Dependent Binding Research Articles

DHX36 binding induces RNA structurome remodeling and regulates RNA abundance via m6A reader YTHDF1

RNA structure constitutes a new layer of gene regulatory mechanisms. RNA binding proteins can modulate RNA secondary structures, thus participating in post-transcriptional regulation. The DEAH-box helicase 36 (DHX36) is known to bind and unwind RNA G-quadruplex (rG4) structure but the transcriptome-wide RNA structure remodeling induced by DHX36 binding and the impact on RNA fate remain poorly understood. Here, we investigate the RNA structurome alteration induced by DHX36 depletion. Our findings reveal that DHX36 binding induces structural remodeling not only at the localized binding sites but also on the entire mRNA transcript most pronounced in 3’UTR regions. DHX36 binding increases structural accessibility at 3’UTRs which is correlated with decreased post-transcriptional mRNA abundance. Further analyses and experiments uncover that DHX36 binding sites are enriched for N6-methyladenosine (m6A) modification and YTHDF1 binding; and DHX36 induced structural changes may facilitate YTHDF1 binding to m6A sites leading to RNA degradation. Altogether, our findings uncover the structural remodeling effect of DHX36 binding and its impact on RNA abundance through regulating m6A dependent YTHDF1 binding.

Preclinical characterization of MTX-101: a novel bispecific CD8 Treg modulator that restores CD8 Treg functions to suppress pathogenic T cells in autoimmune diseases.

Regulatory CD8 T cells (CD8 Treg) are responsible for the selective killing of self-reactive and pathogenic CD4 T cells. In autoimmune disease, CD8 Treg may accumulate in the peripheral blood but fail to control the expansion of pathogenic CD4 T cells that subsequently cause tissue destruction. This CD8 Treg dysfunction is due in part to the expression of inhibitory killer immunoglobulin-like receptors (KIR; KIR2DL isoforms [KIR2DL1, KIR2DL2, and KIR2DL3]); these molecules serve as autoimmune checkpoints and limit CD8 Treg activation. Here we describe the pre-clinical characterization of MTX-101, a bispecific antibody targeting inhibitory KIR and CD8. Using human peripheral blood mononuculear cells (PBMC) derived from healthy donors and autoimmune patients, humanized mouse models, and human derived tissue organoids, we evaluated the molecular mechanisms and functional effects of MTX-101. By binding to KIR, MTX-101 inhibited KIR signaling that can restore CD8 Treg ability to eliminate pathogenic CD4 T cells. MTX-101 bound and activated CD8 Treg in human peripheral blood mononuclear cells (PBMC), resulting in increased CD8 Treg cytolytic capacity, activation, and prevalence. Enhancing CD8 Treg function with MTX-101 reduced pathogenic CD4 T cell expansion and inflammation, without increasing pro-inflammatory cytokines or activating immune cells that express either target alone. MTX-101 reduced antigen induced epithelial cell death in disease affected tissues, including in tissue biopsies from individuals with autoimmune disease (i.e., celiac disease, Crohn's disease). The effects of MTX-101 were specific to autoreactive CD4 T cells and did not suppress responses to viral and bacterial antigens. In a human PBMC engrafted Graft versus Host Disease (GvHD) mouse model of acute inflammation, MTX-101 bound CD8 Treg and delayed onset of disease. MTX-101 induced dose dependent binding, increased prevalence and cytolytic capacity of CD8 Treg, as well as increased CD4 T cell death. MTX-101 selectively bound CD8 Treg without unwanted immune cell activation or increase of pro-inflammatory serum cytokines and exhibited an antibody-like half-life in pharmacokinetic and exploratory tolerability studies performed using IL-15 transgenic humanized mice with engrafted human lymphocytes, including CD8 Treg at physiologic ratios. Collectively, these data support the development of MTX-101 for the treatment of autoimmune diseases.

