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Mechanism of disease and therapeutic rescue of Dok7 congenital myasthenia

Oury, Julien; Zhang, Wei; Leloup, Nadia; Koide, Akiko; Corrado, Alexis D; Ketavarapu, Gayatri; Hattori, Takamitsu; Koide, Shohei; Burden, Steven J
Congenital myasthenia (CM) is a devastating neuromuscular disease, and mutations in DOK7, an adaptor protein that is crucial for forming and maintaining neuromuscular synapses, are a major cause of CM1,2. The most common disease-causing mutation (DOK71124_1127 dup) truncates DOK7 and leads to the loss of two tyrosine residues that are phosphorylated and recruit CRK proteins, which are important for anchoring acetylcholine receptors at synapses. Here we describe a mouse model of this common form of CM (Dok7CM mice) and a mouse with point mutations in the two tyrosine residues (Dok72YF). We show that Dok7CM mice had severe deficits in neuromuscular synapse formation that caused neonatal lethality. Unexpectedly, these deficits were due to a severe deficiency in phosphorylation and activation of muscle-specific kinase (MUSK) rather than a deficiency in DOK7 tyrosine phosphorylation. We developed agonist antibodies against MUSK and show that these antibodies restored neuromuscular synapse formation and prevented neonatal lethality and late-onset disease in Dok7CM mice. These findings identify an unexpected cause for disease and a potential therapy for both DOK7 CM and other forms of CM caused by mutations in AGRIN, LRP4 or MUSK, and illustrate the potential of targeted therapy to rescue congenital lethality.
PMID: 34163073
ISSN: 1476-4687
CID: 4918552

Identification of the nucleotide-free state as a therapeutic vulnerability for inhibition of selected oncogenic RAS mutants

Khan, Imran; Koide, Akiko; Zuberi, Mariyam; Ketavarapu, Gayatri; Denbaum, Eric; Teng, Kai Wen; Rhett, J Matthew; Spencer-Smith, Russell; Hobbs, G Aaron; Camp, Ernest Ramsay; Koide, Shohei; O'Bryan, John P
RAS guanosine triphosphatases (GTPases) are mutated in nearly 20% of human tumors, making them an attractive therapeutic target. Following our discovery that nucleotide-free RAS (apo RAS) regulates cell signaling, we selectively target this state as an approach to inhibit RAS function. Here, we describe the R15 monobody that exclusively binds the apo state of all three RAS isoforms in vitro, regardless of the mutation status, and captures RAS in the apo state in cells. R15 inhibits the signaling and transforming activity of a subset of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., fast exchange mutants). Intracellular expression of R15 reduces the tumor-forming capacity of cancer cell lines driven by select RAS mutants and KRAS(G12D)-mutant patient-derived xenografts (PDXs). Thus, our approach establishes an opportunity to selectively inhibit a subset of RAS mutants by targeting the apo state with drug-like molecules.
PMID: 35139380
ISSN: 2211-1247
CID: 5156812

High-valency anti-CD99 antibodies toward the treatment of T cell acute lymphoblastic leukemia

Romero, Larizbeth A; Hattori, Takamitsu; A E Ali, Mohamed; Ketavarapu, Gayatri; Koide, Akiko; Park, Christopher Y; Koide, Shohei
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia that currently requires intensive chemotherapy. While childhood T-ALL is associated with high cure rates, adult T-ALL is not, and both are associated with significant short- and long-term morbidities. Thus, less toxic and effective strategies to treat T-ALL are needed. CD99 is overexpressed on T-ALL blasts at diagnosis and at relapse. Although targeting CD99 with cytotoxic antibodies has been proposed, the molecular features required for their activity are undefined. We identified human antibodies that selectively bound to the extracellular domain human CD99 and the most potent clone, 10A1, shared an epitope with a previously described cytotoxic IgM antibody. We engineered clone 10A1 in bivalent, trivalent, tetravalent, and dodecavalent formats. Increasing the antibody valency beyond two had no effects on binding to T-ALL cells. In contrast, a valency of ≥3 was required for cytotoxicity, suggesting a mechanism of action in which an antibody clusters ≥3 CD99 molecules to induce cytotoxicity. We developed a human IgG-based tetravalent version of 10A1 that exhibited cytotoxic activity to T-ALL cells but not to healthy peripheral blood cells. The crystal structure of the 10A1 Fab in complex with a CD99 fragment revealed that the antibody primarily recognizes a proline-rich motif (PRM) of CD99 in a manner reminiscent of SH3-PRM interactions. This work further validates CD99 as a promising therapeutic target in T-ALL and defines a pathway toward the development of a selective therapy against T-ALL.
PMID: 34958778
ISSN: 1089-8638
CID: 5108052

