Impact of Drug Conjugation on Thermal and Metabolic Stabilities of Aglycosylated and N-Glycosylated Antibodies.

N-linked glycosylation is one of the most common and complex posttranslational modifications that govern the biological functions and physicochemical properties of therapeutic antibodies. We evaluated thermal and metabolic stabilities of antibody-drug conjugates (ADCs) with payloads attached to the C'E loop in the immunoglobulin G (IgG) Fc CH2 domain, comparing the glycosylated and aglycosylated Fc ADC variants. Our study revealed that introduction of small-molecule drugs into an aglycosylated antibody can compensate for thermal destabilization originating from structural distortions caused by elimination of N-linked glycans. Depending on the conjugation site, glycans had both positive and negative effects on plasma stability of ADCs. The findings highlight the importance of consideration for selection of conjugation site to achieve desirable physicochemical properties and plasma stability.

MCC950/CRID3 potently targets the NACHT domain of wild-type NLRP3 but not disease-associated mutants for inflammasome inhibition.

The nucleotide-binding-domain (NBD)-and leucine-rich repeat (LRR)-containing (NLR) family, pyrin-domain-containing 3 (NLRP3) inflammasome drives pathological inflammation in a suite of autoimmune, metabolic, malignant, and neurodegenerative diseases. Additionally, NLRP3 gain-of-function point mutations cause systemic periodic fever syndromes that are collectively known as cryopyrin-associated periodic syndrome (CAPS). There is significant interest in the discovery and development of diarylsulfonylurea Cytokine Release Inhibitory Drugs (CRIDs) such as MCC950/CRID3, a potent and selective inhibitor of the NLRP3 inflammasome pathway, for the treatment of CAPS and other diseases. However, drug discovery efforts have been constrained by the lack of insight into the molecular target and mechanism by which these CRIDs inhibit the NLRP3 inflammasome pathway. Here, we show that the NAIP, CIITA, HET-E, and TP1 (NACHT) domain of NLRP3 is the molecular target of diarylsulfonylurea inhibitors. Interestingly, we find photoaffinity labeling (PAL) of the NACHT domain requires an intact (d)ATP-binding pocket and is substantially reduced for most CAPS-associated NLRP3 mutants. In concordance with this finding, MCC950/CRID3 failed to inhibit NLRP3-driven inflammatory pathology in two mouse models of CAPS. Moreover, it abolished circulating levels of interleukin (IL)-1ß and IL-18 in lipopolysaccharide (LPS)-challenged wild-type mice but not in Nlrp3L351P knock-in mice and ex vivo-stimulated mutant macrophages. These results identify wild-type NLRP3 as the molecular target of MCC950/CRID3 and show that CAPS-related NLRP3 mutants escape efficient MCC950/CRID3 inhibition. Collectively, this work suggests that MCC950/CRID3-based therapies may effectively treat inflammation driven by wild-type NLRP3 but not CAPS-associated mutants.

Correction: MCC950/CRID3 potently targets the NACHT domain of wild-type NLRP3 but not disease-associated mutants for inflammasome inhibition.

[This corrects the article DOI: 10.1371/journal.pbio.3000354.].

Primary Amine Tethered Small Molecules Promote the Degradation of X-Linked Inhibitor of Apoptosis Protein.

We hypothesized that the proximity-driven ubiquitylation of E3-interacting small molecules could affect the degradation of E3 ubiquitin ligases. A series of XIAP BIR2 domain-binding small molecules was modified to append a nucleophilic primary amine. This modification transforms XIAP binders into inducers of XIAP degradation. The degradation of XIAP is E1- and proteasome-dependent, dependent on the ligase function of XIAP, and is rescued by subtle modifications of the small molecule that would obviate ubiquitylation. We demonstrate in vitro ubiquitylation of the small molecule that is dependent on its interaction with XIAP. Taken together, these results demonstrate the designed ubiquitylation of an engineered small molecule and a novel approach for the degradation of E3 ubiquitin ligases.

Automated and Faster Affinity Capture Method for Biotransformation Assessment of Site-Specific Antibody Drug Conjugates.

