Cell Chemical Biology

To combat refractory diseases, such as cancer, multitarget-directed ligands (MTDLs) have become an emerging area of research to exploit synthetic lethality (SL) relationships associated with drug resistance. Herein, we present the in silico design of MTDLs for the polypharmaceutical treatment of endometrial adenocarcinoma (EAC) and our discovery of a novel SL in EAC; PTEN loss of function (LOF) and the inhibition of CDK9. We used high-resolution x-ray crystallographic data to chemically engineer, LCI133, to inhibit CDK9, CDK4/6-and AURKA/B kinases. PTEN LOF in EAC results in augmented deregulated transcription and a massive increase in nascent RNA, a phenotype which encodes a high level of apoptotic sensitivity to LCI133 and CDK9 inhibitors. Treatment with LCI133 results in a rapid decline nose-dive in global nRNA, MYC nRNA levels and TS elongation (TE) in PTEN LOF EAC. PTEN LOF is necessary and sufficient to confer sensitivity of EAC cells to LCI133 and other CDK9 inhibitors.

APOBEC3A and APOBEC3B are antiviral cytidine deaminases found to drive cancer-associated mutagenesis, contributing to tumor evolution and therapeutic resistance across multiple cancer types. Inhibiting these enzymes holds promise for prolonging response to a wide range of cancer therapies by delaying development of resistance. However, APOBEC3A and APOBEC3B remain challenging drug targets, with no potent and selective small molecule inhibitors reported. Here, we use a fluorescence polarization-based assay to identify small molecules inhibitors of the APOBEC3A-single-stranded DNA interaction. From a library of 2,400 disulfide compounds, we identified 64 hits (mean polarization +/- 3 sigma, hit rate of 2.7%). Intact protein mass spectrometry revealed that a subset of compounds covalently engages A3A at cysteine 64, including Compounds 1 and 2. Compounds 1 and 2 disrupt APOBEC3A/APOBEC3B-single-stranded DNA interactions and inhibit APOBEC3A/APOBEC3B deaminase activity in a dose-dependent manner, with micromolar IC50. Surprisingly, inhibition of APOBEC3A/APOBEC3B by Compounds 1 and 2 is independent of covalent tethering to cysteine, suggesting a predominantly non-covalent mode of binding. Together, these studies establish an integrated workflow for APOBEC ligand discovery and identify Compounds 1 and 2 as starting points for developing chemical probes to investigate APOBEC-driven mutagenesis and therapeutic resistance.

N-linked Fc galactosylation drives variability in therapeutic mAb quality, affecting complement-dependent cytotoxicity and ADCC. Current control strategies suffer from narrow ranges, productivity loss, or unwanted glycoforms. Here, we combine metabolic and cellular glycoengineering by feeding 2-deoxy-2-fluorogalactose (2FG) to CHO cells engineered for hypergalactosylation via COSMC knockout and {beta}4-galactosyltransferase overexpression (C /GT+), achieving broad, tuneable control of Fc {beta}4-galactosylation. In DP12 C-/GT+ cells, a split-feed regimen spanned a wide galactosylation range (95-46%) but generated substantial aglycosylated and Man5 glycoforms. The same regimen in a second host, VRC01 C-/GT+, gave only a narrow range, indicating 2FG dosing does not transfer directly across cell lines. Based on a proposed mechanism where 2FG is metabolised via the Leloir pathway into inhibitory fluorinated nucleotide-sugar intermediates, we developed a cell-normalised, multi-bolus feeding strategy that substantially broadened control in VRC01 C-/GT+ cells while suppressing aglycosylation and Man5 formation. In a fed-batch Ambr(R) 250 process, this strategy achieved comparable galactosylation reduction with roughly ten-fold less 2FG, showing that the fraction of mAb secreted after feeding is a key determinant of control strategy efficiency. Across the resulting glycoform range, Bio-Layer Interferometry identified Fc {beta}4-galactosylation as the strongest correlate of Fc{gamma}RIIIA (CD16a) binding affinity (R2 = 0.92). Together, these findings establish combined metabolic and cellular glycoengineering as a platform for controlling mAb galactosylation across a broad range, with potential to fine-tune downstream pharmacological activity.

