Anti-C1s humanized monoclonal antibody SAR445088: A classical pathway complement inhibitor specific for the active form of C1s.

The objective of this study was to characterize the complement-inhibiting activity of SAR445088, a novel monoclonal antibody specific for the active form of C1s. Wieslab® and hemolytic assays were used to demonstrate that SAR445088 is a potent, selective inhibitor of the classical pathway of complement. Specificity for the active form of C1s was confirmed in a ligand binding assay. Finally, TNT010 (a precursor to SAR445088) was assessed in vitro for its ability to inhibit complement activation associated with cold agglutinin disease (CAD). TNT010 inhibited C3b/iC3b deposition on human red blood cells incubated with CAD patient serum and decreased their subsequent phagocytosis by THP-1 cells. In summary, this study identifies SAR445088 as a potential therapeutic for the treatment of classical pathway-driven diseases and supports its continued assessment in clinical trials.

Molecular Features of the Zn2+ Binding Site in the Prion Protein Probed by 113Cd NMR.

The cellular prion protein (PrPC) is a zinc-binding protein that contributes to the regulation of Zn2+ and other divalent species of the central nervous system. Zn2+ coordinates to the flexible, N-terminal repeat region of PrPC and drives a tertiary contact between this repeat region and a well-defined cleft of the C-terminal domain. The tertiary structure promoted by Zn2+ is thought to regulate inherent PrPC toxicity. Despite the emerging consensus regarding the interaction between Zn2+ and PrPC, there is little direct spectroscopic confirmation of the metal ion's coordination details. Here, we address this conceptual gap by using Cd2+ as a surrogate for Zn2+. NMR finds that Cd2+ binds exclusively to the His imidazole side chains of the repeat segment, with a dissociation constant of ∼1.2 mM, and promotes an N-terminal-C-terminal cis interaction very similar to that observed with Zn2+. Analysis of 113Cd NMR spectra of PrPC, along with relevant control proteins and peptides, suggests that coordination of Cd2+ in the full-length protein is consistent with a three- or four-His geometry. Examination of the mutation E199K in mouse PrPC (E200K in humans), responsible for inherited Creutzfeldt-Jakob disease, finds that the mutation lowers metal ion affinity and weakens the cis interaction. These findings not only provide deeper insight into PrPC metal ion coordination but they also suggest new perspectives on the role of familial mutations in prion disease.

X-ray Absorption Spectroscopy Investigations of Copper(II) Coordination in the Human Amyloid ß Peptide.

Alzheimer's disease (AD) is the main cause of age-related dementia and currently affects approximately 5.7 million Americans. Major brain changes associated with AD pathology include accumulation of amyloid beta (Aß) protein fragments and formation of extracellular amyloid plaques. Redox-active metals mediate oligomerization of Aß, and the resultant metal-bound oligomers have been implicated in the putative formation of harmful, reactive species that could contribute to observed oxidative damage. In isolated plaque cores, Cu(II) is bound to Aß via histidine residues. Despite numerous structural studies of Cu(II) binding to synthetic Aß in vitro, there is still uncertainty surrounding Cu(II) coordination in Aß. In this study, we used X-ray absorption spectroscopy (XAS) and high energy resolution fluorescence detected (HERFD) XAS to investigate Cu(II) coordination in Aß(1-42) under various solution conditions. We found that the average coordination environment in Cu(II)Aß(1-42) is sensitive to X-ray photoreduction, changes in buffer composition, peptide concentration, and solution pH. Fitting of the extended X-ray absorption fine structure (EXAFS) suggests Cu(II) is bound in a mixture of coordination environments in monomeric Aß(1-42) under all conditions studied. However, it was evident that on average only a single histidine residue coordinates Cu(II) in monomeric Aß(1-42) at pH 6.1, in addition to 3 other oxygen or nitrogen ligands. Cu(II) coordination in Aß(1-42) at pH 7.4 is similarly 4-coordinate with oxygen and nitrogen ligands, although an average of 2 histidine residues appear to coordinate at this pH. At pH 9.0, the average Cu(II) coordination environment in Aß(1-42) appears to be 5-coordinate with oxygen and nitrogen ligands, including two histidine residues.

Cyclopentadienones via a tandem C-cyclopropylnitrone cyclization-cycloreversion sequence.

Aldonitrones derived from spiro[2.4]hepta-4,6-diene-1-carbaldehyde and its benzo analog undergo a tandem uncatalyzed intramolecular cyclopropane-nitrone cyclization-5,6-dihydro-1,2-oxazine cycloreversion to give cyclopentadienones. Similarly, the NH-nitrone generated in situ from spiro[cyclopropane-1,1'-indene]carbaldehyde oxime leads to benzocyclopentadienone (1H-inden-1-one) by the same mechanism. DFT calculations are in favor of a concerted yet highly asynchronous pathway for the cyclizations. Control experiments with the dihydro and tetrahydro derivatives show that the spirocyclopentadiene unit is essential for the success of the reaction, invoking spiroconjugative effects for increased cyclopropane reactivity.

