Triphosphate Group Research Articles

Oncolytic virus-mediated p53 overexpression promotes immunogenic cell death and efficacy of PD-1 blockade in pancreatic cancer

Immune checkpoint inhibitors, including anti-programmed cell death 1 (PD-1) antibody, provide improved clinical outcome in certain cancers. However, pancreatic ductal adenocarcinoma (PDAC) is refractory to PD-1 blockade therapy due to poor immune response. Oncolytic virotherapy is a novel approach for inducing immunogenic cell death (ICD). We demonstrated the therapeutic potential of p53-expressing telomerase-specific oncolytic adenovirus OBP-702 to induce ICD and anti-tumor immune responses in human PDAC cells with different p53 status (Capan-2, PK-59, PK-45H, Capan-1, MIA PaCa-2, BxPC-3) and murine PDAC cells (PAN02). OBP-702 significantly enhanced ICD with secretion of extracellular adenosine triphosphate and high-mobility group box protein B1 by inducing p53-mediated apoptosis and autophagy. OBP-702 significantly promoted the tumor infiltration of CD8+ T cells and the anti-tumor efficacy of PD-1 blockade in a subcutaneous PAN02 syngeneic tumor model. Our results suggest that oncolytic adenovirus-mediated p53 overexpression augments ICD and the efficacy of PD-1 blockade therapy against cold PDAC tumors. Further in vivo experiments would be warranted to evaluate the survival benefit of tumor-bearing mice in combination therapy with OBP-702 and PD-1 blockade.

Induction of immunogenic cell death in murine colon cancer cells by ferrocene-containing redox phospholipid polymers.

Immunogenic cell death (ICD), activated by damage‐associated molecular patterns (DAMPs), is an apoptotic cell death process that elicits antitumor immunity. Although anticancer drugs that can induce ICD are promising for cancer treatment, the design strategy for ICD inducers remains unclear. In this study, we demonstrated the cell‐penetrating redox phospholipid polymer poly(2‐methacryloyloxyethyl phosphorylcholine‐co‐vinyl ferrocene) (pMFc) inducing ICD in murine colon cancer CT26 cells. pMFc produced oxidative stress by extracting electrons from CT26 cells and induced the release of DAMPs, such as calreticulin, adenosine triphosphate, and high‐mobility group box 1. Moreover, the injection of pMFc‐treated CT26 cells inhibited tumor formation in subsequently challenged CT26 cells, indicating that pMFc elicited antitumor immunity through ICD. Using in vivo therapy, intratumoral injections of pMFc induced complete tumor regression in 20% (1/5) of mice. These results suggested that the redox phospholipid polymer provides a new option for ICD‐inducing anticancer polymers.

Huaier Induces Immunogenic Cell Death Via CircCLASP1/PKR/eIF2α Signaling Pathway in Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most lethal breast cancer subtype owing to the lack of targeted therapeutic strategies. Immunogenic cell death (ICD), a modality of regulated cancer cell death, offered a novel option for TNBC via augmenting tumor immunogenic microenvironment. However, few ICD-inducing agents are currently available. Here, we showed that Trametes robiniophila Murr (Huaier) triggered ICD in TNBC cells by promoting cell surface calreticulin (CRT) exposure, and increasing release of adenosine triphosphate (ATP) and high-mobility group protein B1 (HMGB1). Co-culturing with Huaier-treated TNBC cells efficiently enhanced the maturation of dendritic cells (DCs), which was further validated via cell-based vaccination assay. In the xenograft mouse model, oral administration of Huaier led to tumor-infiltrating lymphocytes (TILs) accumulation and significantly delayed tumor growth. Besides, depletion of endogenous T cells obviously abrogated the effect. Mechanically, Huaier could elicit endoplasmic reticulum (ER) stress-associated ICD through eIF2α signaling pathway. Further studies revealed that circCLASP1 was involved in the Huaier-induced immunogenicity by binding with PKR in the cytoplasm and thus blocking its degradation. Taken together, we highlighted an essential role of circCLASP1/PKR/eIF2α axis in Huaier-induced ICD. The findings of our study carried significant translational potential that Huaier might serve as a promising option to achieve long-term tumor remission in patients with TNBC.

