Adenosine Triphosphate Hydrolysis Research Articles

Genome-Wide Identification and Analysis of Plasma Membrane H+-ATPases Associated with Waterlogging in Prunus persica (L.) Batsch

Plant plasma membrane H+-ATPase is a transport protein that is generally located on the plasma membrane and generates energy by hydrolyzing adenosine triphosphate (ATP) to pump hydrogen ions (H+) in the cytoplasm out of the cell against a concentration gradient. The plasma membrane H+-ATPases in plants are encoded by a multigene family and potentially play a fundamental role in regulating plant responses to various abiotic stresses, thus contributing to plant adaptation under adverse conditions. To understand the characteristics of the plasma membrane H+-ATPase family in peach (Prunus persica), this study analyzed the plasma membrane H+-ATPase family genes in peach. The results showed that there were 27 members of the plasma membrane H+-ATPase family in peach with amino acid sequences ranging from 943 to 1327. Subcellular localization showed that 23 of the 27 members were located on the cell membrane, and the phylogenetic tree analysis indicated that peach plasma membrane H+-ATPase members were divided into five groups. There were four genes with tandem repeat relationships, and six plasma membrane H+-ATPase genes were differentially expressed after 5 days of flooding and under non-flooding conditions based on the RNA-seq and RT-qPCR analyses. This study also investigated the characteristics and possible functions of the plasma membrane H+-ATPase family members in peach. The results provide theoretical support for further studies on their biological functions in peach.

F-actin architecture determines the conversion of chemical energy into mechanical work

Mechanical work serves as the foundation for dynamic cellular processes, ranging from cell division to migration. A fundamental driver of cellular mechanical work is the actin cytoskeleton, composed of filamentous actin (F-actin) and myosin motors, where force generation relies on adenosine triphosphate (ATP) hydrolysis. F-actin architectures, whether bundled by crosslinkers or branched via nucleators, have emerged as pivotal regulators of myosin II force generation. However, it remains unclear how distinct F-actin architectures impact the conversion of chemical energy to mechanical work. Here, we employ in vitro reconstitution of distinct F-actin architectures with purified components to investigate their influence on myosin ATP hydrolysis (consumption). We find that F-actin bundles composed of mixed polarity F-actin hinder network contraction compared to non-crosslinked network and dramatically decelerate ATP consumption rates. Conversely, linear-nucleated networks allow network contraction despite reducing ATP consumption rates. Surprisingly, branched-nucleated networks facilitate high ATP consumption without significant network contraction, suggesting that the branched network dissipates energy without performing work. This study establishes a link between F-actin architecture and myosin energy consumption, elucidating the energetic principles underlying F-actin structure formation and the performance of mechanical work.

Generation and characterization of antagonistic anti-human CD39 nanobodies.

CD39 is the major enzyme controlling the levels of extracellular adenosine triphosphate (ATP) via the stepwise hydrolysis of ATP to adenosine diphosphate (ADP) and adenosine monophosphate (AMP). As extracellular ATP is a strong promoter of inflammation, monoclonal antibodies (mAbs) blocking CD39 are utilized therapeutically in the field of immune-oncology. Though anti-CD39 mAbs are highly specific for their target, they lack deep penetration into the dense tissue of solid tumors, due to their large size. To overcome this limitation, we generated and characterized nanobodies that targeted and blocked human CD39. From cDNA-immunized alpacas we selected 16 clones from seven nanobody families that bind to two distinct epitopes of human CD39. Among these, clone SB24 inhibited the enzymatic activity of CD39. Of note, SB24 blocked ATP degradation by both soluble and cell surface CD39 as a 15kD monomeric nanobody. Dimerization via fusion to an immunoglobulin Fc portion further increased the blocking potency of SB24 on CD39-transfected HEK cells. Finally, we confirmed the CD39 blocking properties of SB24 on human PBMCs. In summary, SB24 provides a new small biological antagonist of human CD39 with potential application in cancer therapy.