Interaction between Prion Protein's Copper-Bound Octarepeat Domain and a Charged C-Terminal Pocket Suggests a Mechanism for N-Terminal Regulation.

Copper plays a critical role in prion protein (PrP) physiology. Cu(2+) binds with high affinity to the PrP N-terminal octarepeat (OR) domain, and intracellular copper promotes PrP expression. The molecular details of copper coordination within the OR are now well characterized. Here we examine how Cu(2+) influences the interaction between the PrP N-terminal domain and the C-terminal globular domain. Using nuclear magnetic resonance and copper-nitroxide pulsed double electron-electron resonance, with molecular dynamics refinement, we localize the position of Cu(2+) in its high-affinity OR-bound state. Our results reveal an interdomain cis interaction that is stabilized by a conserved, negatively charged pocket of the globular domain. Interestingly, this interaction surface overlaps an epitope recognized by the POM1 antibody, the binding of which drives rapid cerebellar degeneration mediated by the PrP N terminus. The resulting structure suggests that the globular domain regulates the N-terminal domain by binding the Cu(2+)-occupied OR within a complementary pocket.

Imaging active urokinase plasminogen activator in prostate cancer.

The increased proteolytic activity of membrane-bound and secreted proteases on the surface of cancer cells and in the transformed stroma is a common characteristic of aggressive metastatic prostate cancer. We describe here the development of an active site-specific probe for detecting a secreted peritumoral protease expressed by cancer cells and the surrounding tumor microenvironment. Using a human fragment antigen-binding phage display library, we identified a human antibody termed U33 that selectively inhibited the active form of the protease urokinase plasminogen activator (uPA, PLAU). In the full-length immunoglobulin form, U33 IgG labeled with near-infrared fluorophores or radionuclides allowed us to noninvasively detect active uPA in prostate cancer xenograft models using optical and single-photon emission computed tomography imaging modalities. U33 IgG labeled with (111)In had a remarkable tumor uptake of 43.2% injected dose per gram (%ID/g) 72 hours after tail vein injection of the radiolabeled probe in subcutaneous xenografts. In addition, U33 was able to image active uPA in small soft-tissue and osseous metastatic lesions using a cardiac dissemination prostate cancer model that recapitulated metastatic human cancer. The favorable imaging properties were the direct result of U33 IgG internalization through an uPA receptor-mediated mechanism in which U33 mimicked the function of the endogenous inhibitor of uPA to gain entry into the cancer cell. Overall, our imaging probe targets a prostate cancer-associated protease, through a unique mechanism, allowing for the noninvasive preclinical imaging of prostate cancer lesions.

Synthesis and structure-activity relationship studies of 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide derivatives as potent and selective inhibitors of 12-lipoxygenase.

Human lipoxygenases (LOXs) are a family of iron-containing enzymes which catalyze the oxidation of polyunsaturated fatty acids to provide the corresponding bioactive hydroxyeicosatetraenoic acid (HETE) metabolites. These eicosanoid signaling molecules are involved in a number of physiologic responses such as platelet aggregation, inflammation, and cell proliferation. Our group has taken a particular interest in platelet-type 12-(S)-LOX (12-LOX) because of its demonstrated role in skin diseases, diabetes, platelet hemostasis, thrombosis, and cancer. Herein, we report the identification and medicinal chemistry optimization of a 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide-based scaffold. Top compounds, exemplified by 35 and 36, display nM potency against 12-LOX, excellent selectivity over related lipoxygenases and cyclooxygenases, and possess favorable ADME properties. In addition, both compounds inhibit PAR-4 induced aggregation and calcium mobilization in human platelets and reduce 12-HETE in ß-cells.

Tumor-specific activation of an EGFR-targeting probody enhances therapeutic index.

Target-mediated toxicity constitutes a major limitation for the development of therapeutic antibodies. To redirect the activity of antibodies recognizing widely distributed targets to the site of disease, we have applied a prodrug strategy to create an epidermal growth factor receptor (EGFR)-directed Probody therapeutic-an antibody that remains masked against antigen binding until activated locally by proteases commonly active in the tumor microenvironment. In vitro, the masked Probody showed diminished antigen binding and cell-based activities, but when activated by appropriate proteases, it regained full activity compared to the parental anti-EGFR antibody cetuximab. In vivo, the Probody was largely inert in the systemic circulation of mice, but was activated within tumor tissue and showed antitumor efficacy that was similar to that of cetuximab. The Probody demonstrated markedly improved safety and increased half-life in nonhuman primates, enabling it to be dosed safely at much higher levels than cetuximab. In addition, we found that both Probody-responsive xenograft tumors and primary tumor samples from patients were capable of activating the Probody ex vivo. Probodies may therefore improve the safety profile of therapeutic antibodies without compromising efficacy of the parental antibody and may enable the wider use of empowered antibody formats such as antibody-drug conjugates and bispecifics.