Molecular editing, rooted in synthetic chemistry, has emerged as a powerful tool for structure optimization in medicinal chemistry but remains underutilized in targeted protein degradation. Here, we demonstrate the utility of molecular editing by repurposing the NSD2 degrader LLC0424 with GSPT1 neosubstrate engagement to generate two structurally analogous molecules with divergent modes of action: 424-ND, a PROTAC selective for NSD2, and 424-GD, a selective GSPT1 molecular glue. 424-ND drove NSD2 degradation selectively in a CRBN- and proteasome-dependent manner and functionally suppressed androgen receptor (AR) signaling in prostate cancer cells. 424-GD induced selective degradation of GSPT1 and robustly upregulated the integrated stress response markers ATF4 and ATF3. Biolayer interferometry revealed distinct ternary complex cooperativity profiles in the presence of NSD2 or GSPT1, which directly correlate with the observed biased degradation activity. Further molecular dynamics and metadynamics simulations showed that the compounds adopt distinct low-energy conformational ensembles with different spatial orientations, likely underlying their divergent cooperativity and selectivity. These findings not only deliver highly selective chemical probes for NSD2 and GSPT1 but also demonstrate that molecular editing enables precision control over target selectivity, providing a generalizable strategy to tune selectivity in targeted protein degradation.

The Fanconi anaemia (FA) DNA repair pathway is an emerging target for precision cancer therapy. Using a high-throughput FANCD2-monoubiquitination assay, we identified a class of small molecules, including MSG010, that inhibit the FA E3 ligase complex in vitro. Because these molecules, and the metabolite, palmitoyl-CoA, are known to engage allosteric drug and metabolite (ADaM) binding site in AMP-activated kinase (AMPK), we hypothesised that a similar pocket exists within the FA complex. Supporting this, long-chain, but not short-chain, fatty acyl-CoA molecules inhibited the FA E3 ligase complex activity, and sequence analysis revealed similarity between the AMPK ADaM site and a WD40 repeat in the FA subunit FANCX. Targeted mutagenesis of this FANCX region disrupted E3 ligase activity or abolished inhibition by MSG010, suggesting the presence of an ADaM-like site in FANCX. Moreover, MSG010 preferentially killed BRCA1-deficient cells in vitro. These findings identify a putative small-molecule binding site in the FA pathway that may be developed further to test for exploitation as anticancer therapeutics.

Small-molecule drug discovery relies on identifying compounds that modulate specific protein targets, a process often hindered by cellular complexity. Through phenotypic screening of a kinase-focused diazaquinazoline library, we serendipitously identified CEM198 as the first high-affinity ligand of tubulin-tyrosine ligase (TTL). Functional assays combining live-cell TTL inhibition, microtubule polymerization, cell cycle analysis, and proteomics revealed that CEM198 acts through a dual mechanism: directly binding to TTL and altering /{beta}-tubulin conformation. This interaction restricts -tubulin tyrosination and disrupts tubulin polymerization, leading to microtubule destabilization. The differential effects observed between SH-SY5Y and HEK293T cells indicate that effective TTL inhibition depends on both direct binding and structural modulation of the tubulin heterodimer. These findings introduce CEM198 as a chemical probe for investigating the tubulin tyrosination-detyrosination and demonstrate the potential of chemoproteomics to uncover novel modulators of microtubule dynamics.

Macrophages are critical regulators of inflammation and tissue homeostasis, yet aberrant macrophage activation contributes to a wide spectrum of inflammatory and malignant diseases. Therapeutic strategies that directly reduce macrophage numbers have shown promise, but macrophage survival pathways remain incompletely defined, limiting the development of targeted therapeutic strategies. Here, we establish a high-throughput screening platform to identify small molecule inhibitors that impair macrophage survival. Screening a library of more than 2,000 targeted compounds in a cell survival assay, combined with in silico and in vitro analysis of macrophage specificity, revealed the identification of three potent inhibitors: BIX-01294, GSK-J4, and Masitinib. All three compounds downregulated leukemia inhibitory factor receptor (LIFR), whose inhibition markedly reduced macrophage viability. In vivo, these inhibitors effectively depleted large peritoneal macrophages, ameliorated key symptoms of macrophage activation syndrome (MAS), and suppressed tumor growth in a syngeneic transplanted melanoma model as well as in an autochthonous lung cancer model. Together, these findings identify small molecule-mediated macrophage depletion as a promising therapeutic strategy and establish an experimental approach to uncover regulators of macrophage survival.

