Specific Binding Proteins Research Articles

PepSeq: a fully in vitro platform for highly multiplexed serology using customizable DNA-barcoded peptide libraries.

PepSeq is an in vitro platform for building and conducting highly multiplexed proteomic assays against customizable targets by using DNA-barcoded peptides. Starting with a pool of DNA oligonucleotides encoding peptides of interest, this protocol outlines a fully in vitro and massively parallel procedure for synthesizing the encoded peptides and covalently linking each to a corresponding cDNA tag. The resulting libraries of peptide/DNA conjugates can be used for highly multiplexed assays that leverage high-throughput sequencing to profile the binding or enzymatic specificities of proteins of interest. Here, we describe the implementation of PepSeq for fast and cost-effective epitope-level analysis of antibody reactivity across hundreds of thousands of peptides from <1 µl of serum or plasma input. This protocol includes the design of the DNA oligonucleotide library, synthesis of DNA-barcoded peptide constructs, binding of constructs to sample, preparation for sequencing and data analysis. Implemented in this way, PepSeq can be used for a number of applications, including fine-scale mapping of antibody epitopes and determining a subject's pathogen exposure history. The protocol is divided into two main sections: (i) design and synthesis of DNA-barcoded peptide libraries and (ii) use of libraries for highly multiplexed serology. Once oligonucleotide templates are in hand, library synthesis takes 1-2 weeks and can provide enough material for hundreds to thousands of assays. Serological assays can be conducted in 96-well plates and generate sequencing data within a further ~4 d. A suite of software tools, including the PepSIRF package, are made available to facilitate the design of PepSeq libraries and analysis of assay data.

Multiomics Analysis Confirms Effective Target Engagement for RVU120 - a First-in-Class CDK8/19 Kinase Inhibitor in AML and MR-MDS Patients and Reveals the Mechanism of Action

RVU120 is a first-in-class CDK8/19 kinase inhibitor. The primary objective of an ongoing Phase Ib study of RVU120 in AML and HR-MDS patients is to determine the preliminary safety profile and the recommended phase 2 dose of RVU120 as a single agent. Characterization of pharmacodynamic (PD) effects is a secondary objective of the trial. PD analysis supports the determination of the recommended phase 2 dose, further clinical development and ultimately positioning of this novel class of targeted agents in the treatment strategies for AML and MDS patients. A battery of tests has been used to establish the relationship between pharmacokinetics (PK) and PD activity, confirm the on-target activity of RVU120, and reveal a mechanism of action of CDK8/19 inhibitors in preclinical models. Specific protein binding of RVU120 in leukemic and normal blood cells has been verified by capturing CDK8/19 kinases after a competition with ATP acyl-phosphate biotin-labeled probes. Changes in CDK8- dependent phosphorylation of STAT5 were used as proximal biomarkers and were measured by flow cytometry. Transcriptome changes were used as downstream biomarkers and were determined by the integrated analysis of bulk RNAseq, ChIPseq and ATACseq. The molecular activity of RVU120 was correlated with anti-leukemic activity and surface marker changes. In patients, PD activity of RVU120 was established by measuring changes in pSTAT5 levels, directly in blast cells and ex vivo in established AML cell lines, exposed to plasma collected from patients treated with RVU120. Downstream transcriptomic effects in patients were assessed by RNAseq and phenotypic changes were tracked by flow cytometry surface marker panels. Preclinical profiling indicated effective CDK8/19 target engagement and inhibition of pSTAT5 levels at low nanomolar concentrations of RVU120, in both established cell lines and primary leukemic cells. The antileukemic activity was tightly correlated with levels of biomarker inhibition, however, in a group of super-responders enriched for NPM1 mutants and leukemic stem cells, efficacy was achieved at partial biomarker inhibition levels. Downstream profiling revealed inhibition of homeobox genes and induction of STAT5-dependent genes as two major transcriptomic programs regulated by RVU120. Homeobox genes inhibited by RVU120 are essential for the viability and self-renewal of leukemic cells, whereas many STAT5 -dependent genes are known as master regulators of erythropoiesis. Consistently, treatment of healthy and transformed CD34+ cells with RVU120 results in the commitment to erythropoiesis. Profiling of patient samples largely confirmed preclinical findings. Inhibition of pSTAT5 in CD34+ blasts from AML and MDS patients tightly correlated with achieved exposures after the first dose and at a steady state. At the time of submission pSTAT5 inhibition has already reached >50%, a threshold that based on preclinical predictions is sufficient for robust efficacy in selected groups of super-responder patients. Further increase at higher doses, is expected to result in deeper, more durable responses in broader populations of patients. Broad transcriptomic changes were observed in patients after treatment with RVU120. Notably, potent inhibition of homeobox genes was observed in NPM1 mutant blasts and induction of erythroid master regulator genes was observed in SF3B1 mutant cells with erythroid arrest. Clear hallmarks of erythroid differentiation were further identified in several patients by specific changes in surface markers. These patients achieved meaningful clinical responses such as reduction of blasts and transfusion independence, respectively.