Antibody–drug conjugates harboring a kinesin spindle protein inhibitor with immunostimulatory properties

ABSTRACT Antibody–drug conjugates (ADCs) are used to target cancer cells by means of antibodies directed to tumor-associated antigens, causing the incorporation of a cytotoxic payload into target cells. Here, we characterized the mode of action of ADC costing of a TWEAKR-specific monoclonal antibody conjugated to a small molecule kinesin spindle protein (KSP) inhibitor (KSPi). These TWEAKR-KSPi-ADCs showed strong efficacy in a TWEAKR expressing CT26 colon cancer model in mice. TWEAKR-KSPi-ADCs controlled the growth of CT26 colon cancers in immunodeficient as well as in immunocompetent mice. However, when treated with suboptimal doses, TWEAKR-KSPi-ADCs were still active in immunocompetent but not in immunodeficient mice, indicating that TWEAKR-KSPi-ADCs act – in addition to the cytotoxic mode of action – through an immunological mechanism. Indeed, in vitro experiments performed with a cell-permeable small molecule KSPi closely related to the active payload released from the TWEAKR-KSPi-ADCs revealed that KSPi was capable of stimulating several hallmarks of immunogenic cell death (ICD) on three different human cancer cell lines: cellular release of adenosine triphosphate (ATP) and high mobility group B1 protein (HMGB1), exposure of calreticulin on the cell surface as well as a transcriptional type-I interferon response. Further, in vivo experiments confirmed that treatment with TWEAKR-KSPi-ADCs activated immune responses via enhancing the infiltration of CD4+ and CD8+ T lymphocytes in tumors and the local production of interferon-γ, interleukin-2, and tumor necrosis factor-α. In conclusion, the antineoplastic effects of TWEAKR-KSPi-ADCs can partly be attributed to its ICD-stimulatory properties.

The role of the phosphate groups of trinitrophenyl adenosine 5′-triphosphate (TNP-ATP) in allosteric activation of pyruvate carboxylase and the inhibition of acetyl CoA-dependent activation

A previous study showed that 2′-3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) was a weak allosteric activator of Rhizobium etli pyruvate carboxylase (RePC) in the absence of acetyl-CoA. On the other hand, TNP-ATP inhibited the allosteric activation of RePC by acetyl-CoA. Here, we aimed to study the role of triphosphate group of TNP-ATP on its allosteric activation of the enzyme and inhibition of acetyl-CoA-dependent activation of RePC using TNP-ATP and its derivatives, including TNP-ADP, TNP-AMP and TNP-adenosine. The pyruvate carboxylation activity was assayed to determine the effect of reducing the number of phosphate groups in TNP-ATP derivatives on allosteric activation and inhibition of acetyl-CoA activation of RePC and chicken liver pyruvate carboxylase (CLPC). Reducing the number of phosphate groups in TNP-ATP derivatives decreased the activation efficacy for both RePC and CLPC compared to TNP-ATP. The apparent binding affinity and inhibition of activation of the enzymes by acetyl-CoA were also diminished when the number of phosphate groups in the TNP-ATP derivatives was reduced. Whilst TNP-AMP activated RePC, it did not activate CLPC, but it did inhibit acetyl-CoA activation of both RePC and CLPC. Similarly, TNP-adenosine did not activate RePC; however, it did inhibit acetyl-CoA activation using a different mechanism compared to phosphorylated TNP-derivatives. These findings indicate that mechanisms of PC activation and inhibition of acetyl-CoA activation by TNP-ATP and its derivatives are different. This study provides the basis for possible drug development for treatment of metabolic diseases and cancers with aberrant expression of PC.

Structural insights into dpCoA-RNA decapping by NudC

ABSTRACT Various kinds of cap structures, such as m7G, triphosphate groups, NAD and dpCoA, protect the 5′ terminus of RNA. The cap structures bond covalently to RNA and affect its stability, translation, and transport. The removal of the caps is mainly executed by Nudix hydrolase family proteins, including Dcp2, RppH and NudC. Numerous efforts have been made to elucidate the mechanism underlying the removal of m7G, triphosphate group, and NAD caps. In contrast, few studies related to the cleavage of the RNA dpCoA cap have been conducted. Here, we report the hydrolytic activity of Escherichia coli NudC towards dpCoA and dpCoA-capped RNA in vitro. We also determined the crystal structure of dimeric NudC in complex with dpCoA at 2.0 Å resolution. Structural analysis revealed that dpCoA is recognized and hydrolyzed in a manner similar to NAD. In addition, NudC may also remove other dinucleotide derivative caps of RNA, which comprise the AMP moieties. NudC homologs in Saccharomyces cerevisiae and Arabidopsis thaliana exhibited similar dpCoA decapping (deCoAping) activity. These results together indicate a conserved mechanism underpinning the hydrolysis of dpCoA-capped RNA in both prokaryotes and eukaryotes.

