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1.
Nat Commun ; 15(1): 1813, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38418820

RESUMO

Widespread adoption of mirror-image biological systems presents difficulties in accessing the requisite D-protein substrates. In particular, mirror-image phage display has the potential for high-throughput generation of biologically stable macrocyclic D-peptide binders with potentially unique recognition modes but is hindered by the individualized optimization required for D-protein chemical synthesis. We demonstrate a general mirror-image phage display pipeline that utilizes automated flow peptide synthesis to prepare D-proteins in a single run. With this approach, we prepare and characterize 12 D-proteins - almost one third of all reported D-proteins to date. With access to mirror-image protein targets, we describe the successful discovery of six macrocyclic D-peptide binders: three to the oncoprotein MDM2, and three to the E3 ubiquitin ligase CHIP. Reliable production of mirror-image proteins can unlock the full potential of D-peptide drug discovery and streamline the study of mirror-image biology more broadly.


Assuntos
Peptídeos , Proteínas , Ligantes , Descoberta de Drogas
2.
Nat Commun ; 14(1): 6992, 2023 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-37914719

RESUMO

Molecules that induce novel interactions between proteins hold great promise for the study of biological systems and the development of therapeutics, but their discovery has been limited by the complexities of rationally designing interactions between three components, and because known binders to each protein are typically required to inform initial designs. Here, we report a general and rapid method for discovering α-helically constrained (Helicon) polypeptides that cooperatively induce the interaction between two target proteins without relying on previously known binders or an intrinsic affinity between the proteins. We show that Helicons are capable of binding every major class of E3 ubiquitin ligases, which are of great biological and therapeutic interest but remain largely intractable to targeting by small molecules. We then describe a phage-based screening method for discovering "trimerizer" Helicons, and apply it to reprogram E3s to cooperatively bind an enzyme (PPIA), a transcription factor (TEAD4), and a transcriptional coactivator (ß-catenin).


Assuntos
Peptídeos , Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/metabolismo , Peptídeos/metabolismo , Ubiquitinação
3.
Proc Natl Acad Sci U S A ; 119(52): e2210435119, 2022 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-36534810

RESUMO

The α-helix is one of the most common protein surface recognition motifs found in nature, and its unique amide-cloaking properties also enable α-helical polypeptide motifs to exist in membranes. Together, these properties have inspired the development of α-helically constrained (Helicon) therapeutics that can enter cells and bind targets that have been considered "undruggable", such as protein-protein interactions. To date, no general method for discovering α-helical binders to proteins has been reported, limiting Helicon drug discovery to only those proteins with previously characterized α-helix recognition sites, and restricting the starting chemical matter to those known α-helical binders. Here, we report a general and rapid screening method to empirically map the α-helix binding sites on a broad range of target proteins in parallel using large, unbiased Helicon phage display libraries and next-generation sequencing. We apply this method to screen six structurally diverse protein domains, only one of which had been previously reported to bind isolated α-helical peptides, discovering 20 families that collectively comprise several hundred individual Helicons. Analysis of 14 X-ray cocrystal structures reveals at least nine distinct α-helix recognition sites across these six proteins, and biochemical and biophysical studies show that these Helicons can block protein-protein interactions, inhibit enzymatic activity, induce conformational rearrangements, and cause protein dimerization. We anticipate that this method will prove broadly useful for the study of protein recognition and for the development of both biochemical tools and therapeutics for traditionally challenging protein targets.