Selective and noncovalent targeting of RAS mutants for inhibition and degradation

Teng, Kai Wen; Tsai, Steven T; Hattori, Takamitsu; Fedele, Carmine; Koide, Akiko; Yang, Chao; Hou, Xuben; Zhang, Yingkai; Neel, Benjamin G; O'Bryan, John P; Koide, Shohei
Activating mutants of RAS are commonly found in human cancers, but to date selective targeting of RAS in the clinic has been limited to KRAS(G12C) through covalent inhibitors. Here, we report a monobody, termed 12VC1, that recognizes the active state of both KRAS(G12V) and KRAS(G12C) up to 400-times more tightly than wild-type KRAS. The crystal structures reveal that 12VC1 recognizes the mutations through a shallow pocket, and 12VC1 competes against RAS-effector interaction. When expressed intracellularly, 12VC1 potently inhibits ERK activation and the proliferation of RAS-driven cancer cell lines in vitro and in mouse xenograft models. 12VC1 fused to VHL selectively degrades the KRAS mutants and provides more extended suppression of mutant RAS activity than inhibition by 12VC1 alone. These results demonstrate the feasibility of selective targeting and degradation of KRAS mutants in the active state with noncovalent reagents and provide a starting point for designing noncovalent therapeutics against oncogenic RAS mutants.
PMID: 33976200
ISSN: 2041-1723
CID: 4867382

The γδ IEL effector API5 masks genetic susceptibility to Paneth cell death

Matsuzawa-Ishimoto, Yu; Yao, Xiaomin; Koide, Akiko; Ueberheide, Beatrix M; Axelrad, Jordan E; Reis, Bernardo S; Parsa, Roham; Neil, Jessica A; Devlin, Joseph C; Rudensky, Eugene; Dewan, M Zahidunnabi; Cammer, Michael; Blumberg, Richard S; Ding, Yi; Ruggles, Kelly V; Mucida, Daniel; Koide, Shohei; Cadwell, Ken
Loss of Paneth cells and their antimicrobial granules compromises the intestinal epithelial barrier and is associated with Crohn's disease, a major type of inflammatory bowel disease1-7. Non-classical lymphoid cells, broadly referred to as intraepithelial lymphocytes (IELs), intercalate the intestinal epithelium8,9. This anatomical position has implicated them as first-line defenders in resistance to infections, but their role in inflammatory disease pathogenesis requires clarification. The identification of mediators that coordinate crosstalk between specific IEL and epithelial subsets could provide insight into intestinal barrier mechanisms in health and disease. Here we show that the subset of IELs that express γ and δ T cell receptor subunits (γδ IELs) promotes the viability of Paneth cells deficient in the Crohn's disease susceptibility gene ATG16L1. Using an ex vivo lymphocyte-epithelium co-culture system, we identified apoptosis inhibitor 5 (API5) as a Paneth cell-protective factor secreted by γδ IELs. In the Atg16l1-mutant mouse model, viral infection induced a loss of Paneth cells and enhanced susceptibility to intestinal injury by inhibiting the secretion of API5 from γδ IELs. Therapeutic administration of recombinant API5 protected Paneth cells in vivo in mice and ex vivo in human organoids with the ATG16L1 risk allele. Thus, we identify API5 as a protective γδ IEL effector that masks genetic susceptibility to Paneth cell death.
PMID: 36198790
ISSN: 1476-4687
CID: 5351622