Pulsatilla Decoction Combined with 5-Fluorouracil Triggers Immunogenic Cell Death in Colorectal Cancer Cells.

Background: Our research is designed to explore the role of 5-FU and Pulsatilla decoction (PD) through modulation of Immunogenic cell death (ICD) for the co-treatment of Colorectal cancer (CRC). Materials and Methods: Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assays. Cell apoptosis was assessed using flow cytometry. Phosphorylation of STAT3 and expression of Mcl-1 and Bcl-xl were measured by Western blot assays. The levels of ATP and HMGB1 in the supernatants of the culture medium were analyzed by ATP assays and the HMGB1 enzyme linked immunosorbent assay kit. The cell surface levels of CRT were measured by immunofluorescence assays. The tumor growth was analyzed in mice. Results: PD increased 5-FU-induced ICD in CRC cells, as demonstrated by the extracellular levels of adenosine triphosphate (ATP) and high-mobility group box 1 (HMGB1), and the surface levels of calreticulin (CRT). Our mechanism study showed that PD promoted 5-FU-induced ICD by inactivating signal transducer and activator of transcription 3 (STAT3). Furthermore, the co-treatment of 5-FU and PD further promoted 5-FU-induced CRT expression and T cell infiltration in vivo. Tumorigenicity analysis revealed that 5-FU combined with PD notably reduced tumor growth. Conclusion: This study indicated that PD enhances 5-FU-induced ICD and anti-tumor effect in CRC by inactivating STAT3. The combined application of 5-FU with PD may improve the anti-tumor activity of 5-FU in CRC.

A Threose Nucleic Acid Enzyme with RNA Ligase Activity.

Threose nucleic acid (TNA) has been considered a potential RNA progenitor in evolution due to its chemical simplicity and base pairing property. Catalytic TNA sequences with RNA ligase activities might have facilitated the transition to the RNA world. Here we report the isolation of RNA ligase TNA enzymes by in vitro selection. The identified TNA enzyme T8-6 catalyzes the formation of a 2'-5' phosphoester bond between a 2',3'-diol and a 5'-triphosphate group, with a kobs of 1.1 × 10-2 min-1 (40 mM Mg2+, pH 9.0). For efficient reaction, T8-6 requires UA|GA at the ligation junction and tolerates variations at other substrate positions. Functional RNAs such as hammerhead ribozyme can be prepared by T8-6-catalyzed ligation, with site-specific introduction of a 2'-5' linkage. Together, this work provides experimental support for TNA as a plausible pre-RNA genetic polymer and also offers an alternative molecular tool for biotechnology.

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

Disulfiram Copper Complex Enhancing the Radiation Sensitivity and Immunogenicity of Esophageal Cancer Cells

Radiation therapy (RT) is essential for the effective management of esophageal cancer. However, radioresistance of cancer cells limit its efficiency and drives recurrence, thereby leading to poor outcome for patients. Disulfiram is an old alcohol-aversion drug that has been shown to be effective against diverse cancer types in preclinical studies. Disulfiram chelates bivalent metals and forms complexes with copper, which enhances its anti-tumor activity. Here, we provide evidence that the disulfiram copper complex enhances radiation sensitivity and the immunogenicity of esophageal squamous cell carcinoma (ESCC) cells. ESCC cells were treated with either disulfiram/copper gluconate, RT, or a combination of the two in order to test the effects on cell viability. Colony formation assay was performed to evaluate the radiation sensitivity of disulfiram copper complex. The expression of immunogenic substances was analyzed by flow cytometry methods and Immunofluorescence techniques. Then apoptosis analysis was also performed. We conclude by assessing the potential molecular mechanisms involved in enhanced antitumor activity of the disulfiram copper complex. Disulfiram copper complex exhibits antitumor activity in ESCC cells. This complex was applied at low concentrations, about one fifth of the IC50 dosage in each cancer cells. The outcomes revealing significant improvement of radiation sensitivity by disulfiram copper complex. Immunogenic cell death (ICD), occurring through the translocation of calreticulin from the cytoplasm to the tumor surface in addition to extracellular release of adenosine triphosphate (ATP) and high mobility group protein 1 (HMGB1), was found to occur upon the combined treatment. In addition, the combined strategies induced tumor cell apoptosis, necrosis, and autophagy. Further analyses revealed that the mechanism responsible for radioresistance was due to the ability of tumor cells to overcome moderate endoplasmic reticulum stress induced by RT alone. However, RT combined with disulfiram copper complex successfully activated endoplasmic reticulum stress signaling pathway, leading to a robust stress-response thereby inducing cancer cell death. In summary, our results indicate that RT acts as an endoplasmic reticulum stress inducer and ICD inducer. The combination of RT with disulfiram copper complex induces a severe stress-response, promotes ICD and enhances the efficacy of RT. Our study demonstrates the potential of drug repurposing, providing novel mechanistic insights, identification of new cancer-relevant targets and encouragement for further analysis.