Synthesis, characterization and comparative biological activity of a novel set of Cu(II) complexes containing azole-based ligand frames

The synthesis and spectroscopic characterization of three complexes containing a substituted 2-(2-pyridyl)benzothiazole (PyBTh) group in the ligand frame are reported along with the comparative biological activity. The ligands have been substituted at the 6-position with either a methoxy (Py(OMe)BTh) or a methyl group (Py(Me)BTh). Reaction of Py(OMe)BTh with either CuCl2 or Cu(NO3)2·2.5 H2O yielded the monomeric [Cu(Py(OMe)BTh))2(NO3)]NO3·1.5 MeOH, (1·1.5 MeOH) complex or the dimeric [Cu(Py(OMe)BTh)Cl2]2 (2), respectively, with the nuclearity of the complex dependent on the starting Cu(II) salt. Reaction between the methyl substituted ligand and Cu(NO3)2·2.5 H2O resulted in the isolation of Cu(Py(Me)BTh)(NO3)2·0.5 THF (3·0.5 THF). Complexes 1–3 were fully characterized. Cyclic voltammetry measurements were performed on all three complexes as well as on [Cu(PyBTh)2(H2O)](BF4)2 (4), a compound previously reported by us which contains the unsubstituted 2-(2-pyridyl)benzothiazole ligand. The biological activity was studied and included concentration dependent DNA binding and cleavage, antibacterial activity, and cancer cell toxicity. All complexes exhibited DNA cleavage activity, however 2 and 4 were found to be the most potent. Mechanistic studies revealed that the nuclease activity is dependent on an oxidative mechanism reliant principally on O2−. Antibacterial studies revealed complex 4 was more potent compared to 1–3. Cancer cell toxicity studies were carried out on HeLa, PC-3, and MCF7 cells with 1–4, Cu(QBTh)(NO3)2(H2O) and Cu(PyBIm)3(BF4)2. The differences in the observed toxicities suggests the importance of the ligand and its substituents in modulating cell death.

Functional characterization of prenyltransferases involved in de novo synthesis of isoprenoids in the leaf beetle Monolepta hieroglyphica

Prenyltransferases play a pivotal role in the isoprenoid biosynthesis and transfer in insects. In the current study, two classes of prenyltransferases (MhieFPPS1 and MhieFPPS2, MhiePFT-β and MhiePF/GGT-α) were identified in the leaf beetle, Monolepta hieroglyphica. Phylogenetic analysis revealed that MhieFPPS1, MhieFPPS2, MhiePFT-β and MhiePF/GGT-α were clustered in one clade with homologous in insects. Moreover, MhieFPPS2 lacked one aspartate-rich motif SARM. Molecular docking and kinetic analysis indicated that the (E)-GPP displayed higher affinity with MhieFPPS1 compared to DMAPP within the binding pocket containing metal binding sites (MG). The other class of prenyltransferases (MhiePFT-β and MhiePF/GGT-α) lack the aspartate-rich motif. Docking results indicated that binding site of MhiePFT-β involved divalent metal ions (Zn) and bound farnesyl or geranylgeranyl. In vitro, only recombiant MhieFPPS1 could catalyze the formation of (E)-farnesol against different combination of substrates, including IPP/DMAPP and IPP/(E)-GPP, highlighting the importance of SARM for enzyme activities. Kinetic analysis further indicated that MhiePFT-β operated via Zn2+-dependent substrate binding, while MhiePF/GGT-α stabilized the β-subunit during catalytic reaction. These findings contribute to a valuable insight in to understanding of the mechanisms involved in the biosynthesis and delivery of isoprenoid products in beetles.

The m6A methyltransferase METTL3 modifies Kcnk6 promoting on inflammation associated carcinogenesis is essential for colon homeostasis and defense system through histone lactylation dependent YTHDF2 binding

Inflammation induces tumor formation and plays a crucial role in tumor progression and prognosis. KCNK6, by regulating K(+) efflux to reduce NLRP3 Inflammasome-induced lung injury, relaxes the aorta. This study aims to elucidate the effects and biological mechanism of KCNK6 in inflammation-associated carcinogenesis, which may be essential for colon homeostasis and the defense system. To induce colitis, mice were given 3.0% Dextran Sodium Sulfate (DSS) in their drinking water for 7 days. The Azoxymethane (AOM) +DSS method was used to induce colon cancer in the mice model. Bone marrow-derived macrophages (BMDM) from Kcnk6-/- mice, AW264.7 cells, and human colon cancer HCT116 and Caco2 cells were used as in vitro models. The loss of Kcnk6 prevented spontaneous colitis and restored mucosal integrity and homeostatic molecules. Additionally, the loss of Kcnk6 reduced the severity of AOM/DSS-induced carcinogenesis. Kcnk6 promoted cell viability and proliferation in HCT-116 or Caco-2 cells. The loss of Kcnk6 inhibited the levels of inflammatory factors in BMDM cells. Kcnk6 accelerated potassium channel activity, inducing NLRP3 inflammasome activation. METTL3-mediated m6A modification increased Kcnk6 stability in a YTHDF2-dependent manner. Histone lactylation activated the transcription of YTHDF2/Kcnk6. Our study revealed the important role of Kcnk6 in inflammation-associated carcinogenesis progression. The m6A methyltransferase METTL3 and histone lactylation increased Kcnk6 stability in a YTHDF2-dependent manner, providing a potential strategy for inflammation-associated carcinogenesis or colorectal cancer therapy.