KRAS G12C inhibitors have demonstrated meaningful clinical benefit in KRAS G12C-mutant non-small cell lung cancer (NSCLC), yet responses remain heterogeneous and treatment-associated toxicities persist for reasons that are incompletely understood. Cysteine profiling indicates that these covalent inhibitors are highly selective for mutant KRAS; however, such approaches cannot detect noncovalent engagement of additional non-RAS proteins. Here, we used a protein-folding stability profiling technique, stability of proteins from rates of oxidation (SPROX), to identify protein targets of the clinical KRAS G12C inhibitor, divarasib (GDC-6036), in KRAS-mutant NSCLC lysates. SPROX revealed a focused set of candidate interactors, including the essential splicing factor RBM39, which was reproducibly stabilized at both divarasib concentrations tested. We subsequently confirmed that divarasib directly and noncovalently binds to RBM39 protein. In NSCLC cells, divarasib increased RBM39 protein abundance and antagonized RBM39 degradation induced by the aryl-sulfonamide molecular glue indisulam through a post-transcriptional mechanism. Divarasib and RBM39 degraders reciprocally antagonized each other's cytotoxicity, and RBM39 knockdown modestly reduced divarasib-induced cell death. Mechanistically, divarasib-mediated RBM39 stabilization regulated both INSR expression and alternative splicing, altered downstream insulin receptor signaling, and contributed to divarasib-associated cytotoxicity. Consistent with these findings, RBM39 and INSR expression were positively correlated across multiple human cancer types. Collectively, these findings identify RBM39 as a previously unrecognized noncovalent target of divarasib and uncover an RBM39-INSR signaling axis that modulates cellular responses to both divarasib and RBM39 degraders.

Transcription factors remain among the most challenging therapeutic targets in part because they lack well-defined ligandable binding pockets. We recently showed that aberrantly reactive cysteines in transcription factors can be directly targeted with electrophilic small molecules to induce selective transcription factor destabilization and degradation. Here, we extend this strategy to the lineage-defining oncogenic transcription factor PAX8, a critical driver of ovarian cancer. Screening of a chemically diverse library of more than 3,000 cysteine-reactive compounds against an endogenously HiBiT-tagged PAX8 reporter identified a sulfinyl aziridine chemotype that selectively reduced PAX8 abundance. Structure-activity and stereochemical analyses revealed highly enantio- and diastereoselective activity, identifying KL6-159A as the lead compound. Quantitative proteomics demonstrated selective loss of PAX8, while cellular thermal shift analysis and chemoproteomic profiling established direct covalent engagement of PAX8 at cysteine C57. Mutation of C57 completely abolished KL6-159A-induced PAX8 depletion, demonstrating that this residue is essential for compound activity. Transcriptomic profiling revealed broad suppression of the PAX8 transcriptional program, with FOXM1 emerging as the most significantly downregulated regulatory network together with numerous established PAX8 target genes. Collectively, these studies establish direct covalent engagement, transcriptional inhibition, and destabilization of PAX8 and further demonstrate the generality of covalent chemoproteomic approaches for drugging previously intractable transcription factors.

Inhibitor of DNA binding (ID) proteins are key regulators of tumor cell stemness, therapy resistance and pathological angiogenesis in multiple cancer types and other diseases. Here, we characterize the coumarin-derived compound X6632 as a pan-ID inhibitor with dual activity against tumor cells and the tumor-associated microvasculature in a number of human and murine models. X6632 efficiently suppressed ID protein expression, inhibited the proliferation, migration, invasion of melanoma cells, and impaired multiple endothelial cell functions, including proliferation, migration, invasion, tube formation and sprouting in vitro. In back-to-back comparisons, X6632 exhibited an approximately ten-fold higher efficacy compared to the first-generation ID antagonist AGX51. In vivo, X6632 potently reduced pathological (neo)vascularization in established angiogenesis models, including oxygen-induced retinopathy and in Matrigel plug assays. It also significantly decreased blood vessel density in syngeneic melanoma models, delayed tumor growth and, when combined with immune checkpoint blockade, achieved superior tumor control compared with either monotherapy. Moreover, X6632 inhibited clonogenic growth in several breast cancer models, and robustly suppressed the growth of triple negative breast cancer in vivo, both in the highly aggressive 4T1 syngeneic model and in patient-derived xenografts. Collectively, these data establish X6632 as a second-generation, pan-ID protein inhibitor that can simultaneously target malignant cells and the tumor-supporting vasculature, and support the further pre-clinical development of the compound for the treatment of melanoma, breast cancer and potentially additional ID-dependent malignancies, as well as diseases driven by pathological neoangiogenesis.

