The serine protease factor VIIa (FVIIa) in complex with its cellular cofactor tissue factor (TF) initiates the blood coagulation reactions. TF·FVIIa is also implicated in thrombosis-related disorders ...and constitutes an appealing therapeutic target for treatment of cardiovascular diseases. To this end, we generated the FVIIa active site inhibitor G17905, which displayed great potency toward TF·FVIIa (Ki = 0.35 ± 0.11 nm). G17905 did not appreciably inhibit 12 of the 14 examined trypsin-like serine proteases, consistent with its TF·FVIIa-specific activity in clotting assays. The crystal structure of the FVIIa·G17905 complex provides insight into the molecular basis of the high selectivity. It shows that, compared with other serine proteases, FVIIa is uniquely equipped to accommodate conformational disturbances in the Gln217–Gly219 region caused by the ortho-hydroxy group of the inhibitor's aminobenzamidine moiety located in the S1 recognition pocket. Moreover, the structure revealed a novel, nonstandard conformation of FVIIa active site in the region of the oxyanion hole, a “flipped” Lys192-Gly193 peptide bond. Macromolecular substrate activation assays demonstrated that G17905 is a noncompetitive, slow-binding inhibitor. Nevertheless, G17905 effectively inhibited thrombus formation in a baboon arterio-venous shunt model, reducing platelet and fibrin deposition by ∼70% at 0.4 mg/kg + 0.1 mg/kg/min infusion. Therefore, the in vitro potency of G17905, characterized by slow binding kinetics, correlated with efficacious antithrombotic activity in vivo.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a ...therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Bruton’s tyrosine kinase (Btk) inhibitor ibrutinib has shown impressive clinical efficacy in a range of B-cell malignancies. However, acquired resistance has emerged, and second generation ...therapies are now being sought. Ibrutinib is a covalent, irreversible inhibitor that modifies Cys481 in the ATP binding site of Btk and renders the enzyme inactive, thereby blocking B-cell receptor signal transduction. Not surprisingly, Cys481 is the most commonly mutated Btk residue in cases of acquired resistance to ibrutinib. Mutations at other sites, including Thr474, a gatekeeper residue, have also been detected. Herein, we describe noncovalent Btk inhibitors that differ from covalent inhibitors like ibrutinib in that they do not interact with Cys481, they potently inhibit the ibrutinib-resistant Btk C481S mutant in vitro and in cells, and they are exquisitely selective for Btk. Noncovalent inhibitors such as GNE-431 also show excellent potency against the C481R, T474I, and T474M mutants. X-ray crystallographic analysis of Btk provides insight into the unique mode of binding of these inhibitors that explains their high selectivity for Btk and their retained activity against mutant forms of Btk. This class of noncovalent Btk inhibitors may provide a treatment option to patients, especially those who have acquired resistance to ibrutinib by mutation of Cys481 or Thr474.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
The human epidermal growth factor receptor (HER) family of tyrosine kinases is deregulated in multiple cancers either through amplification, overexpression, or mutation. ERBB3/HER3, the only member ...with an impaired kinase domain, although amplified or overexpressed in some cancers, has not been reported to carry oncogenic mutations. Here, we report the identification of ERBB3 somatic mutations in ∼11% of colon and gastric cancers. We found that the ERBB3 mutants transformed colonic and breast epithelial cells in a ligand-independent manner. However, the mutant ERBB3 oncogenic activity was dependent on kinase-active ERBB2. Furthermore, we found that anti-ERBB antibodies and small molecule inhibitors effectively blocked mutant ERBB3-mediated oncogenic signaling and disease progression in vivo.