Piperlongumine, a natural alkaloid from Piper longum L. ameliorates metabolic-associated fatty liver disease by antagonizing the thromboxane A2 receptor

Metabolic dysfunction-associated fatty liver disease (MAFLD) encompasses a broad spectrum of hepatic disorders, including hyperglycemia, hepatic steatosis, and insulin resistance. Piperlongumine (PL), a natural amide alkaloid extracted from the fruits of Piper longum L., exhibited hepatoprotective effects in zebrafish and liver injury mice. This study aimed to investigate the therapeutic potential of PL on MAFLD and its underlying mechanisms. The findings demonstrate that PL effectively combats MAFLD induced by a high-fat diet (HFD) and improves metabolic characteristics in mice. Additionally, our results suggest that the anti-MAFLD effect of PL is attributed to the suppression of excessive hepatic gluconeogenesis, inhibition of de novo lipogenesis, and alleviation of insulin resistance. Importantly, the results indicate that, on the one hand, the hypoglycemic effect of PL is closely associated with CREB-regulated transcriptional coactivators (CRTC2)-dependent cyclic AMP response element binding protein (CREB) phosphorylation; on the other hand, the lipid-lowering effect of PL is attributed to reducing the nuclear localization of sterol regulatory element-binding proteins 1c (Srebp-1c). Mechanistically, PL could alleviate insulin resistance induced by endoplasmic reticulum stress by antagonizing the thromboxane A2 receptor (TP)/Ca2+ signaling, and the TP receptor serves as the potential target for PL in the treatment of MAFLD. Therefore, our results suggested PL effectively improved the major hallmarks of MAFLD induced by HFD, highlighting a potential therapeutic strategy for MAFLD.

Self-Assembled Generation of Multi-zonal Liver Organoids from Human Pluripotent Stem Cells.

Distinct hepatocyte subpopulations are spatially segregated along the portal-central axis and critical to understanding metabolic homeostasis and liver injury. While several bioactive molecules have been described to play a role in directing zonal fates, including ascorbate and bilirubin, in vitro replication of zonal liver architecture has not been achieved to date. In order to evaluate hepatic zonal polarity, we developed a self-assembling zone-specific liver organoid culture by co-culturing ascorbate and bilirubin enriched hepatic progenitors derived from human induced pluripotent stem cells. We found that preconditioned hepatocyte-like cells exhibited zone-specific functions associated with urea cycle, glutathione synthesis and glutamate synthesis. Single nucleus RNA sequencing analysis of these zonally patterned organoids identifies hepatoblast differentiation trajectory that mimics periportal-, interzonal-, and pericentral human hepatocytes. Epigenetic and transcriptomic analysis showed that zonal identity is orchestrated by ascorbate or bilirubin dependent binding of histone acetyltransferase p300 (EP300) to methylcytosine dioxygenase TET1 or hypoxia-inducible factor 1-alpha (HIF1α). Transplantation of the self-assembled zonally patterned human organoids improved survival of immunodeficient rats who underwent bile duct ligation by ameliorating the hyperammonemia and hyperbilirubinemia. Overall, this multi-zonal organoid system serves as an in vitro human model to better recapitulate hepatic architecture relevant to liver development and disease.

Thermally Driven Catch-and-Release of CoCl2.