Non-specificity as the sticky problem in therapeutic antibody development.

Antibodies are highly potent therapeutic scaffolds with more than a hundreddifferent products approved on the market. Successful development of antibody-based drugs requires a trade-off between high target specificity and target binding affinity. In order to better understand this problem, we here review non-specific interactions and explore their fundamental physicochemical origins. We discuss the role of surface patches - clusters of surface-exposed amino acid residues with similar physicochemical properties - as inducers of non-specific interactions. These patches collectively drive interactions including dipole-dipole, π-stacking and hydrophobic interactions to complementary moieties. We elucidate links between these supramolecular assembly processes and macroscopic development issues, such as decreased physical stability and poor in vivo half-life. Finally, we highlight challenges and opportunities for optimizing protein binding specificity and minimizing non-specificity for future generations of therapeutics.

Evolutionary trajectory of receptor binding specificity and promiscuity of the spike protein of SARS-CoV-2.

SARS-CoV-2 infects cells by attachment to its receptor-the angiotensin converting enzyme 2 (ACE2). Regardless of the wealth of structural data, little is known about the physicochemical mechanism of interactions of the viral spike (S) protein with ACE2 and how this mechanism has evolved during the pandemic. Here, we applied experimental and computational approaches to characterize the molecular interaction of S proteins from SARS-CoV-2 variants of concern (VOC). Data on kinetics, activation-, and equilibrium thermodynamics of binding of the receptor binding domain (RBD) from VOC with ACE2 as well as data from computational protein electrostatics revealed a profound remodeling of the physicochemical characteristics of the interaction during the evolution. Thus, as compared to RBDs from Wuhan strain and other VOC, Omicron RBD presented as a unique protein in terms of conformational dynamics and types of non-covalent forces driving the complex formation with ACE2. Viral evolution resulted in a restriction of the RBD structural dynamics, and a shift to a major role of polar forces for ACE2 binding. Further, we investigated how the reshaping of the physicochemical characteristics of interaction affects the binding specificity of S proteins. Data from various binding assays revealed that SARS-CoV-2 Wuhan and Omicron RBDs manifest capacity for promiscuous recognition of unrelated human proteins, but they harbor distinct reactivity patterns. These findings might contribute for mechanistic understanding of the viral tropism and capacity to evade immune responses during evolution.

The protocol of CUT&Tag for metabolic tissue cells.

Cleavage under target and tagment (CUT&Tag) is a technology that utilizes the fusion protein of Tn5 transposase and protein A/G which can guide Tn5 enzyme to the antibody bound to target protein and cleave the chromatin regions adjacent to target protein. Chromatin libraries are then tagged and sequenced by the high-throughput sequencing to obtain chromatin information at specific sites or protein binding locations. CUT&Tag technology plays an important role in the research of DNA and protein interactions. It can be used to understand the modifications of histone and the bindings of transcription factors. Compared with the traditional chromatin immunoprecipitation-sequencing (ChIP-seq) technology, the CUT&Tag has the strengths of high signal-to-noise ratio, good repeatability, short experimental period, and low cell input. It shows great advantages in early embryonic development, stem cells, cancer, epigenetics and other research fields. In this article, we described the protocol of CUT&Tag for metabolic tissue cells (mouse primary islet cells), to provide an epigenetic method for studying metabolic cells.

