin may be a novel mitochondria-targeting compound that selectively induces apoptosis in cancer cells and significantly reduces tumor growth.

Structure activity studies on the crinane alkaloid apoptosis-inducing pharmacophore.

The apoptosis-inducing ability of alpha-ethano bridged crinane alkaloids was investigated using natural and semi-synthetic derivatives uncovering novel structural requirements of this cytotoxic pharmacophore. An alpha-ethano bridge is required; an alpha- or beta-methoxy or hydroxyl H-bond acceptor are all tolerated at C-3; a small substituent (H, or OH) alone is tolerated at C-11; and a C-1 to C-2 double bond is shown to modulate, but is not a requirement for, apoptosis-inducing activity.

Structure-activity studies on the lycorine pharmacophore: A potent inducer of apoptosis in human leukemia cells.

The direct chemoselective differential functionalization of the ring-C hydroxyl groups present in the Amaryllidaceae alkaloid lycorine is described allowing for selective manipulation of the 1,2-hydroxyl groups. A mini-library comprised of synthetic and natural lycorane alkaloids was prepared and their apoptosis-inducing activity investigated in human leukemia (Jurkat) cells. Further insights into the nature of this interesting apoptosis-inducing pharmacophore are described, including the requirement of both free hydroxyl groups in ring-C.

synthesis and biological evaluation of fully functionalized seco-pancratistatin analogues.

The total synthesis of fully functionalized polyhydroxyamide B,C- seco-analogues of the anticancer compound pancratistatin (PST) ( 1) is reported. Key steps include an Evans' MgCl 2-promoted anti-aldol reaction between a functionalized l-threose derivative and ( R)-(+)-oxazolidinone to stereoselectively form the C-1/C-10b bond and a regiospecific radical-mediated oxidative fragmentation of a 1,3-benzylidene. The B,C- seco compounds 25 and 26 exhibited low activity (ED 50 > 30 microg/mL) for inducing apoptosis in human cancer cells.

Synergy of Pancratistatin and Tamoxifen on breast cancer cells in inducing apoptosis by targeting mitochondria.

Pancratistatin (PST), a natural compound obtained from the Hawaiian spider lily, is known to be specific and selective in inducing apoptosis in multiple cancer cell lines while sparing noncancerous cells and cell lines. Here we report the ability of PST to induce apoptosis specifically in human breast cancer cell lines MCF-7 and Hs-578-T compared to their non cancerous counterparts. In cancer cells PST caused increased levels of reactive oxygen species (ROS), decreased ATP and mitochondrial membrane permeabilization indicating the activation of the mitochondrial pathway of apoptosis. In combination with the anti-estrogen Tamoxifen, PST had a synergic effect. Both compounds caused increased production of ROS when applied to isolated mitochondria from these cancer cell lines supporting the observation that Tamoxifen might work through mechanisms distinct from the canonical estrogen receptor antagonism.

Discovery of the apoptosis-inducing activity and high accumulation of the butenolides, menisdaurilide and aquilegiolide in Dicentra spectabilis.

The occurrence of two butenolides, menisdaurilide and aquilegiolide, in commercial specimens of Dicentra spectabilis is reported for the first time; a rapid and direct isolation protocol is described. The ability of these lactones to induce apoptosis in human tumour cell lines at 10 microM concentration is also described. The high abundance and apoptosis-inducing activity reported here indicates that these constituents play a more significant role than the hormonal action previously attributed to them.