Co-delivery of Paclitaxel/Atovaquone/Quercetin to regulate energy metabolism to reverse multidrug resistance in ovarian cancer by PLGA-PEG nanoparticles

Ovarian cancer is a malignant tumor that seriously endangers the lives of women, with chemotherapy being the primary clinical treatment. However, chemotherapy encounters the problem of generating multidrug resistance (MDR), mainly due to drug efflux induced by P-glycoprotein (P-gp), which decreases intracellular accumulation of chemotherapeutic drugs. The drugs efflux mediated by P-gp requires adenosine triphosphate (ATP) hydrolysis to provide energy. Therefore, modulating energy metabolism pathways and inhibiting ATP production may be a potential strategy to reverse MDR. Herein, we developed a PTX-ATO-QUE nanoparticle (PAQNPs) based on a PLGA-PEG nanoplatform capable of loading the mitochondrial oxidative phosphorylation (OXPHOS) inhibitor atovaquone (ATO), the glycolysis inhibitor quercetin (QUE), and the chemotherapeutic drug paclitaxel (PTX) to reverse MDR by inhibiting energy metabolism through multiple pathways. Mechanistically, PAQNPs could effectively inhibit the OXPHOS and glycolytic pathways of A2780/Taxol cells by suppressing the activities of mitochondrial complex III and hexokinase II (HK II), respectively, ultimately decreasing intracellular ATP levels in tumor cells. Energy depletion can effectively inhibit cell proliferation and reduce P-gp activity, increasing the chemotherapeutic drug PTX accumulation in the cells. Moreover, intracellular reactive oxygen species (ROS) is increased with PTX accumulation and leads to chemotherapy-resistant cell apoptosis. Furthermore, PAQNPs significantly inhibited tumor growth in the A2780/Taxol tumor-bearing NCG mice model. Immunohistochemical (IHC) analysis of tumor tissues revealed that P-gp expression was suppressed, demonstrating that PAQNPs are effective in reversing MDR in tumors by inducing energy depletion. In addition, the safety study results, including blood biochemical indices, major organ weights, and H&E staining images, showed that PAQNPs have a favorable in vivo safety profile. In summary, the results suggest that the combined inhibition of the two energy pathways, OXPHOS and glycolysis, can enhance chemotherapy efficacy and reverse MDR in ovarian cancer.

Structural basis for CFTR inhibition by CFTRinh-172

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates electrolyte and fluid balance in epithelial tissues. While activation of CFTR is vital to treating cystic fibrosis, selective inhibition of CFTR is a potential therapeutic strategy for secretory diarrhea and autosomal dominant polycystic kidney disease. Although several CFTR inhibitors have been developed by high-throughput screening, their modes of action remain elusive. In this study, we determined the structure of CFTR in complex with the inhibitor CFTRinh-172 to an overall resolution of 2.7 Å by cryogenic electron microscopy. We observe that CFTRinh-172 binds inside the pore near transmembrane helix 8, a critical structural element that links adenosine triphosphate hydrolysis with channel gating. Binding of CFTRinh-172 stabilizes a conformation in which the chloride selectivity filter is collapsed, and the pore is blocked from the extracellular side of the membrane. Single-molecule fluorescence resonance energy transfer experiments indicate that CFTRinh-172 inhibits channel gating without compromising nucleotide-binding domain dimerization. Together, these data reconcile previous biophysical observations and provide a molecular basis for the activity of this widely used CFTR inhibitor.

Unravelling HetC as a peptidase-based ABC exporter driving functional cell differentiation in the cyanobacterium Nostoc PCC 7120.

The export of peptides or proteins is essential for a variety of important functions in bacteria. Among the diverse protein-translocation systems, peptidase-containing ABC transporters (PCAT) are involved in the maturation and export of quorum-sensing or antimicrobial peptides in Gram-positive bacteria and of toxins in Gram-negative organisms. In the multicellular and diazotrophic cyanobacterium Nostoc PCC 7120, the protein HetC is essential for the differentiation of functional heterocysts, which are micro-oxic and non-dividing cells specialized in atmospheric nitrogen fixation. HetC shows similarities to PCAT systems, but whether it actually acts as a peptidase-based exporter remains to be established. In this study, we show that the N-terminal part of HetC, encompassing the peptidase domain, displays a cysteine-type protease activity. The conserved catalytic residues conserved in this family of proteases are essential for the proteolytic activity of HetC and the differentiation of heterocysts. Furthermore, we show that the catalytic residue of the ATPase domain of HetC is also essential for cell differentiation. Interestingly, HetC has a cyclic nucleotide-binding domain at its N-terminus which can bind ppGpp in vitro and which is required for its function in vivo. Our results indicate that HetC is a peculiar PCAT that might be regulated by ppGpp to potentially facilitate the export of a signaling peptide essential for cell differentiation, thereby broadening the scope of PCAT role in Gram-negative bacteria.IMPORTANCEBacteria have a great capacity to adapt to various environmental and physiological conditions; it is widely accepted that their ability to produce extracellular molecules contributes greatly to their fitness. Exported molecules are used for a variety of purposes ranging from communication to adjust cellular physiology, to the production of toxins that bacteria secrete to fight for their ecological niche. They use export machineries for this purpose, the most common of which energize transport by hydrolysis of adenosine triphosphate. Here, we demonstrate that such a mechanism is involved in cell differentiation in the filamentous cyanobacterium Nostoc PCC 7120. The HetC protein belongs to the ATP-binding cassette transporter superfamily and presumably ensures the maturation of a yet unknown substrate during export. These results open interesting perspectives on cellular signaling pathways involving the export of regulatory peptides, which will broaden our knowledge of how these bacteria use two cell types to conciliate photosynthesis and nitrogen fixation.