Traditionally the biotransformation of antibody drug conjugates (ADCs) has been evaluated by affinity capture on streptavidin magnetic beads coated with a biotinylated capture reagent. To reduce the complexity of the analyte, the affinity captured ADCs are digested with enzymes ("on-bead" or after elution), and/or interchain disulfides are reduced to generate LC and HC fragments prior to mass spectrometry analysis. The "on-bead" enzymatic digestion with IdeS and PNGase F is not efficient and requires longer incubation times to achieve complete Fc and N-glycan removal. This results in a prolonged sample preparation time (7-18 h) and is not suitable for labile ADCs due to the possibility of assay-induced artifacts. To address these challenges, we developed an affinity capture method, where the ADCs are first captured onto streptavidin cartridges coated with a biotinylated generic capture reagent, followed by a 15 min "on-cartridge" digestion with IdeS or PNGase F. The ADCs are then eluted and directly analyzed by LC-HRMS. This method was successfully applied for the biotransformation assessment of site-specific ADCs with payload conjugated on the Fab or Fc. The reduced complexity of the analyte (Fc and N-glycan removal) combined with HRMS enabled sensitive and accurate identification of minor mass change catabolites and changes in the DAR distribution. This automated cartridge-based affinity capture method is fast with a total sample preparation time of less than 4 h (hands-on time of less than 1 h) and can be utilized for any human mAb/ADC independent of isotype (IgG1, IgG2, and IgG4).

Universal Affinity Capture Liquid Chromatography-Mass Spectrometry Assay for Evaluation of Biotransformation of Site-Specific Antibody Drug Conjugates in Preclinical Studies.

Antibody drug conjugates (ADCs) can undergo in vivo biotransformation (e.g., payload metabolism, deconjugation) leading to reduced or complete loss of activity. The location/site of conjugation of payload-linker can have an effect on ADC stability and hence needs to be carefully optimized. Affinity capture LC-MS of intact ADCs or ADC subfragments has been extensively used to evaluate ADC biotransformation. However, the current methods have certain limitations such as the requirement of specific capture reagents, limited mass resolution of low mass change metabolites, low sensitivity, and use of capillary or nanoflow LC-MS. To address these challenges, we developed a generic affinity capture LC-MS assay that can be utilized to evaluate the biotransformation of any site-specific ADC independent of antibody type and site of conjugation (Fab and Fc) in preclinical studies. The method involves a combination of some or all of these steps: (1) "mono capture" or "dual capture" of ADCs from serum with streptavidin magnetic beads coated with a generic biotinylated antihuman capture reagent, (2) "on-bead" digestion with IdeS and/or PNGase F, and (3) reduction of interchain disulfide bonds to generate ∼25 kDa ADC subfragments, which are finally analyzed by LC-HRMS on a TOF mass spectrometer. The advantages of this method are that it can be performed using commercially available generic reagents and requires sample preparation time of less than 7 h. Furthermore, by reducing the size of intact ADC (∼150 kDa) to subfragments (∼25 kDa), the identification of conjugated payload and its metabolites can be achieved with excellent sensitivity and resolution (hydrolysis and other small mass change metabolites). This method was successfully applied to evaluate the in vitro and in vivo biotransformation of ADCs conjugated at different sites (LC, HC-Fab, and HC-Fc) with various classes of payload-linkers.

High-Throughput Platform to Identify Antibody Conjugation Sites from Antibody-Drug Conjugate Libraries.

Antibody-drug conjugates (ADCs) are a therapeutic modality that traditionally enable the targeted delivery of highly potent cytotoxic agents to specific cells such as tumor cells. More recently, antibodies have been used to deliver molecules such as antibiotics, antigens, and adjuvants to bacteria or specific immune cell subsets. Site-directed mutagenesis of proteins permits more precise control over the site and stoichiometry of their conjugation, giving rise to homogeneous chemically defined ADCs. Identification of favorable sites for conjugation in antibodies is essential as reaction efficiency and product stability are influenced by the tertiary structure of immunoglobulin G (IgG). Current methods to evaluate potential conjugation sites are time-consuming and labor intensive, involving multistep processes for individually produced reactions. Here, we describe a highly efficient method for identification of conjugatable genetic variants by analyzing pooled ADC libraries using mass spectrometry. This approach provides a versatile platform to rapidly uncover new conjugation sites for site-specific ADCs.

Tbx16 regulates hox gene activation in mesodermal progenitor cells.