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•Recurrent ERBB3 somatic mutations occur in colon and gastric cancers•ERBB3 mutants with ERBB2 promote ligand-independent oncogenic signaling•Somatic ERBB3 mutants promote tumor formation•Targeted therapeutics block ERBB3 mutant-mediated oncogenic signaling
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Accumulation of amyloid-β (Aβ) peptides and amyloid plaque deposition in brain is postulated as a cause of Alzheimer's disease (AD). The precise pathological species of Aβ remains elusive although ...evidence suggests soluble oligomers may be primarily responsible for neurotoxicity. Crenezumab is a humanized anti-Aβ monoclonal IgG4 that binds multiple forms of Aβ, with higher affinity for aggregated forms, and that blocks Aβ aggregation, and promotes disaggregation. To understand the structural basis for this binding profile and activity, we determined the crystal structure of crenezumab in complex with Aβ. The structure reveals a sequential epitope and conformational requirements for epitope recognition, which include a subtle but critical element that is likely the basis for crenezumab's versatile binding profile. We find interactions consistent with high affinity for multiple forms of Aβ, particularly oligomers. Of note, crenezumab also sequesters the hydrophobic core of Aβ and breaks an essential salt-bridge characteristic of the β-hairpin conformation, eliminating features characteristic of the basic organization in Aβ oligomers and fibrils, and explains crenezumab's inhibition of aggregation and promotion of disaggregation. These insights highlight crenezumab's unique mechanism of action, particularly regarding Aβ oligomers, and provide a strong rationale for the evaluation of crenezumab as a potential AD therapy.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Extensive crosstalk among ErbB/HER receptors suggests that blocking signaling from more than one family member may be essential to effectively treat cancer and limit drug resistance. We generated a ...conventional IgG molecule MEHD7945A with dual HER3/EGFR specificity by phage display engineering and used structural and mutational studies to understand how a single antigen recognition surface binds two epitopes with high affinity. As a human IgG1, MEHD7945A exhibited dual action by inhibiting EGFR- and HER3-mediated signaling in vitro and in vivo and the ability to engage immune effector functions. Compared with monospecific anti-HER antibodies, MEHD7945A was more broadly efficacious in multiple tumor models, showing that combined inhibition of EGFR and HER3 with a single antibody is beneficial.
► MEHD7945A is a two-in-one antibody that targets EGFR and HER3 ► MEHD7945A epitopes include ligand recognition sites on domain III ► MEHD7945A exhibits superior efficacy over monospecific HER antibodies
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
A Trigger Squeezed Eigenbrot, Charles
Structure (London),
03/2008, Volume:
16, Issue:
3
Journal Article
Peer reviewed
Open access
Qiu et al. (2008) add new weight to the very highly detailed understanding of how extracellular ligand binding-induced dimerization of a receptor tyrosine kinase is first manifested inside the cell.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Small molecule inhibitors that target the phosphatidylinositol 3-kinase (PI3K) signaling pathway have received significant interest for the treatment of cancers. The class I isoform PI3Kα is most ...commonly associated with solid tumors via gene amplification or activating mutations. However, inhibitors demonstrating both PI3K isoform and mutant specificity have remained elusive. Herein, we describe the optimization and characterization of a series of benzoxazepin-oxazolidinone ATP-competitive inhibitors of PI3Kα which also induce the selective degradation of the mutant p110α protein, the catalytic subunit of PI3Kα. Structure-based design informed isoform-specific interactions within the binding site, leading to potent inhibitors with greater than 300-fold selectivity over the other Class I PI3K isoforms. Further optimization of pharmacokinetic properties led to excellent in vivo exposure and efficacy and the identification of clinical candidate GDC-0077 (inavolisib, 32), which is now under evaluation in a Phase III clinical trial as a treatment for patients with PIK3CA-mutant breast cancer.
Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in ...severe asthma patients independent of type 2 biomarker status. Active β-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human β-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a β-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.
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•Increased tryptase levels in asthma that is independent of type 2 inflammation•Higher active allele β-tryptase count associated with lesser omalizumab response•Discovery, biochemistry, and structure of a noncompetitive inhibitory anti-tryptase•Favorable PK and potent inhibition of tryptase activity in mouse and primate models
Type 2-low asthma represents a critical unmet need and an opportunity for new drug discovery. Here, Maun et al. demonstrate that increased mast cell tryptase levels are associated with asthma severity independent of type 2 inflammation and that increased active tryptase alleles are associated with decreased clinical responses to anti-IgE treatment. They generated a noncompetitive inhibitory anti-tryptase antibody with in vivo activity in humanized mouse and cynomolgus monkey models as a clinical candidate for severe asthma treatment.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Antibody–drug conjugates (ADCs) have become an important therapeutic modality for oncology, with three approved by the FDA and over 60 others in clinical trials. Despite the progress, improvements in ...ADC therapeutic index are desired. Peptide-based ADC linkers that are cleaved by lysosomal proteases have shown sufficient stability in serum and effective payload-release in targeted cells. If the linker can be preferentially hydrolyzed by tumor-specific proteases, safety margin may improve. However, the use of peptide-based linkers limits our ability to modulate protease specificity. Here we report the structure-guided discovery of novel, nonpeptidic ADC linkers. We show that a cyclobutane-1,1-dicarboxamide-containing linker is hydrolyzed predominantly by cathepsin B while the valine–citrulline dipeptide linker is not. ADCs bearing the nonpeptidic linker are as efficacious and stable in vivo as those with the dipeptide linker. Our results strongly support the application of the peptidomimetic linker and present new opportunities for improving the selectivity of ADCs.
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