All‐Optical Thermometry Monitoring Biochemical Kinetics with NV Centers in Diamond

AbstractNanothermometers enable label‐free measurement of biochemical processes with extremely small sample consumption, which is essential to high‐throughput assays for enzyme searching and drug screening. However, high sensitivity and long‐term chemical stability are still crucial challenges for current nanothermometers. Here, the nitrogen‐vacancy centers in diamond are chosen as atomic thermometers and apply the diamond thermometry to kinetic monitoring of biochemical reactions. The performance of all‐optical diamond thermometry are tested under severe chemical conditions with strong acids and bases. Diamond thermometry demonstrates a sensitivity of 80 mK and chemical stability of 62 mK even in the presence of a drastic pH change during neutralization reactions. The thermodynamics of two enzyme‐catalyzed processes: adenosine triphosphate hydrolysis and urea hydrolysis, are further analyzed. The enthalpy changes obtained at the submicron scale by diamond thermometry are in good agreement with commercial macroscopic isothermal titration calorimetry. This method could offer a universal characterization technique for biochemical studies at the femtoliter scale.

Controlled transport by molecular machines: exploring biological motors and their physics

Molecular motors are fascinating biological machines that play a crucial role in a variety of cellular processes, including mass transport, muscle contraction, DNA replication, transcription and repair, and RNA translation. These structures convert chemical energy from adenosine triphosphate (ATP) hydrolysis into mechanical work.

Integrated Functions of Cardiac Energetics, Mechanics, and Purine Nucleotide Metabolism.

Purine nucleotides play central roles in energy metabolism in the heart. Most fundamentally, the free energy of hydrolysis of the adenine nucleotide adenosine triphosphate (ATP) provides the thermodynamic driving force for numerous cellular processes including the actin-myosin crossbridge cycle. Perturbations to ATP supply and/or demand in the myocardium lead to changes in the homeostatic balance between purine nucleotide synthesis, degradation, and salvage, potentially affecting myocardial energetics and, consequently, myocardial mechanics. Indeed, both acute myocardial ischemia and decompensatory remodeling of the myocardium in heart failure are associated with depletion of myocardial adenine nucleotides and with impaired myocardial mechanical function. Yet there remain gaps in the understanding of mechanistic links between adenine nucleotide degradation and contractile dysfunction in heart disease. The scope of this article is to: (i) review current knowledge of the pathways of purine nucleotide depletion and salvage in acute ischemia and in chronic heart disease; (ii) review hypothesized mechanisms linking myocardial mechanics and energetics with myocardial adenine nucleotide regulation; and (iii) highlight potential targets for treating myocardial metabolic and mechanical dysfunction associated with these pathways. It is hypothesized that an imbalance in the degradation, salvage, and synthesis of adenine nucleotides leads to a net loss of adenine nucleotides in both acute ischemia and under chronic high-demand conditions associated with the development of heart failure. This reduction in adenine nucleotide levels results in reduced myocardial ATP and increased myocardial inorganic phosphate. Both of these changes have the potential to directly impact tension development and mechanical work at the cellular level. © 2024 American Physiological Society. Compr Physiol 14:5345-5369, 2024.

Adjustable luminescence copper nanoclusters nanoswitch based on competitive coordination of samarium ions for cascade detection of adenosine triphosphate and acid phosphatase activity.