66P DDB2 influences epithelial-to-mesenchymal transition and sensitivity to chemotherapy in pancreatic ductal adenocarcinoma cells

Damage specific DNA binding protein 2 (DDB2) is originally implied in the recognition of ultraviolet-induced DNA damage and the initiation of nucleotide excision repair (NER) process. This protein also demonstrated dual roles in several cancers, acting either as an oncogene or a tumor suppressor gene depending on cancer localization. In this study, we investigated the unresolved role of DDB2 in pancreatic ductal adenocarcinoma (PDAC).

Interplay of receptor-ligand binding and lipid domain formation during cell adhesion.

Cell adhesion involved in biological processes such as cell migration, immune responses, and cancer metastasis, is mediated by the specific binding of receptor and ligand proteins. Some of these proteins exhibit affinity for nanoscale lipid clusters in cell membranes. A key question is how these nanoscale lipid clusters influence and react to the receptor-ligand binding during cell adhesion. In this article, we review recent computational studies that shed new light on the interplay of the receptor-ligand binding and the formation of lipid domains in adhering membranes. These studies indicate that the receptor-ligand binding promotes coalescence of lipid clusters into mesoscale domains, which, in turn, enhances both the affinity and cooperativity of the receptor-ligand binding in cell-cell adhesion with mobile ligands. In contrast, in the case of cell-extracellular matrix adhesion with immobile ligands, the receptor-ligand binding and the lipid cluster coalescence can be correlated or anti-correlated, depending strongly on the ligand distribution. These findings deepen our understanding of correlations between cell adhesion and membrane heterogeneities.

Preexisting antibodies targeting SARS-CoV-2 S2 cross-react with commensal gut bacteria and impact COVID-19 vaccine induced immunity

ABSTRACT The origins of preexisting SARS-CoV-2 cross-reactive antibodies and their potential impacts on vaccine efficacy have not been fully clarified. In this study, we demonstrated that S2 was the prevailing target of the preexisting S protein cross-reactive antibodies in both healthy human and SPF mice. A dominant antibody epitope was identified on the connector domain of S2 (1147-SFKEELDKYFKNHT-1160, P144), which could be recognized by preexisting antibodies in both human and mouse. Through metagenomic sequencing and fecal bacteria transplant, we demonstrated that the generation of S2 cross-reactive antibodies was associated with commensal gut bacteria. Furthermore, six P144 reactive monoclonal antibodies were isolated from naïve SPF mice and were proven to cross-react with commensal gut bacteria collected from both human and mouse. A variety of cross-reactive microbial proteins were identified using LC-MS, of which E. coli derived HSP60 and HSP70 proteins were confirmed to be able to bind to one of the isolated monoclonal antibodies. Mice with high levels of preexisting S2 cross-reactive antibodies mounted higher S protein specific binding antibodies, especially against S2, after being immunized with a SARS-CoV-2 S DNA vaccine. Similarly, we found that levels of preexisting S2 and P144-specific antibodies correlated positively with RBD binding antibody titers after two doses of inactivated SARS-CoV-2 vaccination in human. Collectively, our study revealed an alternative origin of preexisting S2-targeted antibodies and disclosed a previously neglected aspect of the impact of gut microbiota on host anti-SARS-CoV-2 immunity.

The association of testis-specific hTAF7L gene variants with idiopathic azoospermic and severe oligozoospermic male infertility.

Spermatogenesis is regulated by complex tissue specific gene expression in the testis to achieve normal male fertility. X-chromosome specific TATA binding protein (TBP)-associated factor 7L (hTAF7L) is one of the transcriptional regulator genes considered essential for spermatogenesis. The aim of this study was to evaluate the role of variants/mutations in the testis-specific hTAF7L gene in non-obstructive azoospermia and severe oligozoospermia male infertility. We studied 156 idiopathic non-obstructive azoospermic, severe oligozoospermic infertile males and 50 fertile proven controls. Infertile males and control subjects were genotyped for variants of the hTAF7L gene using polymerase chain reaction and a direct Sanger sequencing approach. The odds ratio evaluated the association of hTAF7L gene variants with idiopathic male infertility. The variants found in the hTAF7L gene were subjected to an in-silico analysis study. In infertile study subjects, we observed 11 single base pair nucleotide changes at various exons and three frameshift variants at exon 10 in the hTAF7L gene. We also found more than one variant in some non-obstructive azoospermia and severe oligozoospermia infertile males along with control subjects. All these variants changed the amino acid sequences in the hTAF7L gene. However, similar changes were also seen in fertile subjects, and the differences were not statistically significant. In-silico tools also predicted that the variants were likely to be benign. The variants in cDNA of the hTAF7L gene were typical SNPs. It is found that the hTAF7L gene is highly polymorphic and these missense variants are not directly associated with male infertility. However, we feel that more studies are needed to elucidate the role of multiple variants of the hTAF7L gene in the process of normal spermatogenesis.