DNA mismatch repair (MMR) maintains genome stability but paradoxically drives expansion of pathogenic repeats responsible for trinucleotide repeat disorders. Attenuating repeat instability through targeted inhibition of MMR is therefore a therapeutic strategy of interest; however, progress is constrained by a lack of robust methods for defining and isolating MMR subpathways. We exploited features of prime editing to profile the effects of core MMR genes on thousands of programmed DNA mispairs in cells and resolved distinct MMR processes, including a noncanonical, loop-directed mechanism. Guided by these results, we built a set of scalable reporters that detect loss of separable MMR processes and screened ~72,000 small molecules for activity against MutS{beta}-directed MMR. This effort identified a novel series of MutS{beta} inhibitors that reduced (CAG)n expansion in a cell model. Altogether, this study establishes several methods for systematically interrogating MMR, with demonstrated capacity to identify inhibitors of therapeutic value.

Isocitrate dehydrogenase 1 (IDH1) catalyses the interconversion of isocitrate and -ketoglutarate and is overexpressed in several cancers, supporting tumour survival and treatment resistance. Inhibitors targeting the oncogenic mutant forms of IDH1 also inhibit the wild-type (WT) protein, but lose substantial potency due to greater competition with native substrates (isocitrate and Mg2+). We hypothesised that a covalent inhibitor may be more effective at overcoming the higher substrate affinity of wild-type IDH1. Here, a covalent fragment-based approach was used to develop IDH1-C269 selective inhibitors which prevent the key regulatory segment from forming an -helix required for catalytic competency. Following the identification of C269-selective fragments, we used X-ray crystallography to characterise IDH1-fragment bound complexes and guide structure-based design efforts. Inspired by the unexpected detection of bound isocitrate molecule in an X-ray crystal structure, fragment expansion yielded a series of compounds exploiting both an adjacent site and a unique water-mediated hydrogen bonding network. This series achieved strong potency (reaching IC50 = 47.5 nM, a 155-fold improvement in potency relative to the original fragment), retained activity in the presence of competing Mg2+, showed selectivity against IDH2 and reduced NADPH concentrations in a relevant PDAC cell model. Together, these findings present a mechanistic rationale for the covalent targeting of wild-type IDH1 and provide structurally validated inhibitors for further development.

Dysregulation of insulin-like growth factor (IGF) signaling contributes to diverse human diseases. Therapeutic strategies have largely targeted the IGF-1 receptor, but the complexity of the IGF system has limited their clinical efficacy. Pregnancy-associated plasma protein-A (PAPP-A) is a secreted metalloproteinase that regulates local IGF signaling through proteolytic cleavage of IGF-binding proteins (IGFBPs) and represents an attractive therapeutic target. Here, we report the first synthetic PAPP-A inhibitors designed to target the enzyme active site using an IGFBP-5-derived anchor sequence. Replacing the scissile bond with phosphinate or phosphonate moieties yielded potent and selective PAPP-A inhibitors. Cryo-electron microscopy revealed the molecular basis of inhibition and key active-site interactions. The compounds inhibited cleavage of IGFBP-4 and IGFBP-5, two physiological PAPP-A substrates. They suppressed IGF-1 signaling in A549 lung cancer cells, with efficacy comparable to the endogenous PAPP-A inhibitor stanniocalcin-1. They also inhibited Papp-aa-dependent IGF signaling and cell proliferation in wild-type and stc1a-/- mutant zebrafish in vivo in a dose-dependent manner. Together, these findings establish the first class of synthetic, selective PAPP-A inhibitors capable of modulating IGF signaling. They provide new chemical tools for studying and modulating pathological IGF signaling and offer new directions for developing PAPP-A-targeted therapeutic strategies.