Benefit from the strong coordination property, lanthanide metal ions have been used as competitive reagents to modulate the fluorescence changes of the system. However, lanthanide metal ions as inducers for aggregation-induced emission enhancement in nanosystems is rare. Herein, we report a "turn on-off-on" fluorescent switch for cascade detection of acid phosphatase (ACP) and adenosine triphosphate (ATP) based on the competitive coordination of samarium ions (Sm3+). Novel copper nanoclusters (CuNCs) with long wavelength emission (614nm) stabilized by glutathione (GSH) and glycylglycine (Gly-Gly) have been confirmed to have AIE property. With the continuous aggregation of GSH/Gly-Gly CuNCs under the induction of Sm3+, the fluorescence of the system increased to achieve the "turn-on" process. The coordinated behaviour between Sm3+ and GSH/Gly-Gly CuNCs is discussed. Due to the strong metal coordination ability of ATP, the Sm3+ coordinated with the GSH/Gly-Gly CuNCs is competed out, resulting in the fluorescence "turn-off" process of the system. As the substrate of enzymatic hydrolysis of ACP, with the continuous hydrolysis of ATP by ACP, Sm3+ coordinates with GSH/Gly-Gly CuNCs again, which leads to the AIE effect and realize the fluorescence "turn-on" process of the system. This strategy results inATP linear range of 0.508 ~ 120.0μM with a detection limit of 0.508μM (S/N = 3) and ACP linear range of 0.011 ~ 30.0 U·L-1 with a detection limit of 0.011 U·L-1 (S/N = 3). Application tobiologic samples wassuccessful.

Mechanisms for cardiac calcium pump activation by its substrate and a synthetic allosteric modulator using fluorescence lifetime imaging.

The discovery of allosteric modulators is an emerging paradigm in drug discovery, and signal transduction is a subtle and dynamic process that is challenging to characterize. We developed a time-correlated single photon-counting imaging approach to investigate the structural mechanisms for small-molecule activation of the cardiac sarcoplasmic reticulum Ca2+-ATPase, a pharmacologically important pump that transports Ca2+ at the expense of adenosine triphosphate (ATP) hydrolysis. We first tested whether the dissociation of sarcoplasmic reticulum Ca2+-ATPase from its regulatory protein phospholamban is required for small-molecule activation. We found that CDN1163, a validated sarcoplasmic reticulum Ca2+-ATPase activator, does not have significant effects on the stability of the sarcoplasmic reticulum Ca2+-ATPase-phospholamban complex. Time-correlated single photon-counting imaging experiments using the nonhydrolyzable ATP analog β,γ-Methyleneadenosine 5'-triphosphate (AMP-PCP) showed ATP is an allosteric modulator of sarcoplasmic reticulum Ca2+-ATPase, increasing the fraction of catalytically competent structures at physiologically relevant Ca2+ concentrations. Unlike ATP, CDN1163 alone has no significant effects on the Ca2+-dependent shifts in the structural populations of sarcoplasmic reticulum Ca2+-ATPase, and it does not increase the pump's affinity for Ca2+ ions. However, we found that CDN1163 enhances the ATP-mediated modulatory effects to increase the population of catalytically competent sarcoplasmic reticulum Ca2+-ATPase structures. Importantly, this structural shift occurs within the physiological window of Ca2+ concentrations at which sarcoplasmic reticulum Ca2+-ATPase operates. We demonstrated that ATP is both a substrate and modulator of sarcoplasmic reticulum Ca2+-ATPase and showed that CDN1163 and ATP act synergistically to populate sarcoplasmic reticulum Ca2+-ATPase structures that are primed for phosphorylation. This study provides novel insights into the structural mechanisms for sarcoplasmic reticulum Ca2+-ATPase activation by its substrate and a synthetic allosteric modulator.

How Do Instructors Explain The Mechanism by which ATP Drives Unfavorable Processes?

Concerns regarding students' difficulties with the concept of energy date back to the 1970s. They become particularly apparent for systems involving adenosine triphosphate (ATP), which plays a central role in maintaining the nonequilibrium state of biological systems and in driving energetically unfavorable processes. One of the most well-documented misconceptions related to ATP is the idea that breaking bonds releases energy, when the opposite is true. This misconception is often attributed to language used in biology referring to the "high-energy bonds" in ATP. We interviewed chemistry, biology, and biochemistry instructors to learn how they think about and teach the mechanism(s) by which ATP is used as an energy source in biological systems. Across 15 interviews, we found that instructors relied primarily on two mechanisms to explain the role of ATP: 1) energy release, focused on ATP hydrolysis and bond energies; and/or 2) energy transfer, focused on phosphorylation and common intermediates. Many instructors shared negative and uncomfortable experiences related to teaching ATP and energy release. Based on these findings, we suggest instructional strategies that: 1) aim to ease the concerns expressed by introductory biology instructors, and 2) emphasize the role of ATP so as to support students' understanding of molecular mechanisms.