Organic Afterglow Emulsions Exhibiting 2.4 s Phosphorescence Lifetimes and Specific Protein Binding Property

AbstractAqueous afterglow dispersions display promising biomedical applications due to their long‐lived excited states, whereas it is a common problem in the research field that processing and transferring solid‐state afterglow materials into aqueous medium can cause significant drop and even complete loss of their afterglow properties. Here, aqueous afterglow dispersions are fabricated from liquid precursors by dopant–matrix strategy and emulsion polymerization, which avoids the processing procedures on solid‐state materials and achieves 2.4 s long phosphorescence lifetimes in aqueous medium. Luminescent difluoroboron β‐diketonate dopants are first designed with small rate constant of phosphorescence decay and then dissolved in monomers. After emulsion polymerization, the as‐formed polymer latex particles not only assist dopants’ intersystem crossing but also provide a glassy microenvironment to largely suppress nonradiative decay of dopants’ triplets, leading to in situ formation of aqueous afterglow dispersions. This provides a robust method for constructing afterglow emulsions with long‐term colloidal stability, relatively high solid contents, tunable sizes, and diverse compositions. Through emulsion copolymerization, afterglow emulsions decorated with specific ligands are obtained to exhibit specific protein binding properties. Such decorated afterglow emulsions address the issue that afterglow materials lack biorecognition property and can pave the way for their applications in immunoassay and immunoimaging.

The adipocyte microenvironment and cancer.

Many epithelial tumors grow in the vicinity of or metastasize to adipose tissue. As tumors develop, crosstalk between adipose tissue and cancer cells leads to changes in adipocyte function and paracrine signaling, promoting a microenvironment that supports tumor growth. Over the last decade, it became clear that tumor cells co-opt adipocytes in the tumor microenvironment, converting them into cancer-associated adipocytes (CAA). As adipocytes and cancer cells engage, a metabolic symbiosis ensues that is driven by bi-directional signaling. Many cancers (colon, breast, prostate, lung, ovarian cancer, and hematologic malignancies) stimulate lipolysis in adipocytes, followed by the uptake of fatty acids (FA) from the surrounding adipose tissue. The FA enters the cancer cell through specific fatty acid receptors and binding proteins (e.g., CD36, FATP1) and are used for membrane synthesis, energy metabolism (β-oxidation), or lipid-derived cell signaling molecules (derivatives of arachidonic and linolenic acid). Therefore, blocking adipocyte-derived lipid uptake or lipid-associated metabolic pathways in cancer cells, either with a single agent or in combination with standard of care chemotherapy, might prove to be an effective strategy against cancers that grow in lipid-rich tumor microenvironments.

Evolutionary conserved relocation of chromatin remodeling complexes to the mitotic apparatus

BackgroundATP-dependent chromatin remodeling complexes are multi-protein machines highly conserved across eukaryotic genomes. They control sliding and displacing of the nucleosomes, modulating histone-DNA interactions and making nucleosomal DNA more accessible to specific binding proteins during replication, transcription, and DNA repair, which are processes involved in cell division. The SRCAP and p400/Tip60 chromatin remodeling complexes in humans and the related Drosophila Tip60 complex belong to the evolutionary conserved INO80 family, whose main function is promoting the exchange of canonical histone H2A with the histone variant H2A in different eukaryotic species. Some subunits of these complexes were additionally shown to relocate to the mitotic apparatus and proposed to play direct roles in cell division in human cells. However, whether this phenomenon reflects a more general function of remodeling complex components and its evolutionary conservation remains unexplored.ResultsWe have combined cell biology, reverse genetics, and biochemical approaches to study the subcellular distribution of a number of subunits belonging to the SRCAP and p400/Tip60 complexes and assess their involvement during cell division progression in HeLa cells. Interestingly, beyond their canonical chromatin localization, the subunits under investigation accumulate at different sites of the mitotic apparatus (centrosomes, spindle, and midbody), with their depletion yielding an array of aberrant outcomes of mitosis and cytokinesis, thus causing genomic instability. Importantly, this behavior was conserved by the Drosophila melanogaster orthologs tested, despite the evolutionary divergence between fly and humans has been estimated at approximately 780 million years ago.ConclusionsOverall, our results support the existence of evolutionarily conserved diverse roles of chromatin remodeling complexes, whereby subunits of the SRCAP and p400/Tip60 complexes relocate from the interphase chromatin to the mitotic apparatus, playing moonlighting functions required for proper execution of cell division.