A heat-driven catch-and-release strategy for CoCl2 capture is described. It is based on the use of an immobilized neutral dicyclohexylacetamide-based receptor L supported on polystyrene (PS-L). An X-ray diffraction analysis of a single crystal of L·CoCl2 revealed an ion-pair complex comprising a hexacoordinated cobalt cation [L·Co]2+ and a tetrachlorocobaltate anion [CoCl4]2-. Temperature dependent binding was seen, as inferred from UV-vis spectroscopic studies. Fits to the van't Hoff equation yielded values of ΔH° = 12.4 kJ/mol and ΔS° = 56.0 J/K·mol for L + CoCl2, and ΔH° = 16.5 kJ/mol and ΔS° = 85.0 J/K·mol for PS-L + CoCl2 in 95% ethanol. Consequently, cobalt capture and release are mediated by heating and cooling, respectively. The material PS-L exhibits a preference for binding cobalt over manganese and nickel as inferred from Langmuir-Freundlich isotherm analyses that revealed binding constants of KLF = 88.5 M-1 for CoCl2, 52.7 M-1 for MnCl2, and 49.7 M-1 for NiCl2. In a simulated ion mixture containing equimolar CoCl2, MnCl2, and NiCl2, ICP-MS analyses served to confirm that cobalt was selectively enriched to 52 mol % (from an initial level of ca. 32 mol %) after one catch-and-release cycle and 76.6% after three cycles. Our experimental results were validated by density functional theory calculations, which also show stronger binding of Co over Mn and Ni to L.

Abstract Mo062: Cpne5 is Necessary for Normal Sinoatrial Node Function

Background: Cpne5 (Copine 5) is a Ca2+-dependent phospholipid binding protein implicated in membrane trafficking but has no known role within the heart to date. In single-cell RNA-sequencing (scRNA-seq) of the developing mouse heart, we found Cpne5 gene expression to be highly enriched throughout the cardiac conduction system (CCS). Further, variants in CPNE5 were identified in 6 independent GWAS related to heart rate variability in humans. We therefore hypothesize that Cpne5 is necessary for normal CCS function. Objective: Determine whether Cpne5 is necessary for the normal development and/or function of the CCS. Methods: To assess the necessity of Cpne5, homozygous systemic knockout (KO) mice were generated using CRISPR/Cas9. Cpne5KO and littermate wild-type (WT) control mice were characterized using surface electrocardiogram (ECG), continuous telemetry and echocardiogram. Protein expression of Cpne5 and CCS-related ion channels were assessed by immunofluorescence of tissue sections, isolated adult murine SAN cells and whole mount immunostaining with 3D volumetric imaging (iDISCO+). Further, CRISPR-mediated deletion of CPNE5 was performed in human induced pluripotent stem cells (hiPSC). Differentiated SAN-like cells were phenotyped via calcium transient (CaT) dynamics and microelectrode array. Results: Successful deletion of Cpne5 in CRISPR-generated KO mice was confirmed by PCR immunofluorescence. Homozygous Cpne5KO mice were viable, showed normal growth and allele frequencies segregated in a normal Mendelian fashion. No gross anatomic defects of the CCS were observed in Cpne5KO mice and transthoracic echocardiogram studies showed slight reduction in left ventricular fractional shortening but otherwise a structurally normal heart. Notably, while surface electrocardiogram (ECG) demonstrated normal intervals (PR, QRS, QTc), Cpne5KO mice exhibited pronounced sinus arrhythmia, sinus pauses, and increased heart rate variability compared to WT controls on long term telemetry. Additionally, CPNE5KO hiPSC-derived SAN-like cells exhibited irregular CaT as well as significant beat period variability compared to isogenic WT controls. Conclusion: Systemic loss-of-function of Cpne5 in mice resulted in sinus node dysfunction, consistent with an hiPSC-derived SAN model and human GWAS data, implicating a novel, cell-intrinsic role for Cpne5 in CCS function and/or development. Additional phenotyping and molecular studies are ongoing to further elucidate the underlying mechanism.

Arabidopsis CaLB1 undergoes phase separation with the ESCRT protein ALIX and modulates autophagosome maturation

Autophagy is relevant for diverse processes in eukaryotic cells, making its regulation of fundamental importance. The formation and maturation of autophagosomes require a complex choreography of numerous factors. The endosomal sorting complex required for transport (ESCRT) is implicated in the final step of autophagosomal maturation by sealing of the phagophore membrane. ESCRT-III components were shown to mediate membrane scission by forming filaments that interact with cellular membranes. However, the molecular mechanisms underlying the recruitment of ESCRTs to non-endosomal membranes remain largely unknown. Here we focus on the ESCRT-associated protein ALG2-interacting protein X (ALIX) and identify Ca2+-dependent lipid binding protein 1 (CaLB1) as its interactor. Our findings demonstrate that CaLB1 interacts with AUTOPHAGY8 (ATG8) and PI(3)P, a phospholipid found in autophagosomal membranes. Moreover, CaLB1 and ALIX localize with ATG8 on autophagosomes upon salt treatment and assemble together into condensates. The depletion of CaLB1 impacts the maturation of salt-induced autophagosomes and leads to reduced delivery of autophagosomes to the vacuole. Here, we propose a crucial role of CaLB1 in augmenting phase separation of ALIX, facilitating the recruitment of ESCRT-III to the site of phagophore closure thereby ensuring efficient maturation of autophagosomes.