Abstract PR015: Determining the mechanism of action of the anti-ENTPD2 antibody, KAZ954

Abstract Background: Ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2) is expressed in gastrointestinal malignancies and catalyzes the hydrolysis of adenosine triphosphate (ATP), creating an immunosuppressive microenvironment by reducing the cytotoxic antitumor immune response, enhancing the proliferation and polarization of immunosuppressive cells, and increasing neovascularization. Blocking the hydrolase activity of ENTPD2 increases extracellular ATP and triggers a sustained proinflammatory response in the tumor microenvironment. In this report, we describe the development and characterization of a first-in-class monoclonal anti-ENTPD2 antibody, KAZ954, and surrogate anti-mouse ENTPD2 (mENTPD2) antibody, capable of potently inhibiting the ATP hydrolase activity of ENTPD2 and inducing antibody-dependent cellular cytotoxicity (ADCC). Methods: The ability of KAZ954 to inhibit ENTPD2-induced conversion of ATP to adenosine diphosphate (ADP) was evaluated. In vitro, NIH/3T3 cells engineered to express human or cyno-ENTPD2 and RKO cancer cell lines with endogenous ENTPD2 expression were used. In vivo, 4T1 syngeneic cell lines expressing mENTPD2 were generated, which have a luciferase molecule attached to the plasma membrane to measure extracellular ATP. The shift in ATP:ADP ratio was verified by matrix-assisted laser desorption/ionization imaging analysis. To assess the ADCC-inducing ability of KAZ954, a lactate dehydrogenase release assay was run using peripheral blood mononuclear cell (PBMCs) and RKO as effector and target cells, respectively. Two different ENTPD2-engineered syngeneic tumor models were used to study pharmacodynamics and the potential for single agent, and combinatorial, efficacy. Endpoint measurements included flow cytometry of tumor and blood and serum cytokine analysis. RASLseq was used to further identify RNA changes in mouse syngeneic tumor models treated with anti-mENTPD2. Results: KAZ954 potently inhibited the hydrolysis of ATP to ADP in NIH/3T3 cells, expressing exogenous human or cyno ENTPD2, and in RKO cell lines. KAZ954 IgG was able to bind ENTPD2 on RKO cell lines and Fcγ receptors on PBMCs, inducing RKO cell lysis and LDH release by ADCC. However, the KAZ954 F(ab’)2 fragment, although able to bind ENTPD2, was unable to induce RKO cell lysis due to its inability to bind to Fcγ receptors. Preclinical syngeneic tumor models treated with a surrogate mENTPD2 antibody demonstrated that the blockade of ENTPD2 hydrolase activity results in a delay in tumor growth which is further augmented in combination with immune checkpoint inhibitors or further blockade of the adenosine pathway. Evaluation of the in vivo responses to KAZ954 treatment revealed an increase in ATP:ADP ratio; an increase in intra-tumoral monocytes, NK cells, peripheral CD8+ T cells, macrophages, and CD11b+ dendritic cells, and a gene signature consistent with immune activation in the tumor. Conclusion: KAZ954 binds to and inhibits the hydrolase activity of ENTPD2, leading to an increased intratumoral ATP:ADP ratio and a systemic inflammatory response. Citation Format: Samantha Zaharevitz, Richard Salamone, Carie Jackson, Kerri Grove, Sergio Briones, David Kim, Jian Shi, Kenneth Ng, Marina Harris, Chris Lee, Eric Peters, Anna Galkin, Lisa Guerrettaz, Deborah A Knee. Determining the mechanism of action of the anti-ENTPD2 antibody, KAZ954 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR015.

Abstract B114: A Phase 1/1b study of KAZ954 alone and in combination with spartalizumab (PDR001) and taminadenant (NIR178) in patients with advanced gastrointestinal malignancies