Sensing Adenosine and ATP by Aptamers and Gold Nanoparticles: Opposite Trends of Color Change from Domination of Target Adsorption Instead of Aptamer Binding.

The 27 mer DNA aptamer for adenosine and adenosine 5'-triphosphate (ATP) is a popular model system for designing biosensors. Various strategies have been reported for label-free colorimetric detection using gold nanoparticles (AuNPs). It is generally accepted that free aptamers can protect AuNPs against salt-induced aggregation, whereas target-bound aptamers cannot. However, these studies only considered the aptamer binding to its target, and the adsorption of the aptamer on AuNPs, but none considered the adsorption of target molecules by AuNPs. We herein report that the adsorption of adenosine destabilized citrate-capped AuNPs with an apparent Kd of just 7.7 μM adenosine, whereas that of ATP stabilized the AuNPs because of the negative charges from the triphosphate group. The adsorbed ATP inhibited the adsorption of DNA. Using the aptamer and a nonbinding mutant, ATP and guanosine 5'-triphosphate (GTP) had the same colorimetric response, and so did adenosine and guanosine, regardless of the DNA sequence, indicating that the color change mainly reflected the adsorption of the nucleosides and nucleotides instead of aptamer binding. The related literature examples using this aptamer were classified into three types and individually analyzed, where the reported color changes can all be explained by the adsorption of target analytes.

Structural Basis of 7SK RNA 5′-Gamma-Phosphate Methylation and Retention by MePCE

7SK non-coding RNA is an essential regulator of eukaryotic transcription elongation through dynamic RNP assemblies that sequester or release the positive transcription elongation factor b (P-TEFb). All 7SK RNPs include a core comprising 7SK RNA, methylphosphate capping enzyme (MePCE), and La-related protein 7 (Larp7). In addition to its non-enzymatic role as a core component of 7SK RNPs, MePCE catalyzes the formation of a gamma-phosphate monomethylation cap, unique to 7SK and U6 in humans. How MePCE can specifically recognize and modify its RNA substrate and remain bound to 7SK to form the core 7SK RNP has not been previously known. We report 2.0 and 2.1 Å X-ray crystal structures of human MePCE methyltransferase domain bound to S-adenosylhomocysteine (SAH) and uncapped or capped 7SK substrates, respectively. We find that 7SK recognition is achieved by an extensive network of protein contacts to a 5' hairpin-single-stranded RNA region, involving the triphosphate group, two terminal basepairs and six single-stranded nucleotides, thus explaining MePCE specificity for 7SK and U6. The large size of the RNA binding site required for recognition and catalysis is unique among RNA methyltransferases studied to date. The structures reveal SAH and product RNA in a near-transition state geometry, where the cofactor SAH is buried inside the active site by the RNA-interacting residues. Unexpectedly, binding experiments show that MePCE has higher affinity for capped vs uncapped 7SK, with kinetic data supporting a slow product release model. Taken together, these data describe the features of an unusual RNA capping enzyme, and reveal the molecular mechanism of methyl transfer and 7SK retention by MePCE for subsequent assembly of the core 7SK RNP.

ATP-Charged Nanoclusters Enable Intracellular Protein Delivery and Activity Modulation for Cancer Theranostics.