The transcription factor T-box 16 (Tbx16, or Spadetail) is an essential regulator of paraxial mesoderm development in zebrafish (Danio rerio). Mesodermal progenitor cells (MPCs) fail to differentiate into trunk somites in tbx16 mutants and instead accumulate within the tailbud in an immature state. However, the mechanisms by which Tbx16 controls mesoderm patterning have remained enigmatic. We describe here the use of photoactivatable morpholino oligonucleotides to determine the Tbx16 transcriptome in MPCs. We identified 124 Tbx16-regulated genes that were expressed in zebrafish gastrulae, including several developmental signaling proteins and regulators of gastrulation, myogenesis and somitogenesis. Unexpectedly, we observed that a loss of Tbx16 function precociously activated posterior hox genes in MPCs, and overexpression of a single posterior hox gene was sufficient to disrupt MPC migration. Our studies support a model in which Tbx16 regulates the timing of collinear hox gene activation to coordinate the anterior-posterior fates and positions of paraxial MPCs.

The BAH domain of ORC1 links H4K20me2 to DNA replication licensing and Meier-Gorlin syndrome.

The recognition of distinctly modified histones by specialized 'effector' proteins constitutes a key mechanism for transducing molecular events at chromatin to biological outcomes. Effector proteins influence DNA-templated processes, including transcription, DNA recombination and DNA repair; however, no effector functions have yet been identified within the mammalian machinery that regulate DNA replication. Here we show that ORC1--a component of ORC (origin of replication complex), which mediates pre-DNA replication licensing--contains a bromo adjacent homology (BAH) domain that specifically recognizes histone H4 dimethylated at lysine 20 (H4K20me2). Recognition of H4K20me2 is a property common to BAH domains present within diverse metazoan ORC1 proteins. Structural studies reveal that the specificity of the BAH domain for H4K20me2 is mediated by a dynamic aromatic dimethyl-lysine-binding cage and multiple intermolecular contacts involving the bound peptide. H4K20me2 is enriched at replication origins, and abrogating ORC1 recognition of H4K20me2 in cells impairs ORC1 occupancy at replication origins, ORC chromatin loading and cell-cycle progression. Mutation of the ORC1 BAH domain has been implicated in the aetiology of Meier-Gorlin syndrome (MGS), a form of primordial dwarfism, and ORC1 depletion in zebrafish results in an MGS-like phenotype. We find that wild-type human ORC1, but not ORC1-H4K20me2-binding mutants, rescues the growth retardation of orc1 morphants. Moreover, zebrafish depleted of H4K20me2 have diminished body size, mirroring the phenotype of orc1 morphants. Together, our results identify the BAH domain as a novel methyl-lysine-binding module, thereby establishing the first direct link between histone methylation and the metazoan DNA replication machinery, and defining a pivotal aetiological role for the canonical H4K20me2 mark, via ORC1, in primordial dwarfism.

Post-transcriptional mechanisms contribute to Etv2 repression during vascular development.

etv2 is an endothelial-specific ETS transcription factor that is essential for vascular differentiation and morphogenesis in vertebrates. While recent data suggest that Etv2 is dynamically regulated during vascular development, little is known about the mechanisms involved in this process. Here, we find that etv2 transcript and protein expression are highly dynamic during zebrafish vascular development, with both apparent during early somitogenesis and subsequently down-regulated as development proceeds. Inducible knockdown of Etv2 in zebrafish embryos prior to mid-somitogenesis stages, but not later, caused severe vascular defects, suggesting a specific role in early commitment of lateral mesoderm to the endothelial linage. Accordingly, Etv2-overexpressing cells showed an enhanced ability to commit to endothelial lineages in mosaic embryos. We further find that the etv2 3' untranslated region (UTR) is capable of repressing an endothelial autonomous transgene and contains binding sites for members of the let-7 family of microRNAs. Ectopic expression of let-7a could repress the etv2 3'UTR in sensor assays and was also able to block endogenous Etv2 protein expression, leading to concomitant reduction of endothelial genes. Finally, we observed that Etv2 protein levels persisted in maternal-zygotic dicer1 mutant embryos, suggesting that microRNAs contribute to its repression during vascular development. Taken together, our results suggest that etv2 acts during early development to specify endothelial lineages and is then down-regulated, in part through post-transcriptional repression by microRNAs, to allow normal vascular development.

Sequential gene silencing using wavelength-selective caged morpholino oligonucleotides.