Abstract Background:KAZ954 (KAZ), a humanized immunoglobulin G1 monoclonal antibody, is a potent, selective inhibitor of anti-ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2). Preclinical data show that KAZ blocks the hydrolysis of adenosine triphosphate, enhances immune activation in the tumor microenvironment, and augments the efficacy of PDR001 (PDR, anti-PD-1), and NIR178 (NIR, adenosine A2aR inhibitor). Methods: This is an open-label, multicenter, dose-escalation (ESC)/expansion study (NCT04237649). We present the dose-ESC results of KAZ single-agent (SA) and with PDR or NIR in patients (pts) with advanced gastrointestinal (GI) malignancies. Dose ESC was guided by Bayesian logistic regression models and comprised 3 arms: (1) SA KAZ administered intravenously (IV) at 30, 50, 100, and 150 mg biweekly (Q2W) flat dosing; KAZ 100 mg priming dose followed by 300, 600, and 1200 mg experimental dose; (2) KAZ 100 mg priming dose followed by 300, 600, and 1200 mg experimental dose with PDR 400 mg IV every 4 weeks; and (3) KAZ 100 mg priming dose followed by 300 and 600 mg experimental dose with NIR 160 and 240 mg twice daily orally. Primary objectives were to evaluate safety and tolerability and determine the maximum tolerated dose (MTD)/recommended dose for expansion (RDE). Secondary objectives were to assess antitumor activity and pharmacokinetics (PK). Tumor biopsies and blood samples were taken for biomarker analysis. Results: As of March 20, 2023, 77 pts were enrolled in ESC KAZ (n=43), KAZ+PDR (n=18), and KAZ+NIR (n=16) arms. Tumor types enrolled were microsatellite-stable colorectal cancer (37, 48%), pancreatic ductal adenocarcinoma (27, 35%), cholangiocarcinoma (10, 13%), and esophageal cancer (3, 4%). At data cutoff, 43 (100%), 15 (83.3%), and 11 (68.8%) pts discontinued treatment, and treatment-related adverse events (TRAEs) were reported in 37 (86%), 12 (66.7%), and 13 (81.3%) pts in KAZ, KAZ+PDR, and KAZ+NIR arms, respectively. KAZ had an acceptable and manageable safety profile. Given the occurrence of cytokine release syndrome (CRS) observed with cycle 1 day 1 (C1D1), prophylactic premedication with acetaminophen, antihistamine, and corticosteroid, together with a priming dose on C1D1, was established as the recommended regimen to administer KAZ. Grade ≥3 TRAEs were reported in 9 pts(20.9%) in KAZ, most commonly CRS and decreased lymphocyte count (n=2 each); 7 pts(38.9%) in KAZ+PDR, most commonly lymphopenia and increased aspartate aminotransferase (n=2 each); and 2 pts(12.5%) in KAZ+NIR (asthenia and lymphopenia [n=1 each]). No MTDs were reached in any treatment arm explored. Although no objective responses were observed, 12(27.9%), 2(11.1%), and 2(12.5%) pts showed stable disease in the KAZ, KAZ+PDR, and KAZ+NIR arms, respectively. Conclusions: KAZ was considered to be safe and well tolerated at all doses, and PK/PD analysis suggest effective ENTPD2 inhibition from a dose range of 150 to 1200 mg Q2W. Although a biologically active dose range was established, further dose-exploration studies are required to establish an RDE. Citation Format: Devalingam Mahalingam, Chia-Chi Lin, Philippe L. Bedard, Massimo Di Nicola, Zev Wainberg, Patricia LoRusso, Shubham Pant, Brigette B.Y. Ma, Wei-Peng Yong, Mia C. Weiss, Alessio Amatu, Kentaro Yamazaki, Lisa Nardi, Jong Bong Lee, Mike Roy, Anhthu Dang, Shu Yang, Bernard Pereira, Javier A. Otero, Teresa Macarulla. A Phase 1/1b study of KAZ954 alone and in combination with spartalizumab (PDR001) and taminadenant (NIR178) in patients with advanced gastrointestinal malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B114.

Exploring the expression of Adenosine Pathway-Related Markers CD73 and CD39 in Colorectal and Pancreatic Carcinomas Characterized by Multiplex Immunofluorescence: A Pilot Study.

Generating high levels of immunosuppressive adenosine in the tumor microenvironment contributes to cancer immune evasion. CD39 and CD73 hydrolyze adenosine triphosphate into adenosine; thus, efforts have been made to target this pathway for cancer immunotherapy. Our objective was optimizing a multiplex immunofluorescence (mIF) panel to explore the role of CD39 and CD73 within the tumor microenvironment. In three-time points, a small cohort (n=8 ) of colorectal and pancreatic adenocarcinomas were automated staining using an mIF panel against CK, CD3, CD8, CD20, CD39, CD73 and CD68 to compare them with individual markers immunohistochemistry (IHC) for internal panel validation. Densities of immune cells and distances from different tumor-associated immune cells to tumor cells were exploratory assessment and compared with clinicopathologic variables and outcomes. Comparing the three-time points and individual IHC staining results, we demonstrated high reproducibility of the mIF panel. CD39 and CD73 expression was low in malignant cells; the exploratory analysis showed higher densities of CD39 expression by various cells, predominantly stromal cells, followed by T cells, macrophages, and B cells. No expression of CD73 by B cells or macrophages was detected. Distance analysis revealed proximity of cytotoxic T cells, macrophages, and T cells expressing CD39 to malignant cells, suggesting a close regulatory signal driven by this adenosine marker. We optimized an mIF panel for detection of markers in the adenosine pathway, an emerging clinically relevant pathway. The densities and spatial distribution demonstrated that this pathway may modulate aspects of the tumor immune microenvironment.