Assuntos
Amidas , Peptídeos , Conformação Proteica em alfa-Hélice , Sítios de Ligação , Peptídeos/química , Biblioteca de Peptídeos
4.
Antibodies (Basel) ; 9(4)2020 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-33172091

RESUMO

Bispecific molecules are biologically significant, yet their complex structures pose important manufacturing and pharmacokinetic challenges. Nevertheless, owing to similarities with monoclonal antibodies (mAbs), IgG-like bispecifics conceptually align well with conventional expression and manufacturing platforms and often exhibit potentially favorable drug metabolism and pharmacokinetic (DMPK) properties. However, IgG-like bispecifics do not possess target bivalency and current designs often require tedious engineering and purification to ensure appropriate chain pairing. Here, we present a near-native IgG antibody format, the 2xVH, which can create bivalency for each target or epitope and requires no engineering for cognate chain pairing. In this modality, two different variable heavy (VH) domains with distinct binding specificities are grafted onto the first constant heavy (CH1) and constant light (CL) domains, conferring the molecule with dual specificity. To determine the versatility of this format, we characterized the expression, binding, and stability of several previously identified soluble human VH domains. By grafting these domains onto an IgG scaffold, we generated several prototype 2xVH IgG and Fab molecules that display similar properties to mAbs. These molecules avoided the post-expression purification necessary for engineered bispecifics while maintaining a capacity for simultaneous dual binding. Hence, the 2xVH format represents a bivalent, bispecific design that addresses limitations of manufacturing IgG-like bispecifics while promoting biologically-relevant dual target engagement.

5.
J Biol Chem ; 295(52): 18379-18389, 2020 12 25.
Artigo em Inglês | MEDLINE | ID: mdl-33122192

RESUMO

The dimeric ectonucleotidase CD73 catalyzes the hydrolysis of AMP at the cell surface to form adenosine, a potent suppressor of the immune response. Blocking CD73 activity in the tumor microenvironment can have a beneficial effect on tumor eradication and is a promising approach for cancer therapy. Biparatopic antibodies binding different regions of CD73 may be a means to antagonize its enzymatic activity. A panel of biparatopic antibodies representing the pairwise combination of 11 parental monoclonal antibodies against CD73 was generated by Fab-arm exchange. Nine variants vastly exceeded the potency of their parental antibodies with ≥90% inhibition of activity and subnanomolar EC50 values. Pairing the Fabs of parents with nonoverlapping epitopes was both sufficient and necessary whereas monovalent antibodies were poor inhibitors. Some parental antibodies yielded potent biparatopics with multiple partners, one of which (TB19) producing the most potent. The structure of the TB19 Fab with CD73 reveals that it blocks alignment of the N- and C-terminal CD73 domains necessary for catalysis. A separate structure of CD73 with a Fab (TB38) which complements TB19 in a particularly potent biparatopic shows its binding to a nonoverlapping site on the CD73 N-terminal domain. Structural modeling demonstrates a TB19/TB38 biparatopic antibody would be unable to bind the CD73 dimer in a bivalent manner, implicating crosslinking of separate CD73 dimers in its mechanism of action. This ability of a biparatopic antibody to both crosslink CD73 dimers and fix them in an inactive conformation thus represents a highly effective mechanism for the inhibition of CD73 activity.


Assuntos
5'-Nucleotidase/química , 5'-Nucleotidase/imunologia , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Neoplasias Pulmonares/imunologia , 5'-Nucleotidase/metabolismo , Domínio Catalítico , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Conformação Proteica , Células Tumorais Cultivadas
6.
Mol Ther ; 28(2): 664-676, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-31843448

RESUMO

Patients with α-dystroglycanopathies, a subgroup of rare congenital muscular dystrophies, present with a spectrum of clinical manifestations that includes muscular dystrophy as well as CNS and ocular abnormalities. Although patients with α-dystroglycanopathies are genetically heterogeneous, they share a common defect of aberrant post-translational glycosylation modification of the dystroglycan alpha-subunit, which renders it defective in binding to several extracellular ligands such as laminin-211 in skeletal muscles, agrin in neuromuscular junctions, neurexin in the CNS, and pikachurin in the eye, leading to various symptoms. The genetic heterogeneity associated with the development of α-dystroglycanopathies poses significant challenges to developing a generalized treatment to address the spectrum of genetic defects. Here, we propose the development of a bispecific antibody (biAb) that functions as a surrogate molecular linker to reconnect laminin-211 and the dystroglycan beta-subunit to ameliorate sarcolemmal fragility, a primary pathology in patients with α-dystroglycan-related muscular dystrophies. We show that the treatment of LARGEmyd-3J mice, an α-dystroglycanopathy model, with the biAb improved muscle function and protected muscles from exercise-induced damage. These results demonstrate the viability of a biAb that binds to laminin-211 and dystroglycan simultaneously as a potential treatment for patients with α-dystroglycanopathy.