Cereblon (CRBN) is widely used in targeted protein degradation, but its ligand space has remained dominated by a narrow set of cyclic imide chemotypes. Here, we show that the accessible CRBN ligand space extends substantially beyond this canonical space. A high-throughput screen of > 40,000 compounds, followed by orthogonal biophysical validation, X-ray crystallography and SAR analyses, identified several chemically distinct ligand classes. These include linear acetyl-based motifs, a phthalide-derived scaffold, steroidal compounds, and a range of bicyclic ligands. They engage CRBN through distinct recognition modes, several of which deviate from the canonical hydrogen-bonding pattern. Steroidal scaffolds were particularly notable: cortisone binds the human CRBN thalidomide-binding domain with an affinity comparable to thalidomide, with its A-ring occupying the tri-tryptophan pocket in a glutarimide-like orientation despite lacking the canonical imide NH donor. SAR within this series showed substantial tolerance for chemical modification and scaffold simplification, raising the possibility that endogenous steroidal metabolites may contribute to the physiological ligand landscape of CRBN. Bicyclic lactams additionally provided synthetically accessible scaffolds with tunable affinity and promising sites for linker attachment. Across the identified ligand classes, none of the tested representatives induced detectable degradation of canonical CRBN neosubstrates, and several showed largely clean proteomic profiles. Together, these findings broaden the chemical, mechanistic and potential physiological landscape of CRBN recognition and provide diverse starting points for alternative, potentially neosubstrate-sparing CRBN recruiters.

Genetic variants throughout the multi-domain protein leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of autosomal dominant Parkinson's disease, and the most prevalent Parkinson's-associated LRRK2 variants enhance kinase activity. Hence, the kinase domain has been extensively targeted for therapeutic development. However, clinical progression of LRRK2 kinase inhibitors has been limited by on-target peripheral toxicities linked to strong kinase suppression, raising the question of whether targeting LRRK2 kinase activity through other means could offer a more tunable therapeutic window. LRRK2 is one of two proteins in the human proteome that possess a Roc-COR GTPase domain in tandem with a kinase domain, and the GTPase domain has been shown to intramolecularly regulate kinase output. Here, we ask whether the Roc-COR GTPase can be targeted as an alternative approach to downregulate kinase activity. We utilize a chemical genetic approach to sensitize LRRK2 to existing GTPase inhibitors and show that pharmacologically engaging the Roc-COR GTPase domain in cells decreases but does not fully inhibit LRRK2-mediated Rab10 T73 phosphorylation. This study lays the groundwork for future efforts directed at the development of pharmacological inhibitors for the LRRK2 GTPase, providing an alternative approach for therapies targeting LRRK2-driven Parkinson's disease.

Site-specific protein modification allows diverse functionalities to be introduced while minimizing perturbations to the protein structure and activity. Considerable efforts have been made to achieve site-specific modification of native proteins to overcome the heterogeneity resulting from conventional stochastic Lys or Cys modification. We have previously achieved the selective modification of Lys65 in a native immunoglobulin G1 (IgG1) antibody (trastuzumab) using EzMTG-pG(Fab), which is an engineered zymogen of microbial transglutaminase (EzMTG) fused to a Fab-binding protein G [pG(Fab)]. However, this approach cannot be widely applied to different types of IgG antibodies. Here, we designed pG(Fab)-EzMTG by fusing pG(Fab) to the N-terminus of EzMTG. Notably, switching the fusion partners dramatically altered the IgG modification site from Lys65 to Lys225, which is located in the hinge site of native IgG1 antibodies. This Lys225-selective labeling was applicable to different IgG1 antibodies. As a functional application, the cytotoxic drug monomethyl auristatin E (MMAE) was conjugated to Lys225 of trastuzumab, and the resulting antibody-drug conjugate exhibited antigen-specific cytotoxicity. These findings demonstrate that fusion-protein architecture determines site selectivity in proximity-directed enzymatic modification, providing a strategy for the site-specific functionalization of native antibodies.