Computational Assessment of Protein-Protein Binding Specificity within a Family of Synaptic Surface Receptors.

Atomic-level information is essential to explain the formation of specific protein complexes in terms of structure and dynamics. The set of Dpr and DIP proteins, which play a key role in the neuromorphogenesis in the nervous system of Drosophila melanogaster, offer a rich paradigm to learn about protein-protein recognition. Many members of the DIP subfamily cross-react with several members of the Dpr family and vice versa. While there exists a total of 231 possible Dpr-DIP heterodimer complexes from the 21 Dpr and 11 DIP proteins, only 57 "cognate" pairs have been detected by surface plasmon resonance (SPR) experiments, suggesting that the remaining 174 pairs have low or unreliable binding affinity. Our goal is to assess the performance of computational approaches to characterize the global set of interactions between Dpr and DIP proteins and identify the specificity of binding between each DIP with their corresponding Dpr binding partners. In addition, we aim to characterize how mutations influence the specificity of the binding interaction. In this work, a wide range of knowledge-based and physics-based approaches are utilized, including mutual information, linear discriminant analysis, homology modeling, molecular dynamics simulations, Poisson-Boltzmann continuum electrostatics calculations, and alchemical free energy perturbation to decipher the origin of binding specificity of the Dpr-DIP complexes examined. Ultimately, the results show that those two broad strategies are complementary, with different strengths and limitations. Biological inter-relations are more clearly revealed through knowledge-based approaches combining evolutionary and structural features, the molecular determinants controlling binding specificity can be predicted accurately with physics-based approaches based on atomic models.

DDX18 prevents R-loop-induced DNA damage and genome instability via PARP-1.

R loops occur frequently in genomes and contribute to fundamental biological processes at multiple levels. Consequently, understanding the molecular and cellular biology of R loops has become an emerging area of research. Here, it is shown that poly(ADP-ribose) polymerase-1 (PARP-1) can mediate the association of DDX18, a putative RNA helicase, with R loops thereby modulating R-loop homeostasis in endogenous R-loop-prone and DNA lesion regions. DDX18 depletion results in aberrant endogenous R-loop accumulation, which leads to DNA-replication defects. In addition, DDX18 depletion renders cells more sensitive to DNA-damaging agents and reduces RPA32 and RAD51 foci formation in response to irradiation. Notably, DDX18 depletion leads to γH2AX accumulation and genome instability, and RNase H1 overexpression rescues all the DNA-repair defects caused by DDX18 depletion. Taken together, these studies uncover a function of DDX18 in R-loop-mediated events and suggest a role for PARP-1 in mediating the binding of specific DDX-family proteins with R loops in cells.

Theoretical insights into water network of B-DNA duplex with Watson-Crick and Hoogsteen base pairing geometries

Solvation is essential for the proper structure and functioning of biomolecules. It has a major role in establishing the B-form as the dominant structure of DNA and strongly affects its biological functions. In our genome, two modes of base pairing (Watson-Crick (WC) and Hoogsteen (HG)) exist in equilibrium and are related to the functional properties of the DNA including ligand recognition and gene regulation. Here, we use molecular dynamics simulation along with topological analysis of the H-bond network to investigate the hydration patterns of DNA structure with WC and HG base pairings. We showed that DNA most significantly disturbs the H-bonding properties of the first hydration shell and slightly the second shell compared to bulk water and that the water network is considerably different in the minor and major grooves. We found that HG base pairing leads to increased solvation of the major groove and a less ordered water structure in the minor groove accompanied with increasing hydrophobicity of the minor groove which could also contribute to the site recognition of specific DNA binding proteins. Our newly developed method to selectively study the solvation of the minor and major grooves opens the door to study in finer details the binding between various form of DNA and different ligands such drugs, RNA and proteins.