Inhibition of RAS-driven signaling and tumorigenesis with a pan-RAS monobody targeting the Switch I/II pocket

Wallon, Lauren; Khan, Imran; Teng, Kai Wen; Koide, Akiko; Zuberi, Mariyam; Li, Jianping; Ketavarapu, Gayatri; Traaseth, Nathaniel J; O'Bryan, John P; Koide, Shohei
RAS mutants are major therapeutic targets in oncology with few efficacious direct inhibitors available. The identification of a shallow pocket near the Switch II region on RAS has led to the development of small-molecule drugs that target this site and inhibit KRAS(G12C) and KRAS(G12D). To discover other regions on RAS that may be targeted for inhibition, we have employed small synthetic binding proteins termed monobodies that have a strong propensity to bind to functional sites on a target protein. Here, we report a pan-RAS monobody, termed JAM20, that bound to all RAS isoforms with nanomolar affinity and demonstrated limited nucleotide-state specificity. Upon intracellular expression, JAM20 potently inhibited signaling mediated by all RAS isoforms and reduced oncogenic RAS-mediated tumorigenesis in vivo. NMR and mutation analysis determined that JAM20 bound to a pocket between Switch I and II, which is similarly targeted by low-affinity, small-molecule inhibitors, such as BI-2852, whose in vivo efficacy has not been demonstrated. Furthermore, JAM20 directly competed with both the RAF(RBD) and BI-2852. These results provide direct validation of targeting the Switch I/II pocket for inhibiting RAS-driven tumorigenesis. More generally, these results demonstrate the utility of tool biologics as probes for discovering and validating druggable sites on challenging targets.
PMID: 36252024
ISSN: 1091-6490
CID: 5352392

Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT

Diver, Melinda M; Pedi, Leanne; Koide, Akiko; Koide, Shohei; Long, Stephen B
The maturation of RAS GTPases and approximately 200 other cellular CAAX proteins involves three enzymatic steps: addition of a farnesyl or geranylgeranyl prenyl lipid to the cysteine (C) in the C-terminal CAAX motif, proteolytic cleavage of the AAX residues and methylation of the exposed prenylcysteine residue at its terminal carboxylate. This final step is catalysed by isoprenylcysteine carboxyl methyltransferase (ICMT), a eukaryote-specific integral membrane enzyme that resides in the endoplasmic reticulum. ICMT is the only cellular enzyme that is known to methylate prenylcysteine substrates; methylation is important for the biological functions of these substrates, such as the membrane localization and subsequent activity of RAS, prelamin A and RAB. Inhibition of ICMT has potential for combating progeria and cancer. Here we present an X-ray structure of ICMT, in complex with its cofactor, an ordered lipid molecule and a monobody inhibitor, at 2.3 Å resolution. The active site spans cytosolic and membrane-exposed regions, indicating distinct entry routes for the cytosolic methyl donor, S-adenosyl-l-methionine, and for prenylcysteine substrates, which are associated with the endoplasmic reticulum membrane. The structure suggests how ICMT overcomes the topographical challenge and unfavourable energetics of bringing two reactants that have different cellular localizations together in a membrane environment-a relatively uncharacterized but defining feature of many integral membrane enzymes.
PMID: 29342140
ISSN: 1476-4687
CID: 2916082