Differential Influences of Endogenous and Exogenous Sensory Neuropeptides on the ATP Metabolism by Soluble Ectonucleotidases in the Murine Bladder Lamina Propria.

Bladder urothelium and suburothelium/lamina propria (LP) have prominent sensory and transducer functions with the active participation of afferent neurons and urothelium-derived purine mediators such as adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine (ADO). Effective concentrations of purines at receptor targets depend significantly on the extracellular degradation of ATP by ectonucleotidases (ENTDs). We recently reported the regulated release of soluble ENTDs (s-ENTDs) in the LP and the consequent degradation of ATP to ADP, AMP, and ADO. Afferent neurons in the LP can be activated by urothelial ATP and release peptides and other transmitters that can alter the activity of cells in their vicinity. Using a murine decentralized ex vivo detrusor-free bladder model, 1,N6-etheno-ATP (eATP) as substrate, and sensitive HPLC-FLD methodologies, we found that exogenous neuropeptides calcitonin gene-related peptide (CGRP), substance P (Sub P), neurokinin A (NKA), and pituitary adenylate cyclase-activating polypeptide [PACAP (1-38)] all increased the degradation of eATP by s-ENTDs that were released in the LP spontaneously and/or during bladder filling. Using antagonists of neuropeptide receptors, we observed that endogenous NKA did not modify the ATP hydrolysis by s-ENTDs, whereas endogenous Sub P increased both the constitutive and distention-induced release of s-ENTDs. In contrast, endogenous CGRP and PACAP (1-38) increased the distention-induced, but not the spontaneous, release of s-ENTDs. The present study puts forward the novel idea that interactions between peptidergic and purinergic signaling mechanisms in the LP have an impact on bladder excitability and functions by regulating the effective concentrations of adenine purines at effector cells in the LP.

Identification of natural Rutaecarpine as a potent tobacco mosaic virus (TMV) helicase candidate for managingintractable plant viral diseases.

Naturally occurring alkaloids are particularly suitable for use as pesticide precursors and further modifications due to their cost-effectiveness, unique mechanism of action, tolerable degradation, and environmental friendliness. The famous tobacco mosaic virus (TMV) is a persistent plant pathogenic virus that can parasitize many plants and severely reduce crop production. To treat TMV disease, TMV helicase acts as a crucial target by hydrolyzing adenosine triphosphate (ATP) to provide energy for double-stranded RNA unwinding. To seek novel framework alkaloid leads targeting TMV helicase, this work successfully established an efficient screening platform for TMV helicase inhibitors based on natural alkaloids. In vivo activity screening, enzyme activity detection, and binding assays showed that Rutaecarpine from Evodia rutaecarpa (Juss.) Benth exhibited excellent TMV helicase inhibitory properties [dissociation constant (Kd ) = 1.1 μm, half maximal inhibitory concentration (IC50 ) = 227.24 μm] and excellent anti-TMV ability. Molecular docking and dynamic simulations depicted that Rutaecarpine could stably bind in active pockets of helicase with low binding energy (ΔGbind = -17.8 kcal/mol) driven by hydrogen bonding and hydrophobic interactions. Given Rutaecarpine's laudable bioactivity and structural modifiability, it can serve as a privileged building block for further pesticide discovery.

Mechanism of a high free-energy transduction efficiency of Bacillus PS3 F1-ATPase from the perspective of solvent entropy

Bacillus PS3 F1-ATPase is a rotary molecular motor with a 100% free-energy transduction efficiency (The 100% efficiency means that the free-energy change upon hydrolysis of adenosine triphosphate (ATP) is almost completely consumed by the external dissipation arising from the rotation of the central stalk (γ subunit) of F1-ATPase.). In this work, to elucidate the mechanism of 100% free-energy transduction efficiency, the rotation of Bacillus PS3 F1-ATPase was investigated from the perspective of solvent entropy. The two F1-ATPase structures of the catalytic- and binding-dwell states recently published were used to compute the solvation entropy (The γ subunit of the F1-ATPase structure at the binding-dwell state is rotated by 44° relative to the structure of the catalytic-dwell state). To compute the solvation entropy, the integral equation theory was used. It was found that the change in the solvent entropy of the F1-ATPase upon 44° rotation of the γ subunit is approximately zero, which is surprising because a large structural change occurs in F1-ATPase upon rotation. The present result indicated the compensation of solvent entropy in the F1-ATPase. The compensation behavior was discussed by decomposing the solvent entropy of F1-ATPase into the components of the solvent entropy of subunits and that at the interfaces between the adjacent subunits. We also discussed that the system energy and conformational entropy would also remain unchanged upon the 44° rotation of the γ subunit. Based on the results and discussion, the following rotation mechanism was proposed: the free energy of Bacillus PS3 F1-ATPase is unchanged upon rotation; and the ATP binding, hydrolysis of ATP, and release of products lead to the rotation. This mechanism is consistent with the 100% free-energy transduction efficiency of F1-ATPase.