Molecular dynamics-based descriptors of 3-O-Sulfated Heparan sulfate as contributors of protein binding specificity

Glycosaminoglycans are linear periodic and anionic polysaccharides found in the extracellular matrix, involved in a range of key biochemical processes as a result of their interactions with a variety of protein partners. Due to the template-less synthesis, high flexibility and charge of GAGs, as well as the multipose binding of GAG ligands to receptors, the specificity of GAG-protein interactions can be difficult to elucidate. In this study we propose a set of MD-based descriptors of unbound Heparan Sulfate hexasaccharides that can be used to characterize GAGs and explain their binding affinity to a set of protein receptors. With the help of experimental data on GAG-protein binding affinity, we were able to further characterize the nature of this interaction in addition to providing a basis for predictor functions of GAG-protein binding specificity.

A pathogenic mechanism associated with myopathies and structural birth defects involves TPM2-directed myogenesis.

Nemaline myopathy (NM) is the most common congenital myopathy, characterized by extreme weakness of the respiratory, limb, and facial muscles. Pathogenic variants in Tropomyosin 2 (TPM2), which encodes a skeletal muscle–specific actin binding protein essential for sarcomere function, cause a spectrum of musculoskeletal disorders that include NM as well as cap myopathy, congenital fiber type disproportion, and distal arthrogryposis (DA). The in vivo pathomechanisms underlying TPM2-related disorders are unknown, so we expressed a series of dominant, pathogenic TPM2 variants in Drosophila embryos and found 4 variants significantly affected muscle development and muscle function. Transient overexpression of the 4 variants also disrupted the morphogenesis of mouse myotubes in vitro and negatively affected zebrafish muscle development in vivo. We used transient overexpression assays in zebrafish to characterize 2 potentially novel TPM2 variants and 1 recurring variant that we identified in patients with DA (V129A, E139K, A155T, respectively) and found these variants caused musculoskeletal defects similar to those of known pathogenic variants. The consistency of musculoskeletal phenotypes in our assays correlated with the severity of clinical phenotypes observed in our patients with DA, suggesting disrupted myogenesis is a potentially novel pathomechanism of TPM2 disorders and that our myogenic assays can predict the clinical severity of TPM2 variants.

Abstract 2640: Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism

Abstract Patients with late-stage and human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) continue to have a very poor prognosis. The development of more effective novel therapies that improve overall survival and overcome drug resistance is an urgent priority. We, and others, have reported that HNSCC tumors significantly overexpress NEDD8 and exhibit high sensitivity to the first-in-class NEDD8 activating enzyme (NAE) inhibitor pevonedistat. Here, we report that disruption of NEDD8-mediated protein turnover with pevonedistat dramatically augments cisplatin-induced DNA damage and apoptosis in HNSCC models. Further analysis revealed that the specific pevonedistat target, CUL4A, played an essential role in driving the synergy of the pevonedistat and cisplatin combination. Targeted inhibition of CUL4A resulted in significant downregulation in Damage Specific DNA binding protein 2 (DDB2), a DNA-damage recognition protein that promotes nucleotide excision repair and resistance to cisplatin. Silencing of CUL4A enhanced cisplatin-induced DNA damage and apoptosis in a manner similar to that of pevonedistat demonstrating that targeted inhibition of CUL4A may be a novel approach to augment cisplatin therapy. Administration of pevonedistat to mice bearing HNSCC tumors significantly decreased DDB2 expression in tumor cells, increased DNA damage and potently enhanced the activity of cisplatin to yield tumor regression and long-term survival of all animals. Our findings provide strong rationale for clinical investigation of CUL4A inhibition with pevonedistat as a novel strategy to augment the efficacy of cisplatin therapy for patients with HNSCC and identify loss of DDB2 as a key pharmacodynamic mediator controlling sensitivity to this regimen. Citation Format: Trace M. Jones, Claudia M. Espitia, Aikseng Ooi, Julie E. Bauman, Jennifer S. Carew, Steffan T. Nawrocki. Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2640.

Construction of ssDNA-Attached LR-Chimera Involving Z-DNA for ZBP1 Binding Analysis.