Inhibition of RAS function through targeting an allosteric regulatory site

Spencer-Smith, Russell; Koide, Akiko; Zhou, Yong; Eguchi, Raphael R; Sha, Fern; Gajwani, Priyanka; Santana, Dianicha; Gupta, Ankit; Jacobs, Miranda; Herrero-Garcia, Erika; Cobbert, Jacqueline; Lavoie, Hugo; Smith, Matthew; Rajakulendran, Thanashan; Dowdell, Evan; Okur, Mustafa Nazir; Dementieva, Irina; Sicheri, Frank; Therrien, Marc; Hancock, John F; Ikura, Mitsuhiko; Koide, Shohei; O'Bryan, John P
RAS GTPases are important mediators of oncogenesis in humans. However, pharmacological inhibition of RAS has proved challenging. Here we describe a functionally critical region, located outside the effector lobe of RAS, that can be targeted for inhibition. We developed NS1, a synthetic binding protein (monobody) that bound with high affinity to both GTP- and GDP-bound states of H-RAS and K-RAS but not N-RAS. NS1 potently inhibited growth factor signaling and oncogenic H-RAS- and K-RAS-mediated signaling and transformation but did not block oncogenic N-RAS, BRAF or MEK1. NS1 bound the alpha4-beta6-alpha5 region of RAS, which disrupted RAS dimerization and nanoclustering and led to blocking of CRAF-BRAF heterodimerization and activation. These results establish the importance of the alpha4-beta6-alpha5 interface in RAS-mediated signaling and define a previously unrecognized site in RAS for inhibiting RAS function.
PMID: 27820802
ISSN: 1552-4469
CID: 2304332

Molecular basis for antibody recognition of multiple drug-peptide/MHC complexes

Maso, Lorenzo; Rajak, Epsa; Bang, Injin; Koide, Akiko; Hattori, Takamitsu; Neel, Benjamin G; Koide, Shohei
The HapImmuneTM platform exploits covalent inhibitors as haptens for creating major histocompatibility complex (MHC)-presented tumor-specific neoantigens by design, combining targeted therapies with immunotherapy for the treatment of drug-resistant cancers. A HapImmune antibody, R023, recognizes multiple sotorasib-conjugated KRAS(G12C) peptides presented by different human leukocyte antigens (HLAs). This high specificity to sotorasib, coupled with broad HLA-binding capability, enables such antibodies, when reformatted as T cell engagers, to potently and selectively kill sotorasib-resistant KRAS(G12C) cancer cells expressing different HLAs upon sotorasib treatment. The loosening of HLA restriction could increase the patient population that can benefit from this therapeutic approach. To understand the molecular basis for its unconventional binding capability, we used single-particle cryogenic electron microscopy to determine the structures of R023 bound to multiple sotorasib-peptide conjugates presented by different HLAs. R023 forms a pocket for sotorasib between the VH and VL domains, binds HLAs in an unconventional, angled way, with VL making most contacts with them, and makes few contacts with the peptide moieties. This binding mode enables the antibody to accommodate different hapten-peptide conjugates and to adjust its conformation to different HLAs presenting hapten-peptides. Deep mutational scanning validated the structures and revealed distinct levels of mutation tolerance by sotorasib- and HLA-binding residues. Together, our structural information and sequence landscape analysis reveal key features for achieving MHC-restricted recognition of multiple hapten-peptide antigens, which will inform the development of next-generation therapeutic antibodies.
PMID: 38781214
ISSN: 1091-6490
CID: 5654922

Proton-coupled transport mechanism of the efflux pump NorA

Li, Jianping; Li, Yan; Koide, Akiko; Kuang, Huihui; Torres, Victor J; Koide, Shohei; Wang, Da-Neng; Traaseth, Nathaniel J
Efflux pump antiporters confer drug resistance to bacteria by coupling proton import with the expulsion of antibiotics from the cytoplasm. Despite efforts there remains a lack of understanding as to how acid/base chemistry drives drug efflux. Here, we uncover the proton-coupling mechanism of the Staphylococcus aureus efflux pump NorA by elucidating structures in various protonation states of two essential acidic residues using cryo-EM. Protonation of Glu222 and Asp307 within the C-terminal domain stabilized the inward-occluded conformation by forming hydrogen bonds between the acidic residues and a single helix within the N-terminal domain responsible for occluding the substrate binding pocket. Remarkably, deprotonation of both Glu222 and Asp307 is needed to release interdomain tethering interactions, leading to opening of the pocket for antibiotic entry. Hence, the two acidic residues serve as a "belt and suspenders" protection mechanism to prevent simultaneous binding of protons and drug that enforce NorA coupling stoichiometry and confer antibiotic resistance.
PMID: 38802368
ISSN: 2041-1723
CID: 5663352