Although outstanding progress has been made in understanding the pathophysiology of thrombotic thrombocytopenic purpura (TTP), knowledge of the immunopathogenesis of the disease is only at an early stage. Anti-ADAMTS13 auto-antibodies were shown to block proteolysis of von Willebrand factor and/or induce ADAMTS13 clearance from the circulation. However, it still remains to identify which immune cells are involved in the production of anti-ADAMTS13 autoantibodies, and therefore account for the remarkable efficacy of the B-cell depleting agents in this disease. The mechanisms leading to the loss of tolerance of the immune system towards ADAMTS13 involve the predisposing genetic factors of the human leukocyte antigen class II locus DRB1*11 and DQB1*03 alleles as well as the protective allele DRB1*04, and modifying factors such as ethnicity, sex and obesity. Future studies have to identify why these identified genetic risk factors are also frequently to be found in the healthy population although the incidence of immune-mediated thrombotic thrombocytopenic purpura (iTTP) is extremely low. Moreover, the development of recombinant ADAMTS13 opens a new therapeutic era in the field. Interactions of recombinant ADAMTS13 with the immune system of iTTP patients will require intensive investigation, especially for its potential immunogenicity. Better understanding of iTTP immunopathogenesis should, therefore, provide a basis for the development of novel therapeutic approaches to restore immune tolerance towards ADAMTS13 and thereby better prevent refractoriness and relapses in patients with iTTP. In this review, we address these issues and the related challenges in this field.
Antibodies that develop in patients with immune thrombotic thrombocytopenic purpura (iTTP) commonly target the spacer epitope R568/F592/R660/Y661/Y665 (RFRYY). In this study we present a detailed contribution of each residue in this epitope for autoantibody binding. Different panels of mutations were introduced here to create a large collection of full-length ADAMTS13 variants comprising conservative (Y←→F), semi-conservative (Y/F→L), non-conservative (Y/F→N) or alanine (Y/F/R→A) substitutions. Previously reported Gain-of-Function (GoF, KYKFF) and truncated ‘MDTCS’ variants were also included. Sera of 18 patients were screened against all variants. Conservative mutations of the aromatic residues did not reduce the binding of autoantibodies. Moderate resistance was achieved by replacing R568 and R660 by lysines or alanines. Semi-conservative mutations of aromatic residues show a moderate effectiveness in autoantibody resistance. Non-conservative asparagine or alanine mutations of aromatic residues are the most effective. In the mixtures of autoantibodies from the majority (89%) of patients screened, autoantibodies targeting the spacer RFRYY epitope have preponderance compared to other epitopes. Reductions in ADAMTS13 proteolytic activity were observed for all full-length mutant variants, in varying degrees. The greatest activity reductions were observed in the most autoantibody-resistant variants (15-35% residual activity in FRETS-VWF73). Among these, a triple-alanine mutant RARAA showed activity in a VWF multimer assay. This study shows that non-conservative and alanine modifications of residues within the exosite-3 spacer RFRYY epitope in full-length ADAMTS13 resist the binding of autoantibodies from iTTP patients, while retaining residual proteolytic activity. Our study provides a framework for the design of autoantibody-resistant ADAMTS13 variants for further therapeutic development.