Spectrally differentiated caged morpholino oligonucleotides (cMOs) and wavelength-selective illumination have been used to sequentially inactivate organismal gene function. The efficacy of these reverse-genetic chemical probes has been demonstrated in zebrafish embryos, and these reagents have been employed to examine the mechanisms of mesoderm patterning.

Synthetic molecules that protect cells from anoikis and their use in cell transplantation.

One of the major problems encountered in cell transplantation is the low level of survival of transplanted cells due to detachment-induced apoptosis, called anoikis. The present study reports on the chemical synthesis and biological evaluation of water-soluble molecules that protect suspended cells from anoikis. The synthetic molecules bind to and induce clusters of integrins and heparan-sulfate-bound syndecans, two classes of receptors that are important for extracellular matrix-mediated cell survival. Molecular biological analysis indicates that such molecules prolong the survival of suspended NIH3T3 cells, at least in part, by promoting clustering of syndecan-4 and integrin ß1 on the cell surface, leading to the activation of small GTPase Rac-1 and Akt. In vivo experiments using animal disease models demonstrated the ability of the molecules to improve cell engraftment. The cluster-inducing molecules may provide a starting point for the design of new synthetic tools for cell-based therapy.

Small-molecule-induced clustering of heparan sulfate promotes cell adhesion.

Adhesamine is an organic small molecule that promotes adhesion and growth of cultured human cells by binding selectively to heparan sulfate on the cell surface. The present study combined chemical, physicochemical, and cell biological experiments, using adhesamine and its analogues, to examine the mechanism by which this dumbbell-shaped, non-peptidic molecule induces physiologically relevant cell adhesion. The results suggest that multiple adhesamine molecules cooperatively bind to heparan sulfate and induce its assembly, promoting clustering of heparan sulfate-bound syndecan-4 on the cell surface. A pilot study showed that adhesamine improved the viability and attachment of transplanted cells in mice. Further studies of adhesamine and other small molecules could lead to the design of assembly-inducing molecules for use in cell biology and cell therapy.

Ternary complex dissociation kinetics contribute to mutant-selective EGFR degradation.

Targeted degradation of proteins by chimeric heterobifunctional degraders has emerged as a major drug discovery paradigm. Despite the increased interest in this approach, the criteria dictating target protein degradation by a degrader remain poorly understood, and potent target engagement by a degrader does not strongly correlate with target degradation. In this study, we present the biochemical characterization of an epidermal growth factor receptor (EGFR) degrader that potently binds both wild-type and mutant EGFR, but only degrades EGFR mutant variants. Mechanistic studies reveal that ternary complex half-life strongly correlates with processive ubiquitination with purified components and mutant-selective degradation in cells. We present cryoelectron microscopy and hydrogen-deuterium exchange mass spectroscopy data on wild-type and mutant EGFR ternary complexes, which demonstrate that potent target degradation can be achieved in the absence of stable compound-induced protein-protein interactions. These results highlight the importance of considering target conformation during degrader development as well as leveraging heterobifunctional ligand binding kinetics to achieve robust target degradation.

Cyclic caged morpholinos: conformationally gated probes of embryonic gene function.

Feeling a bit cagey: morpholino-based antisense reagents have been caged through oligonucleotide cyclization, enabling photocontrol of gene expression in zebrafish embryos and larvae. Using these reagents, the timing of exocrine cell fate commitment in the developing pancreas has been examined.

Identifying transcriptional programs underlying cancer drug response with TraCe-seq.

Genetic and non-genetic heterogeneity within cancer cell populations represent major challenges to anticancer therapies. We currently lack robust methods to determine how preexisting and adaptive features affect cellular responses to therapies. Here, by conducting clonal fitness mapping and transcriptional characterization using expressed barcodes and single-cell RNA sequencing (scRNA-seq), we have developed tracking differential clonal response by scRNA-seq (TraCe-seq). TraCe-seq is a method that captures at clonal resolution the origin, fate and differential early adaptive transcriptional programs of cells in a complex population in response to distinct treatments. We used TraCe-seq to benchmark how next-generation dual epidermal growth factor receptor (EGFR) inhibitor-degraders compare to standard EGFR kinase inhibitors in EGFR-mutant lung cancer cells. We identified a loss of antigrowth activity associated with targeted degradation of EGFR protein and an essential role of the endoplasmic reticulum (ER) protein processing pathway in anti-EGFR therapeutic efficacy. Our results suggest that targeted degradation is not always superior to enzymatic inhibition and establish TraCe-seq as an approach to study how preexisting transcriptional programs affect treatment responses.