SummaryProtein drugs own a large share in the market and hold great prospects for the treatment of many diseases. However, the available protein drugs are limited to the extracellular target, owing to the inefficient transduction and activity modulation of proteins targeting intracellular environment. In this study, we constructed ATP-charged platforms to overcome the above-mentioned barriers for cancer theranostics. The phenylboronic acid-modified polycations (PCD) were synthesized to assemble with enzymes and shield its activity in the blood circulation. When the PCD nanoclusters reached tumor site, they effectively transported the enzymes into the cells, followed by recovering its catalytic activity after being charged with ATP. Importantly, the cascaded enzyme systems (GOx&HRPA) selectively induced starvation therapy as well as photoacoustic imaging of tumor. Our results revealed that the intelligent nanoclusters were broadly applicable for protein transduction and enzyme activity modulation, which could accelerate the clinical translation of protein drugs toward intracellular target.

Immune Modulation by Telomerase-Specific Oncolytic Adenovirus Synergistically Enhances Antitumor Efficacy with Anti-PD1 Antibody

The clinical benefit of monotherapy involving immune checkpoint inhibitors (ICIs) such as anti-programmed death-1 antibody (PD-1 Ab) is limited to small populations. We previously developed a telomerase-specific oncolytic adenovirus, Telomelysin (OBP-301), the safety of which was confirmed in a phase I clinical study. Here, we examined the potential of OBP-502, an OBP-301 variant, as an agent for inducing immunogenic cell death (ICD) and synergistically enhancing the efficacy of OBP-502 with PD-1 Ab using CT26 murine colon cancer and PAN02 murine pancreatic cancer cell lines. OBP-502 induced the release of ICD molecules such as adenosine triphosphate (ATP) and high-mobility group box protein 1 (HMGB1) from CT26 and PAN02 cells, leading to recruitment of CD8-positive lymphocytes and inhibition of Foxp3-positive lymphocyte infiltration into tumors. Combination therapy involving OBP-502 intratumoral administration and PD-1 Ab systemic administration significantly suppressed the growth of not only OBP-502-treated tumors but also tumors not treated with OBP-502 (so-called abscopal effect) in CT26 and PAN02 bilateral subcutaneous tumor models, in which active recruitment of CD8-positve lymphocytes was observed even in tumors not treated with OBP-502. This combined efficacy was similar to that observed in a CT26 rectal orthotopic tumor model involving liver metastases. In conclusion, telomerase-specific oncolytic adenoviruses are promising candidates for combined therapies with ICIs.

Immunogenic Cell Death: An opportunity for Clinical Oncology?

Apoptosis was initially seen as a kind of silent cell death with non-induction of the immune response 1. However, in the recent past, it has been seen that death induced by either infections or actions of certain agents can elicit a specific immune response, namely immunogenic cell death (ICD)2. This ICD activates the immune system against antigens associated with deceased cells with the concomitant exposure and releasing of the so-called damage-associated molecular patterns (DAMPs) by dying cells3. Four principal DAMPs related to ICD have been identified (but not limited to): the endoplasmic reticulum (ER) chaperone calreticulin (CRT), heat shock proteins (HSPs), adenosine triphosphate (ATP) and high mobility group box-1 (HMGB-1)4.

Why Cellular Di/Triphosphates Preferably Bind Mg2+ and Not Ca2.

Di/triphosphates perform a multitude of essential tasks, being important components of many vital organic cofactors such as adenosine/guanosine di/triphosphate (ADP/GDP, ATP/GTP), flavin adenine dinucleotide, and nicotinamide adenine dinucleotide and its phosphate derivative. They are generally bound to cations inside cells, in particular Mg2+ in the case of ATP/GTP. Yet how their metal-binding modes depend on the number, charge, and solvent exposure of the polyphosphate group and how Mg2+and Ca2+ dications that coexist in cellular fluids compete for di/triphosphates in biological systems remain elusive. Using density functional theory calculations combined with a polarizable continuum model, we have determined the relative free energies and stabilities of the different binding modes of di- and triphosphate groups to Mg2+ and Ca2+. We show that the thermodynamic outcome of the competition between Mg2+ and Ca2+ for cellular di/triphosphates depends mainly on the oligomericity/charge and metal-binding mode of the phosphate ligand as well as the solvent exposure of the binding site. Increasing the charge and thus denticity of the phosphate ligand from bi- to tridentate in a buried binding pocket enhances the affinity of the host system for the stronger charge acceptor, Mg2+. The cellular di/triphosphates's intrinsic properties and the protein matrix allowing them to bind a dication bi/tridentately, along with the higher cytosolic concentration of Mg2+ compared to Ca2+, enables Mg2+ to outcompete Ca2+ in binding to these highly charged anions. This suggests an explanation for why nature has chosen Mg2+ but not Ca2+ to perform most of the essential tasks associated with biological triphosphates.