Phonon-induced band gap renormalization by dielectric dependent global hybrid density functional tight binding

Phonon-induced band gap renormalization by dielectric dependent global hybrid density functional tight binding

ATTAINMENT: A phase Ib trial of MDX-124, a first-in-class annexin-A1 targeting antibody, alone and in combination with anti-cancer treatments, in patients with advanced solid tumors.

TPS2671 Background: Annexin-A1 is a Ca2+-dependent phospholipid binding protein that is secreted from both cancer and immune cells in response to several physiological stimuli. Secreted annexin-A1 activates formyl peptide receptors driving cancer cell proliferation, angiogenesis, migration, and drug resistance, as well as modulating the tumor microenvironment. Annexin-A1 overexpression is observed in multiple cancers, including pancreatic, triple-negative breast, colorectal, lung and prostate, correlating with poor prognosis and decreased overall survival. MDX-124 is a novel, first-in-class humanised monoclonal antibody that targets annexin-A1. It significantly reduces cancer cell growth via cell cycle arrest, inhibits tumour growth in-vivo, reduces metastasis and induces antibody-dependent cellular cytotoxicity in annexin-A1 expressing cancer cells. Additionally, MDX-124 has synergistic activity when combined with chemotherapy and immunotherapy in pre-clinical models. These data indicate blocking the action of annexin-A1 has anti-cancer and therapeutic activity. Methods: ATTAINMENT is a modular, multi-arm, First-in-Human trial to evaluate the safety and tolerability of MDX-124 alone and in combination with anti-cancer treatments, in participants with locally advanced, unresectable, or metastatic solid malignancies. Module 1 is a single agent dose escalation using a Bayesian optimal interval (BOIN) design with an expansion cohort, followed by Module 2, which will evaluate MDX-124 in combination with standard of care in indication-specific arms by using a traditional 3+3 dose escalation scheme. Adult patients (≥18 years) with ECOG performance score 0-1 and histologically/cytologically confirmed solid tumors believed to overexpress annexin-A1 which are not amenable to or refractory to standard therapy, or for which no standard therapy exists are eligible. The primary objective is to determine the recommended phase 2 dose (RP2D) of MDX-124 both as a single agent and in combination with anti-cancer treatments. Secondary objectives will assess the safety and tolerability of MDX-124 when given as a single agent or in combination with anti-cancer treatments, characterize pharmacokinetic parameters and assess evidence of preliminary anti-tumor activity per RECIST v1.1 criteria. Exploratory objectives will assess host immune response to MDX-124 (immunogenicity and immunophenotyping), circulating levels of annexin-A1 at baseline and after dosing to correlate with response and outcome to MDX-124, and the impact of MDX-124 on blood/tissue biomarkers. The study began enrolling patients at sites in the UK in August 2023. Cohorts at 1, 2.5 and 5 mg/kg have been completed without DLT and enrollment to cohort 4 (10 mg/kg) began in January 2024. Clinical trial information: ISRCTN78740398.

Secondary Sites of the C-type Lectin-Like Fold.

C-type lectins are a large superfamily of proteins involved in a multitude of biological processes. In particular, their involvement in immunity and homeostasis has rendered them attractive targets for diverse therapeutic interventions. They share a characteristic C-type lectin-like domain whose adaptability enables them to bind a broad spectrum of ligands beyond the originally defined canonical Ca2+-dependent carbohydrate binding. Together with variable domain architecture and high-level conformational plasticity, this enables C-type lectins to meet diverse functional demands. Secondary sites provide another layer of regulation and are often intricately linked to functional diversity. Located remote from the canonical primary binding site, secondary sites can accommodate ligands with other physicochemical properties and alter protein dynamics, thus enhancing selectivity and enabling fine-tuning of the biological response. In this review, we outline the structural determinants allowing C-type lectins to perform a large variety of tasks and to accommodate the ligands associated with it. Using the six well-characterized Ca2+-dependent and Ca2+-independent C-type lectin receptors DC-SIGN, langerin, MGL, dectin-1, CLEC-2 and NKG2D as examples, we focus on the characteristics of non-canonical interactions and secondary sites and their potential use in drug discovery endeavors.