ADAMTS13 (a disintegrin and metalloprotease with a thrombospondin type-1 motif, member 13) and von Willebrand factor (VWF) can be considered as scale weights which control platelet adhesion during primary haemostasis. In a very uncommon condition designated thrombotic thrombocytopenic purpura (TTP), functional absence of ADAMTS13 tips the balance toward VWF-mediated platelet adhesion in the microcirculation. TTP is associated with a high mortality and arises from either a congenital or acquired autoimmune deficiency of the plasma enzyme ADAMTS13. In case of acquired ADAMTS13 deficiency, autoantibodies bind to and inhibit the function of ADAMTS13. Currently available treatments of TTP aim to supply ADAMTS13 through plasma exchange or are aimed at B-cell depletion with rituximab. None of the available therapeutics, however, aims at protection of ADAMTS13 from circulating autoantibodies. In this review, our aim is to describe the structure-function relationship of ADAMTS13 employing homology models and previously published crystal structures. Structural bioinformatics investigation of ADAMTS13 reveals many insights and explains how mutations and autoantibodies may lead to the pathophysiology of TTP. The results of these studies provide a roadmap for the further development of rationally designed therapeutics for the treatment of patients with acquired TTP. In addition, we share our opinion on the state of the art of the open-closed conformations of ADAMTS13 which regulate the activity of this highly specific VWF cleaving protease.Ã On behalf of the PROFILE Consortium, http://www.itn-profile.eu/
Cytochrome P450 102A1 from Bacillus megaterium (BM3) is a fatty acid hydroxylase that has one of the highest turnover rates of any mono-oxygenase. Recent studies have shown how mutants of BM3 can produce metabolites of known drug compounds similar to those observed in humans. Single-point mutations in the binding pocket change the regioselective metabolism of fenamic acids from aromatic hydroxylation to aliphatic hydroxylation. This study is concerned with the individual contribution from accessibility and reactivity for drug metabolism with a future goal to develop fast methods for prediction. For a BM3 M11 mutant as well as the M11 V87F and M11 V87I mutants, we studied the metabolism of the nonsteroidal anti-inflammatory drugs (NSAIDs) mefenamic acid, meclofenamic acid, tolfenamic acid, and diclofenac. Density functional theory (DFT; B3LYP and B3LYP-D3) calculations for all possible reactions were performed using a porphyrin model reacting with the four substrates. Molecular dynamics (MD) simulations were used to determine the potential sites of metabolism that are accessible. Finally, we combine reactivity and accessibility for each potential site to interpret the experimentally determined metabolism. Generally, the 3 and 5 positions (on the ring containing the acidic substituent) and the 2′, 3′, and 4′ positions are most reactive, whereas 4, 5, 3′, and 4′ are most accessible. Combining reactivity and accessibility show that the 5, 3′, and 4′ positions are predicted to be most prone to be metabolized, in agreement with experimentally observed data. Reactivity seems to be the dominant factor in the CYP-mediated metabolism of these NSAIDs, which is consistent with previously published methods based solely on reactivity.
The plasmatic von Willebrand factor (VWF) circulates in a compact form unable to bind platelets. Upon shear stress, the VWF A1 domain is exposed, allowing VWF-binding to platelet glycoprotein Ib-V-IX (GPIbα chain). For a better understanding of the role of this interaction in cardiovascular disease, molecules are needed to specifically interfere with the opened VWF A1 domain interaction with GPIbα. Therefore, we in silico designed and chemically synthetized stable cyclic peptides interfering with the platelet-binding of the VWF A1 domain per se or complexed with botrocetin. Selected peptides (26–34 amino acids) with the lowest-binding free energy were: the monocyclic mono- vOn Willebrand factoR-GPIbα InTerference (ORbIT) peptide and bicyclic bi-ORbIT peptide. Interference of the peptides in the binding of VWF to GPIb-V-IX interaction was retained by flow cytometry in comparison with the blocking of anti-VWF A1 domain antibody CLB-RAg35. In collagen and VWF-dependent whole-blood thrombus formation at a high shear rate, CLB-RAg35 suppressed stable platelet adhesion as well as the formation of multilayered thrombi. Both peptides phenotypically mimicked these changes, although they were less potent than CLB-RAg35. The second-round generation of an improved peptide, namely opt-mono-ORbIT (28 amino acids), showed an increased inhibitory activity under flow. Accordingly, our structure-based design of peptides resulted in physiologically effective peptide-based inhibitors, even for convoluted complexes such as GPIbα-VWF A1.
The horizon of drug discovery is currently expanding to target and modulate protein–protein interactions (PPIs) in globular proteins and intrinsically disordered proteins that are involved in various diseases. To either interrupt or stabilize PPIs, the 3D structure of target protein–protein (or protein–peptide) complexes can be exploited to rationally design PPI modulators (inhibitors or stabilizers) through structure-based molecular design. In this review, we present an overview of experimental and computational methods that can be used to determine 3D structures of protein–protein complexes. Several approaches including rational and in silico methods that can be applied to design peptides, peptidomimetics and small compounds by utilization of determined 3D protein–protein/peptide complexes are summarized and illustrated.
The ADAMTS13 mutations result in a severe ADAMTS13 deficiency explaining the patient's phenotype.
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