Targeting chronic lymphocytic leukemia with N-methylated thrombospondin-1–derived peptides overcomes drug resistance

AbstractChronic lymphocytic leukemia (CLL), the most common adulthood leukemia in Western countries, is a very heterogeneous disease characterized by a peripheral accumulation of abnormal CD5+ B lymphocytes in the immune system. Despite new therapeutic developments, there remains an unmet medical need for CLL. Here, we demonstrate that the use of N-methylated thrombospondin-1 (TSP-1)–derived peptides is an efficient way to kill the malignant CLL cells, including those from high-risk individuals with poor clinical prognosis, del11q, del17p, 2p gain, or complex karyotype. PKT16, our hit N-methylated peptide, triggers the elimination of the leukemic cells, sparing the nontumor cells, including the hematopoietic precursors, and reduces the in vivo tumor burden of a CLL-xenograft mice model. A complementary analysis underscores the improved cytotoxic efficiency of PKT16 compared with the previously described TSP-1–derived probes, such as PKHB1. PKT16 elicits an original caspase-independent programmed necrotic mode of cell death, different from necroptosis or ferroptosis, implicating an intracellular Ca2+ deregulation that provokes mitochondrial damage, cell cycle arrest, and the specific death of the malignant CLL cells. The activation of the Gαi proteins and the subsequent drop of cyclic adenosine monophosphate levels and protein kinase A activity regulate this cytotoxic cascade. Remarkably, PKT16 induces the molecular hallmarks of immunogenic cell death, as defined by the calreticulin plasma membrane exposure and the release of adenosine triphosphate and high-mobility group box 1 protein from the dying CLL cells. Thus, PKT16 appears to be able to stimulate an anticancer in vivo immune response. Collectively, our results pave the way toward the development of an efficient strategy against CLL.

Molecular dynamics investigation of a redox switch in the anti-HIV protein SAMHD1.

HIV-1 is restricted in macrophages and certain quiescent myeloid cells due to a "Scorched Earth" dNTP starvation strategy attributed to the sterile alpha motif and HD domain protein-SAMHD1. Active SAMHD1 tetramers are assembled by GTP-Mg+2-dNTP cross bridges and cleave the triphosphate groups of dNTPs at a K m of ~10 μM, which is consistent with dNTP concentrations in cycling cells, but far higher than the equivalent concentration in quiescent cells. Given the substantial disparity between the dNTP concentrations required to activate SAMHD1 tetramers (~10 μM) and the dNTP concentrations in noncycling cells (~10 nM), the possibility of alternate enzymatically active forms of SAMHD1, including monomers remains open. In particular, the possibility of redox regulation of such monomers is also an open question. There have been experimental studies on the regulation of SAMHD1 by Glutathione driven redox reactions recently. Therefore, in this work, we have performed all-atom molecular dynamics simulations to study the dynamics of monomeric SAMHD1 constructs in the context of the three redox-susceptible Cysteine residues and compared them to monomers assembled within a tetramer. Our results indicate that assembly into a tetramer causes ordering of the catalytic core and increased solvent accessibility of the Catalytic Site. We have also found that glutathionylation of surface exposed C522 causes long range allosteric disruptions extending into the protein core. Finally, we see evidence suggesting a transient interaction between C522 and C341. Such a disulfide linkage has been hypothesized by experimental models, but has never been observed in crystal structures before.