Assuntos
Anticorpos Biespecíficos/farmacologia , Distroglicanas/metabolismo , Laminina/metabolismo , Síndrome de Walker-Warburg/metabolismo , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/metabolismo , Modelos Animais de Doenças , Distroglicanas/imunologia , Expressão Gênica , Humanos , Imuno-Histoquímica , Injeções Intramusculares , Laminina/genética , Laminina/imunologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas/genética , Sarcolema/efeitos dos fármacos , Sarcolema/metabolismo , Síndrome de Walker-Warburg/tratamento farmacológico , Síndrome de Walker-Warburg/etiologia
7.
Alzheimers Res Ther ; 10(1): 117, 2018 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-30486882

RESUMO

BACKGROUND: Anti-amyloid ß (Aß) immunotherapy represents a major area of drug development for Alzheimer's disease (AD). However, Aß peptide adopts multiple conformations and the pathological forms to be specifically targeted have not been identified. Aß immunotherapy-related vasogenic edema has also been severely dose limiting for antibodies with effector functions binding vascular amyloid such as bapineuzumab. These two factors might have contributed to the limited efficacy demonstrated so far in clinical studies. METHODS: To address these limitations, we have engineered SAR228810, a humanized monoclonal antibody (mAb) with limited Fc effector functions that binds specifically to soluble protofibrillar and fibrillar forms of Aß peptide and we tested it together with its murine precursor SAR255952 in vitro and in vivo. RESULTS: Unlike gantenerumab and BAN2401, SAR228810 and SAR255952 do not bind to Aß monomers, low molecular weight Aß oligomers or, in human brain sections, to Aß diffuse deposits which are not specific of AD pathology. Both antibodies prevent Aß42 oligomer neurotoxicity in primary neuronal cultures. In vivo, SAR255952, a mouse aglycosylated IgG1, dose-dependently prevented brain amyloid plaque formation and plaque-related inflammation with a minimal active dose of 3 mg/kg/week by the intraperitoneal route. No increase in plasma Aß levels was observed with SAR255952 treatment, in line with its lack of affinity for monomeric Aß. The effects of SAR255952 translated into synaptic functional improvement in ex-vivo hippocampal slices. Brain penetration and decoration of cerebral amyloid plaques was documented in live animals and postmortem. SAR255952 (up to 50 mg/kg/week intravenously) did not increase brain microhemorrhages and/or microscopic changes in meningeal and cerebral arteries in old APPSL mice while 3D6, the murine version of bapineuzumab, did. In immunotolerized mice, the clinical candidate SAR228810 demonstrated the same level of efficacy as the murine SAR255952. CONCLUSION: Based on the improved efficacy/safety profile in non-clinical models of SAR228810, a first-in-man single and multiple dose administration clinical study has been initiated in AD patients.


Assuntos
Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/imunologia , Anticorpos Monoclonais Humanizados/administração & dosagem , Encéfalo/imunologia , Imunoterapia/métodos , Doença de Alzheimer/imunologia , Peptídeos beta-Amiloides/metabolismo , Animais , Anticorpos Monoclonais Humanizados/efeitos adversos , Encéfalo/metabolismo , Potenciais Pós-Sinápticos Excitadores/imunologia , Feminino , Hipocampo/imunologia , Hipocampo/fisiopatologia , Humanos , Imunoterapia/efeitos adversos , Masculino , Camundongos Endogâmicos C57BL , Imagem Óptica , Cultura Primária de Células , Fatores de Risco
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