The binding of proteins to Z-DNA is hard to analyze, especially for short non-modified DNA, because it is easily transferred to B-DNA. Here, by the hybridization of a larger circular single-stranded DNA (ssDNA) with a smaller one, an LR-chimera (involving a left-handed part and a right-handed one) with an ssDNA loop is produced. The circular ssDNAs are prepared by the hybridization of two ssDNA fragments to form two nicks, followed by nick sealing with T4 DNA ligase. No splint (a scaffold DNA for circularizing ssDNA) is required, and no polymeric byproducts are produced. The ssDNA loop on the LR-chimera can be used to attach it with other molecules by hybridization with another ssDNA. The gel shift binding assay with Z-DNA specific binding antibody (Z22) or Z-DNA binding protein 1 (ZBP1) shows that stable Z-DNA can form under physiological ionic conditions even when the extra ssDNA part is present. Concretely, a 5′-terminal biotin-modified DNA oligonucleotide complementary to the ssDNA loop on the LR-chimera is used to attach it on the surface of a biosensor inlaid with streptavidin molecules, and the binding constant of ZBP1 with Z-DNA is analyzed by BLI (bio-layer interferometry). This approach is convenient for quantitatively analyzing the binding dynamics of Z-DNA with other molecules.

Narazaciclib’s kinase inhibitory activity is differentiated from approved CDK4/6 inhibitors in preclinical models.

e15096 Background: Despite clinical benefit of CDK4/6 inhibitors in HR+Her2-mBC, patients progress. Neutropenia and diarrhea are safety concerns. Narazaciclib (ON 123300) is a multi-targeted kinase inhibitor of CDK4/6, ARK5 (NUAK1), CSF1R, and c-Kit at low nM concentrations designed to enhance efficacy and safety. Narazaciclib is in Ph I trials; NCT04739293 and CXHL1900340; in different regimens. Treatment of tumor cell lines with narazaciclib induces G1; G2 arrest and apoptosis. Mouse models suggest narazaciclib causes less neutropenia than palbociclib (palbo). Since signaling pathways are affected, we explored the activity of narazaciclib in direct comparisons with abemaciclib (abe), palbo and ribociclib (ribo) using in vitro and cell based assays. Methods: Comparison of narazaciclib’s in vitro IC50 profile to abe, palbo and ribo was studied against a panel of 370 kinases (HotSpot). Kd values were determined by KINOMEscan. Intracellular IC50 kinase values were determined by NanoBret technology. Protein specific binding of narazaciclib and palbo was investigated by Cellular Thermal Shift Assay (CETSA). To investigate narazaciclib’s effect on signaling pathways, integrative Inferred Kinase Activity (INKA) analysis was performed. Results: Narazaciclib and abe were found to be the most promiscuous in vitro kinase inhibitors and ribo the most specific. Abe and narazaciclib had similar profiles against the CDK family members. Kd values of CDK4/cyclinD1 binding show similar trends; abe (0.08 nM), narazaciclib (0.18 nM), palbo (0.75 nM) and ribo (1.3 nM). Narazaciclib and abe displayed nM activity against CDK2/cyclinA. The IC50 values against GSK3β, whose inhibition putatively causes diarrhea, was 374 nM for narazaciclib and 13 nM for abe. Although narazaciclib displayed nM IC50 values in the in vitro assays against many CDKs, narazaciclib was very specific in cellular kinase assays with highest activity against CDK4/6, CSF1R and NUAK1. CETSA-MS revealed more potential targets engaged by narazaciclib compared to palbo in both lysates and intact cells such as CHEK1, AAK1, BMP2K, GSK3α and GSK3β, but did not identify binding to NUAK1 or CSF1R. INKA analysis demonstrated that narazaciclib induced unique deregulated phosphorylation compared to palbo, including BUB1 (highly expressed in TNBC), NUAK1, CAMK2D, CDK16 and ULK1. Inhibition of autophagy, both at early and late stages, may sensitize cancer cells to narazaciclib and induce irreversible cell proliferation inhibition, providing a novel therapeutic approach. Conclusions: These studies identify important differences generated from assay models studying kinase inhibition, binding and pathway engagement and will guide future studies with narazaciclib targeting specific kinase driven tumors with the potential for improved safety. These preclinical data await confirmation from current and future clinical trials.