Humanization of a strategic CD3 epitope enables evaluation of clinical T-cell engagers in a fully immunocompetent in vivo model.

T-cell engagers (TCEs) are a growing class of biotherapeutics being investigated in the clinic for treatment of a variety of hematological and solid tumor indications. However, preclinical evaluation of TCEs in vivo has been mostly limited to xenograft tumor models in human T-cell reconstituted immunodeficient mice, which have a number of limitations. To explore the efficacy of human TCEs in fully immunocompetent hosts, we developed a knock-in mouse model (hCD3E-epi) in which a 5-residue N-terminal fragment of murine CD3-epsilon was replaced with an 11-residue stretch from the human sequence that encodes for a common epitope recognized by anti-human CD3E antibodies in the clinic. T cells from hCD3E-epi mice underwent normal thymic development and could be efficiently activated upon crosslinking of the T-cell receptor with anti-human CD3E antibodies in vitro. Furthermore, a TCE targeting human CD3E and murine CD20 induced robust T-cell redirected killing of murine CD20-positive B cells in ex vivo hCD3E-epi splenocyte cultures, and also depleted nearly 100% of peripheral B cells for up to 7 days following in vivo administration. These results highlight the utility of this novel mouse model for exploring the efficacy of human TCEs in vivo, and suggest a useful tool for evaluating TCEs in combination with immuno-oncology/non-immuno-oncology agents against heme and solid tumor targets in hosts with a fully intact immune system.

Adhesamine, a new synthetic molecule, accelerates differentiation and prolongs survival of primary cultured mouse hippocampal neurons.

Attachment to the substrate is essential for both survival and differentiation of various kinds of cells, such as neurons and epithelial cells. We recently found a small synthetic molecule, adhesamine, which boosts adhesion and growth of mammalian cells. In the present study, we applied adhesamine to primary cultured hippocampal neuronal cells and compared its effects with those of PLL (poly-L-lysine), which is widely used as a substrate for cell cultures. Neurons grown on adhesamine-coated coverslips survived for up to 1 month without a feeder layer of glial cells, and had greater viability than cells grown on PLL-coated coverslips. Morphological analysis revealed that neurons cultured with adhesamine exhibited earlier differentiation, i.e. earlier axonal outgrowth and dendritic maturation with enhanced neurite branching, than neurons cultured with PLL. Synaptic formation and postsynaptic responses were evident as early as 4 days in cells cultured with adhesamine. Acceleration of differentiation is mediated by earlier activation of the signalling pathways from heparan sulfate in the extracellular matrix to both FAK (focal adhesion kinase) and MAPK (mitogen-activated protein kinase). Improved survival rates and accelerated maturation of neurons exposed to adhesamine suggest that this completely synthetic molecule may be a useful reagent for culturing neuronal cells.

Heterobifunctional Molecules Induce Dephosphorylation of Kinases-A Proof of Concept Study.

Heterobifunctional molecules have proven powerful tools to induce ligase-dependent ubiquitination of target proteins. We describe here a chemical strategy for controlling a different post-translational modification (PTM): phosphorylation. Heterobifunctional molecules were designed to promote the proximity of a protein phosphatase (PP1) to protein targets. The synthesized molecules induced the PP1-dependent dephosphorylation of AKT and EGFR. To our knowledge, this work represents the first examples of small molecules recruiting non-native partners to induce removal of a PTM.

First total synthesis of 4-methylthio-3-butenyl glucosinolate.

The first total synthesis of 4-methylthio-3-butenyl glucosinolate (MTBG), a natural bioactive compound and a precursor of radish phototropism-regulating substances, was achieved from commercially available 1,4-butanediol. The glucosinolate framework was prepared by coupling of an oximyl chloride derivative and tetraacetyl thioglucose. A methylthio group was introduced to the framework by a Wittig reaction between triphenylphosphonium thiomethylmethylide and an aldehyde intermediate of glucosinolate. The synthetic route should facilitate preparation of various derivatives needed for probe synthesis based on MTBG.