Multifunctional interaction of CihC/FbpC orthologs of relapsing fever spirochetes with host-derived proteins involved in adhesion, fibrinolysis, and complement evasion.

Relapsing fever (RF) remains a neglected human disease that is caused by a number of diverse pathogenic Borrelia (B.) species. Characterized by high cell densities in human blood, relapsing fever spirochetes have developed plentiful strategies to avoid recognition by the host defense mechanisms. In this scenario, spirochetal lipoproteins exhibiting multifunctional binding properties in the interaction with host-derived molecules are known to play a key role in adhesion, fibrinolysis and complement activation. Binding of CihC/FbpC orthologs to different human proteins and conversion of protein-bound plasminogen to proteolytic active plasmin were examined by ELISA. To analyze the inhibitory capacity of CihC/FbpC orthologs on complement activation, a microtiter-based approach was performed. Finally, AlphaFold predictions were utilized to identified the complement-interacting residues. Here, we elucidate the binding properties of CihC/FbpC-orthologs from distinct RF spirochetes including B. parkeri, B. hermsii, B. turicatae, and B. recurrentis to human fibronectin, plasminogen, and complement component C1r. All CihC/FbpC-orthologs displayed similar binding properties to fibronectin, plasminogen, and C1r, respectively. Functional studies revealed a dose dependent binding of plasminogen to all borrelial proteins and conversion to active plasmin. The proteolytic activity of plasmin was almost completely abrogated by tranexamic acid, indicating that lysine residues are involved in the interaction with this serine protease. In addition, a strong inactivation capacity toward the classical pathway could be demonstrated for the wild-type CihC/FbpC-orthologs as well as for the C-terminal CihC fragment of B. recurrentis. Pre-incubation of human serum with borrelial molecules except CihC/FbpC variants lacking the C-terminal region protected serum-susceptible Borrelia cells from complement-mediated lysis. Utilizing AlphaFold2 predictions and existing crystal structures, we mapped the putative key residues involved in C1r binding on the CihC/FbpC orthologs attempting to explain the relatively small differences in C1r binding affinity despite the substitutions of key residues. Collectively, our data advance the understanding of the multiple binding properties of structural and functional highly similar molecules of relapsing fever spirochetes proposed to be involved in pathogenesis and virulence.

Loss of CREBBP and KMT2D cooperate to accelerate lymphomagenesis and shape the lymphoma immune microenvironment

Despite regulating overlapping gene enhancers and pathways, CREBBP and KMT2D mutations recurrently co-occur in germinal center (GC) B cell-derived lymphomas, suggesting potential oncogenic cooperation. Herein, we report that combined haploinsufficiency of Crebbp and Kmt2d induces a more severe mouse lymphoma phenotype (vs either allele alone) and unexpectedly confers an immune evasive microenvironment manifesting as CD8+ T-cell exhaustion and reduced infiltration. This is linked to profound repression of immune synapse genes that mediate crosstalk with T-cells, resulting in aberrant GC B cell fate decisions. From the epigenetic perspective, we observe interaction and mutually dependent binding and function of CREBBP and KMT2D on chromatin. Their combined deficiency preferentially impairs activation of immune synapse-responsive super-enhancers, pointing to a particular dependency for both co-activators at these specialized regulatory elements. Together, our data provide an example where chromatin modifier mutations cooperatively shape and induce an immune-evasive microenvironment to facilitate lymphomagenesis.

Borrelia burgdorferi PlzA is a cyclic-di-GMP dependent DNA and RNA binding protein.

The PilZ domain-containing protein, PlzA, is the only known cyclic di-GMP binding protein encoded by all Lyme disease spirochetes. PlzA has been implicated in the regulation of many borrelial processes, but the effector mechanism of PlzA was not previously known. Here, we report that PlzA can bind DNA and RNA and that nucleic acid binding requires c-di-GMP, with the affinity of PlzA for nucleic acids increasing as concentrations of c-di-GMP were increased. A mutant PlzA that is incapable of binding c-di-GMP did not bind to any tested nucleic acids. We also determined that PlzA interacts predominantly with the major groove of DNA and that sequence length and G-C content play a role in DNA binding affinity. PlzA is a dual-domain protein with a PilZ-like N-terminal domain linked to a canonical C-terminal PilZ domain. Dissection of the domains demonstrated that the separated N-terminal domain bound nucleic acids independently of c-di-GMP. The C-terminal domain, which includes the c-di-GMP binding motifs, did not bind nucleic acids under any tested conditions. Our data are supported by computational docking, which predicts that c-di-GMP binding at the C-terminal domain stabilizes the overall protein structure and facilitates PlzA-DNA interactions via residues in the N-terminal domain. Based on our data, we propose that levels of c-di-GMP during the various stages of the enzootic life cycle direct PlzA binding to regulatory targets.