Liquid Crystal ordering of DNA Dickerson Dodecamer duplexes with different 5’- Phosphate terminations

The onset of liquid crystal (LC) phases in concentrated aqueous solutions of DNA oligomers crucially depends on the end-to-end interaction between the DNA duplexes, which can be provided by the aromatic stacking of the terminal base-pairs or by the pairing of complementary dangling-ends. Here we investigated the LC behavior of three blunt-end 12-base-long DNA duplexes synthesized with hydroxyl, phosphate and triphosphate 5’-termini. We experimentally characterized the concentration-temperature phase diagrams and we quantitatively estimated the end-to-end stacking free energy, by comparing the empirical data with the predictions of coarse-grained linear aggregation models.The preservation of LC ordering, even in presence of the bulky and highly charged triphosphate group, indicates that attractive stacking interactions are still present and capable of induce linear aggregation of the DNA duplexes. This finding strengthens the potential role of chromonic like self-assembly for the prebiotic formation of linear polymeric nucleic acids.

DAMPs‐coated Prussian blue nanoparticles as photothermal‐nanoimmunotherapy agents for cancer

Nanoparticle-based photothermal therapy (PTT) has been widely investigated in cancer therapy as a rapid and minimally invasive tumor ablation technique. In this treatment modality, the intrinsic photothermal conversion property of synthesized nanoparticles allows the conversion of incident light into heat, which generates cell temperature increases, thereby killing cancer cells in contact with the nanoparticles. Here, Prussian blue nanoparticles (PBNPs) are used as photothermal agents, as they exhibit strong absorbance at near-infrared (NIR) wavelengths, are stable, non-toxic, and have 20.5% photothermal conversion efficiencies. In studies using PBNPs to administer photothermal therapy (PBNP-PTT) to localized tumors in a neuroblastoma model, PBNP-PTT generates local heating, resulting in decreased tumor growth and significantly improved survival. Importantly, we have shown that under certain conditions, PBNP-PTT generates immunogenic cell death (ICD), a favorable cell death phenotype characterized by concurrent cytotoxicity and immune cell engagement, enabling a potentially robust and long-lasting immune response and allowing for immunological memory against recurrent or metastatic disease. ICD is defined by the release or exposure of danger-associated molecular patterns (DAMPs), such as calreticulin, adenosine triphosphate (ATP), and high mobility group box 1 (HMGB1) from dying cancer cells. DAMPs are critical for the maturation, antigen uptake, and presentation of dendritic cells, and serve as powerful immunological adjuvants to activate a cytotoxic T lymphocyte response; these markers must be present for a cell to be classified as undergoing ICD. Previous studies in our lab show that the PBNP-PTT-stimulated release of DAMPs from dying cancer cells is both disease- and stimulus-specific; that is, depending on the cell type and conditions of PBNP-PTT, DAMPs may or may not be released sufficiently to generate downstream responses. To ensure that PBNP-PTT induces the release and expression of the correct DAMPs to induce ICD, we synthesized DAMPs-coated PBNPs to use in PBNP-PTT. Our hypothesis is that PBNP-PTT allows to heat cancer cells, causing exposure of some DAMPs, while simultaneously releasing other DAMPs from the PBNPs, augmenting the immunogenic effect. Here, we show the synthesis strategy for coating PBNPs with DAMPs, the number of DAMPs bound to PBNPS using the Bio-Red Protein Assay, and the stability of the DAMPs-PBNPs over several days. We present consistent data showing the cytotoxic capability of DAMPs-PBNP-based PTT using CellTiter-Glo® Luminescent Cell Viability Assay. Additionally, we compare the effects of applying PBNP-based PTT on the surface versus interstitially, to further optimize our photothermal nanoimmunotherapy. Support or Funding Information UPR-Ponce RISE (NIH-NIGMS R25GM096955) Hypothesis The stability of PBNPs and PBNPs-DAMPs measured over 7 days using a Dynamic Light Scattering. Cytotoxicity capabilities of PBNPs and DAMPs-PBNPs.The viability of treated neuroblastoma cells with PBNPs and DAMPs-PBNPs at different concentrations using CellTiter-Glo® Luminescent Cell Viability Assay. Cytotoxicity capabilities of PBNP-PTT and DAMPs-PBNP-PTTThe viability of tumor cells of neuroblastoma under PBNPs-PTT and DAMPs-PBNPs-PTT, after 24h of incubation, treated at different concentrations of the nanoparticles (0–0.1 mg/mL) using a 0.75W (A) and 1.00W laser power (B). Reached temperatures at 0.75W (C) and 1.00W (D) laser power for the treated tumor cells under PTT for 10 min. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.