A nonequilibrium allosteric model for receptor-kinase complexes: The role of energy dissipation in chemotaxis signaling

The Escherichia coli chemotaxis signaling pathway has served as a model system for the adaptive sensing of environmental signals by large protein complexes. The chemoreceptors control the kinase activity of CheA in response to the extracellular ligand concentration and adapt across a wide concentration range by undergoing methylation and demethylation. Methylation shifts the kinase response curve by orders of magnitude in ligand concentration while incurring a much smaller change in the ligand binding curve. Here, we show that the disproportionate shift in binding and kinase response is inconsistent with equilibrium allosteric models. To resolve this inconsistency, we present a nonequilibrium allosteric model that explicitly includes the dissipative reaction cycles driven by adenosine triphosphate (ATP) hydrolysis. The model successfully explains all existing joint measurements of ligand binding, receptor conformation, and kinase activity for both aspartate and serine receptors. Our results suggest that the receptor complex acts as an enzyme: Receptor methylation modulates the ON-state kinetics of the kinase (e.g., phosphorylation rate), while ligand binding controls the equilibrium balance between kinase ON/OFF states. Furthermore, sufficient energy dissipation is responsible for maintaining and enhancing the sensitivity range and amplitude of the kinase response. We demonstrate that the nonequilibrium allosteric model is broadly applicable to other sensor-kinase systems by successfully fitting previously unexplained data from the DosP bacterial oxygen-sensing system. Overall, this work provides a nonequilibrium physics perspective on cooperative sensing by large protein complexes and opens up research directions for understanding their microscopic mechanisms through simultaneous measurements and modeling of ligand binding and downstream responses.

Empagliflozin improves cardiac energetics during ischaemia/reperfusion by directly increasing cardiac ketone utilization.

Empagliflozin (EMPA), a potent inhibitor of the renal sodium-glucose cotransporter 2 and an effective treatment for Type 2 diabetes, has been shown to have cardioprotective effects, independent of improved glycaemic control. Several non-canonical mechanisms have been proposed to explain these cardiac effects, including increasing circulating ketone supply to the heart. This study aims to test whether EMPA directly alters cardiac ketone metabolism independent of supply. The direct effects of EMPA on cardiac function and metabolomics were investigated in Langendorff rat heart perfused with buffer containing 5 mM glucose, 4 mM β-hydroxybutyrate (βHb) and 0.4 mM intralipid, subject to low flow ischaemia/reperfusion. Cardiac energetics were monitored in situ using 31P NMR spectroscopy. Steady-state 13C labelling was performed by switching 12C substrates for 13C1 glucose or 13C4 βHb and 13C incorporation into metabolites determined using 2D 1H-13C HSQC NMR spectroscopy. EMPA treatment improved left ventricular-developed pressure during ischaemia and reperfusion compared to vehicle-treated hearts. In EMPA-treated hearts, total adenosine triphosphate (ATP) and phosphocreatine (PCr) levels, and Gibbs free energy for ATP hydrolysis were significantly higher during ischaemia and reperfusion. EMPA treatment did not alter the incorporation of 13C from glucose into glycolytic products lactate or alanine neither during ischaemia nor reperfusion. In ischaemia, EMPA led to a decrease in 13C1 glucose incorporation and a concurrent increase in 13C4 βHb incorporation into tricarboxylic acid (TCA) cycle intermediates succinate, citrate, and glutamate. During reperfusion, the concentration of metabolites originating from 13C1 glucose was similar to vehicle but those originating from 13C4 βHb remained elevated in EMPA-treated hearts. Our findings indicate that EMPA causes a switch in metabolism away from glucose oxidation towards increased ketone utilization in the rat heart, thereby improving function and energetics both during ischaemia and recovery during reperfusion. This preference of ketone utilization over glucose was observed under conditions of constant supply of substrate, suggesting that EMPA acts directly by modulating cardiac substrate preference, independent of substrate availability. The mechanisms underlying our findings are currently unknown, warranting further study.