Abstract 1907: MYTX-011: A pH-dependent anti-cMET ADC with increased payload delivery in vivo and potent activity against cMET-expressing tumors of various epithelial origins

Abstract cMET is an oncogenic receptor tyrosine kinase overexpressed in multiple types of solid tumors, including non-small cell lung cancer (NSCLC), gastric, head and neck, liver cancer and others. Several cMET-directed therapies including antibody-drug conjugates (ADCs) have been developed that may benefit a subset of patients with tumors that have high cMET expression, MET amplification, or dependency on cMET signaling. MYTX-011 is an investigational, pH-sensitive, vcMMAE-based ADC that is designed to potentially benefit not only patients whose tumors express high levels of cMET, but also a broader set of patients whose tumors express low to moderate levels of cMET. MYTX-011 is engineered to rapidly dissociate from cMET only at the acidic pH of endolysosomes. We have previously demonstrated that MYTX-011 drives increased internalization and cytotoxicity in tumor cells expressing moderate cMET levels compared to a matched parent non-pH sensitive ADC. MYTX-011 also demonstrated markedly superior efficacy in NSCLC xenografts with moderate cMET expression as compared to either the parent ADC or benchmark ADCs representing clinical-stage anti-cMET ADCs with vcMMAE and maytansinoid payloads. MYTX-011 was efficacious in NSCLC PDX models with moderate cMET expression of adenocarcinoma/squamous histology and in heterogeneous cMET-expressing EGFR mutant models. In these studies, we demonstrate that MYTX-011 exerts enhanced efficacy by delivering increased levels of payload to cMET+ tumors compared to the parent ADC. MYTX-011 treatment induced significantly increased levels of phospho-histone H3 (pHH3), a pharmacodynamic marker of MMAE cytotoxicity, in moderate cMET expressing NCI-H1975 NSCLC xenograft tumors compared to the parent ADC. Further, increased levels of free MMAE were detected in tumors from mice treated with MYTX-011 compared to a matched dose of the parent ADC, indicating that the pH dependent binding engineered into MYTX-011 translated to increased payload delivery to tumors in vivo. To explore the potential efficacy of MYTX-011 in other cMET-expressing tumor types, we tested the activity of MYTX-011 in a panel of cell lines derived from gastric and head and neck cancers. MYTX-011 demonstrated increased cMET-dependent cytotoxicity compared to the parent ADC. MYTX-011 was also highly active in cMET+ xenograft models derived from gastric (non-MET amplified), esophageal, and head and neck cancers. Together, these findings highlight the potential of MYTX-011 as a therapeutic candidate for treating a broader range of cMET-expressing malignancies. Citation Format: Deepak Kanojia, William Comb, William Israelsen, Federico Colombo, Lin Nie, Nimish Gera, Thomas Chittenden, Brian Fiske. MYTX-011: A pH-dependent anti-cMET ADC with increased payload delivery in vivo and potent activity against cMET-expressing tumors of various epithelial origins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1907.

Differential scanning fluorimetry to assess PFAS binding to bovine serum albumin protein.

The rapid screening of protein binding affinity for poly- and perfluoroalkyl substances (PFAS) benefits risk assessment and fate and transport modelling. PFAS are known to bioaccumulate in livestock through contaminated food and water. One excretion pathway is through milk, which may be facilitated by binding to milk proteins such as bovine serum albumin (BSA). We report a label-free differential scanning fluorimetry approach to determine PFAS-BSA binding over a broad temperature range. This method utilizes the tryptophan residue within the protein binding pocket as an intrinsic fluorophore, eliminating the need for fluorophore labels that may influence binding. BSA association constants were determined by (a) an equilibrium-based model at the melting temperature of BSA and (b) the Hill adsorption model to account for temperature dependent binding and binding cooperativity. Differences in binding between PFAS and fatty acid analogs revealed that a combination of size and hydrophobicity drives PFAS binding.