Triple-negative breast cancer (TNBC) remains a major clinical challenge due to a lack of effective interventions and severe drug resistance. Disrupting tumor metabolism using nitric oxide (NO) donors to drive ferroptosis represents a promising therapeutic strategy. Here, we developed 3A72, a novel furan-coumarin-based NO donor with potent anti-TNBC efficacy. In vitro and in vivo studies demonstrated that 3A72 significantly suppresses TNBC cell proliferation and restricts xenograft tumor growth. Notably, its efficacy matches the first-line drug paclitaxel but with enhanced safety and reduced systemic toxicity. Mechanistically, 3A72 induces ferroptosis in triple negative breast cancer (TNBC) cells via a synergistic dual-pathway mechanism. Initially, 3A72 releases intracellular NO to trigger cytoplasmic calcium overload, followed by high-affinity binding to both inositol 1,4,5-trisphosphate receptor (IPR) and acyl-CoA synthetase long-chain family member 4 (ACSL4). Binding to IP3R provokes mitochondrial calcium overload and a massive burst of reactive oxygen species (ROS), while concurrent ACSL4 activation drives essential membrane phospholipid remodeling. Crucially, our transcriptomic and structural predictions reveal that while ACSL4 exhibits a broader distribution across organelle membranes, its functional association with IP3R spatially converges at the endoplasmic reticulum membranes associated with mitochondria (MAMs). This subcellular niche closely couples IPR-mediated ROS generation with ACSL4-driven lipid remodeling, accelerating lethal malondialdehyde (MDA) accumulation and triggering irreversible ferroptosis. Overall, these findings highlight the MAMs calcium-lipid metabolic hub as a promising structural scaffold for antitumor drug development.

Targeted protein stabilization by deubiquitinase-targeting chimeras (DUBTACs) has emerged as a promising therapeutic strategy for preserving the function of essential proteins. DUBTACs are bifunctional molecules that recruit deubiquitinating enzymes (DUBs) to remove ubiquitin chains from substrate proteins, thereby preventing their proteasomal degradation. A key requirement for DUBTACs development is the identification of ligands that bind DUBs without impairing their catalytic activity. However, such noninhibitory ligands are exceedingly rare. Here, we report a novel proximity-driven on-bead functional screening platform for the discovery of noninhibitory DUB ligands. Using this approach, we discovered a previously unreported noninhibitory ligand for USP7. Furthermore, we generated DUBTACs by conjugating this USP7 ligand to a CFTR-binding ligand. Our lead compound, WJ045, effectively stabilized CFTR protein levels in cells, demonstrating the potential of this platform for discovering DUB recruiters and facilitating the development of targeted protein stabilization therapeutics.

Brutons tyrosine kinase (BTK) transduces B-cell receptor (BCR), Toll-like receptor (TLR), and Fc receptor (FcR) signaling, and overactivation of these pathways drives B-cell malignancies and antibody-mediated autoimmune disease. Small molecule inhibitors block the enzymatic functions of BTK, but this inhibition is undermined by resistance mutations, several of which abolish BTKs kinase activity yet promote oncogenic signaling through BTK scaffolding functions. We report the discovery and characterization of bexobrutideg (NX-5948), a heterobifunctional degrader that recruits cereblon (CRBN) to selectively degrade BTK while sparing molecular glue neosubstrates. We demonstrate that bexobrutideg acts catalytically, degrading thousands of copies of BTK per molecule per hour, and this event-driven pharmacology renders it resilient to mutations that confer resistance to both covalent- and noncovalent-inhibitors. Bexobrutideg is orally bioavailable, driving deep and durable BTK degradation across species. Bexobrutideg demonstrates strong efficacy in wild-type and ibrutinib-resistant lymphoma models and robustly suppresses pathway activation in models of autoimmune disease.

Deubiquitinase-targeting chimeras (DUBTACs) have emerged as a promising strategy for targeted protein stabilization, but their broader application remains limited by the scarcity of ligandable deubiquitinase recruiters. Here, we report a previously unexplored four-membered-ring OTUB1 recruiter chemotype. Through systematic structure-activity relationship studies, we identified compound 21 (MS2159) as a potent and selective covalent OTUB1 ligand. Biochemical and intact protein mass spectrometric analyses demonstrated that MS2159 selectively engages the non-catalytic C23 residue of OTUB1, shows minimal reactivity toward other tested proteins, and preserves OTUB1 deubiquitinase activity. Conjugation of MS2159 with the CFTR ligand lumacaftor yielded compound 25 (MS2134), which effectively stabilized {Delta}F508-CFTR. Collectively, these findings establish a new OTUB1 recruiter scaffold, expand the ligandable chemical space of OTUB1, and provide additional opportunities for developing next-generation DUBTACs.