Deciphering aging-associated molecular mechanisms in bone marrow derived hematopoietic stem cells in the elderly using NGS data

Aging is a complex process that is not yet fully understood. Despite advancements in research, a deeper understanding of the underlying biological mechanisms is necessary to develop interventions that promote healthy longevity. The aim of this study was to elucidate the complex mechanisms associated with healthy aging and longevity in healthy elderly individuals. The RNA sequencing (RNA-seq) data used in this study was obtained from the Gene Expression Omnibus (GEO) database (accession number GSE104406), which was collected from Fluorescent Activated Cell Sorting (FACS) of human bone marrow derived human hematopoietic stem cells (BM-HSCs) (Lineage-, CD34+, CD38-) young (18-30 years old) and aged (65-75 years old) donors who had no known hematological malignancy, with 10 biological replicates per group. The GEO RNA-seq Experiments Interactive Navigator (GREIN) software was used to obtain raw gene-level counts and filtered metadata for this dataset. Next generation knowledge discovery (NGKD) tools provided by BioJupies were used to obtain differentially regulated pathways, gene ontologies (GO), and gene signatures in the HSCs. Finally, theL1000 Characteristic Direction Signature Search Engine (L1000CDS2) tool was used to identify specific drugs that reverse aging-associated gene signatures in old but healthy individuals. The down-regulation of signaling pathways such as longevity regulation, proteasome, notch, apoptosis, nuclear factor kappa B (NFkB), and peroxisome proliferator-activated receptors (PPAR) signaling pathways in the HSCs of healthy elderly. GO functions related to negative regulation of bone morphogenetic protein (BMP), telomeric DNA binding, nucleoside binding, calcium -dependent protein binding, chromatin-DNA binding, SMAD binding, and demethylase activity were significantly downregulated in the BM-HSCs of the elderly compared to the healthy young group. Importantly, we identified potential drugs such as salermide, celestrol, cercosporin, dorsomorphin dihydrochloride, and LDN-193189 monohydrochloride that can reverse the aging-associated signatures in HSCs from healthy elderly. The analysis of RNA-seq data based on NGKD techniques revealed a plethora of differentially regulated pathways, gene ontologies, and drugs with anti-aging potential to promote healthspan in the elderly.

Deciphering aging-associated molecular mechanisms in bone marrow derived hematopoietic stem cells in the elderly using NGS data.

Aging is a complex process that is not yet fully understood. Despite advancements in research, a deeper understanding of the underlying biological mechanisms is necessary to develop interventions that promote healthy longevity. The aim of this study was to elucidate the complex mechanisms associated with healthy aging and longevity in healthy elderly individuals. The RNA sequencing (RNA-seq) data used in this study was obtained from the Gene Expression Omnibus (GEO) database (accession number GSE104406), which was collected from Fluorescent Activated Cell Sorting (FACS) of human bone marrow derived human hematopoietic stem cells (BM-HSCs) (Lineage-, CD34+, CD38-) young (18-30 years old) and aged (65-75 years old) donors who had no known hematological malignancy, with 10 biological replicates per group. The GEO RNA-seq Experiments Interactive Navigator (GREIN) software was used to obtain raw gene-level counts and filtered metadata for this dataset. Next generation knowledge discovery (NGKD) tools provided by BioJupies were used to obtain differentially regulated pathways, gene ontologies (GO), and gene signatures in the BM-HSCs. Finally, the L1000 Characteristic Direction Signature Search Engine (L1000CDS2) tool was used to identify specific drugs that reverse aging-associated gene signatures in old but healthy individuals. The down-regulation of signaling pathways such as longevity regulation, proteasome, Notch, apoptosis, nuclear factor kappa B (NFkB), and peroxisome proliferator-activated receptors (PPAR) signaling pathways in the BM-HSCs of healthy elderly. GO functions related to negative regulation of bone morphogenetic protein (BMP), telomeric DNA binding, nucleoside binding, calcium -dependent protein binding, chromatin-DNA binding, SMAD binding, and demethylase activity were significantly downregulated in the BM-HSCs of the elderly compared to the healthy young group. Importantly, potential drugs such as salermide, celestrol, cercosporin, dorsomorphin dihydrochloride, and LDN-193189 monohydrochloride that can reverse the aging-associated signatures in HSCs from healthy elderly were identified. The analysis of RNA-seq data based on NGKD techniques revealed a plethora of differentially regulated pathways, gene ontologies, and drugs with anti-aging potential to promote healthspan in the elderly.