Harnessing the Therapeutic Potential of Antibodies Targeting Connexin Hemichannels

Connexin hemichannels have been implicated in pathology-promoting conditions, including inflammation, numerous widespread human diseases, including cancer and diabetes, and several rare diseases linked to pathological point mutations

Cues to opening mechanisms from in silico electric field excitation of cx26 hemichannel and in vitro mutagenesis studies in HeLa transfectans

Connexin channels play numerous essential roles in virtually every organ by mediating solute exchange between adjacent cells, or between cytoplasm and extracellular milieu. Our understanding of the structure-function relationship of connexin channels relies on X-ray crystallographic data for human connexin 26 (hCx26) intercellular gap junction channels. Comparison of experimental data and molecular dynamics simulations suggests that the published structures represent neither fully-open nor closed configurations. To facilitate the search for alternative stable configurations, we developed a coarse grained (CG) molecular model of the hCx26 hemichannel and studied its responses to external electric fields. When challenged by a field of 0.06 V/nm, the hemichannel relaxed toward a novel configuration characterized by a widened pore and an increased bending of the second transmembrane helix (TM2) at the level of the conserved Pro87. A point mutation that inhibited such transition in our simulations impeded hemichannel opening in electrophysiology and dye uptake experiments conducted on HeLa tranfectants. These results suggest that the hCx26 hemichannel uses a global degree of freedom to transit between different configuration states, which may be shared among the whole connexin family

A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function

Connexin hemichannels, which are plasma membrane hexameric channels (connexons) composed of connexin protein protomers, have been implicated in a host of physiological processes and pathological conditions. A number of single point pathological mutations impart a "leaky" character to the affected hemichannels, i.e., make them more active or hyperactive, suggesting that normal physiological condition could be recovered using selective hemichannel inhibitors. Recently, a human-derived monoclonal antibody named abEC1.1 has been shown to inhibit both wild type and hyperactive hemichannels composed of human (h) connexin 26 (hCx26) subunits. The aims of this work were (1) to characterize further the ability of abEC1.1 to selectively modulate connexin hemichannel function and (2) to assess its in vitro stability in view of future translational applications. In silico analysis of abEC1.1 interaction with the hCx26 hemichannel identified critically important extracellular domain amino acids that are conserved in connexin 30 (hCx30) and connexin 32 (hCx32). Patch clamp experiments performed in HeLa DH cells confirmed the inhibition efficiency of abEC1.1 was comparable for hCx26, hCx30 and hCx32 hemichannels. Of note, even a single amino acid difference in the putative binding region reduced drastically the inhibitory effects of the antibody on all the other tested hemichannels, namely hCx30.2/31.3, hCx30.3, hCx31, hCx31.1, hCx37, hCx43 and hCx45. Plasma membrane channels composed of pannexin 1 were not affected by abEC1.1. Finally, size exclusion chromatography assays showed the antibody does not aggregate appreciably in vitro. Altogether, these results indicate abEC1.1 is a promising tool for further translational studies

The p.Cys169Tyr variant of connexin 26 is not a polymorphism

Mutations in the GJB2 gene, which encodes the gap junction protein connexin 26 (Cx26), are the primary cause of hereditary prelingual hearing impairment. Here, the p.Cys169Tyr missense mutation of Cx26 (Cx26C169Y), previously classified as a polymorphism, has been identified as causative of severe hearing loss in two Qatari families. We have analyzed the effect of this mutation using a combination of confocal immunofluorescence microscopy and molecular dynamics simulations. At the cellular level, our results show that the mutant protein fails to form junctional channels in HeLa transfectants despite being correctly targeted to the plasma membrane. At the molecular level, this effect can be accounted for by disruption of the disulfide bridge that Cys169 forms with Cys64 in the wild-type structure (Cx26WT). The lack of the disulfide bridge in the Cx26C169Y protein causes a spatial rearrangement of two important residues, Asn176 and Thr177. In the Cx26WT protein, these residues play a crucial role in the intra-molecular interactions that permit the formation of an intercellular channel by the head-to-head docking of two opposing hemichannels resident in the plasma membrane of adjacent cells. Our results elucidate the molecular pathogenesis of hereditary hearing loss due to the connexin mutation and facilitate the understanding of its role in both healthy and affected individuals

Design and Characterization of a Human Monoclonal Antibody that Modulates Mutant Connexin 26 Hemichannels Implicated in Deafness and Skin Disorders

Background: Mutations leading to changes in properties, regulation, or expression of connexin-made channels have been implicated in 28 distinct human hereditary diseases. Eight of these result from variants of connexin 26 (Cx26), a protein critically involved in cell-cell signaling in the inner ear and skin. Lack of non-toxic drugs with defined mechanisms of action poses a serious obstacle to therapeutic interventions for diseases caused by mutant connexins. In particular, molecules that specifically modulate connexin hemichannel function without affecting gap junction channels are considered of primary importance for the study of connexin hemichannel role in physiological as well as pathological conditions. Monoclonal antibodies developed in the last three decades have become the most important class of therapeutic biologicals. Recombinant methods permit rapid selection and improvement of monoclonal antibodies from libraries with large diversity.Methods: By screening a combinatorial library of human single-chain fragment variable (scFv) antibodies expressed in phage, we identified a candidate that binds an extracellular epitope of Cx26. We characterized antibody action using a variety of biochemical and biophysical assays in HeLa cells, organotypic cultures of mouse cochlea and human keratinocyte-derived cells.Results: We determined that the antibody is a remarkably efficient, non-toxic, and completely reversible inhibitor of hemichannels formed by connexin 26 and does not affect direct cell-cell communication via gap junction channels. Importantly, we also demonstrate that the antibody efficiently inhibits hyperative mutant Cx26 hemichannels implicated in autosomal dominant non-syndromic hearing impairment accompanied by keratitis and hystrix-like ichthyosis-deafness (KID/HID) syndrome. We solved the crystal structure of the antibody, identified residues that are critical for binding and used molecular dynamics to uncover its mechanism of action.Conclusions: Although further studies will be necessary to validate the effect of the antibody in vivo, the methodology described here can be extended to select antibodies against hemichannels composed by other connexin isoforms and, consequently, to target other pathologies associated with hyperactive hemichannels. Our study highlights the potential of this approach and identifies connexins as therapeutic targets addressable by screening phage display libraries expressing human randomized antibodies

Development of computational methods for the analysis of conductance and unitary permeability of channels formed by connexins involved in hereditary deafness

This Ph.D. research project focused on the study of channels composed of connexin 26 (Cx26) protein subunits. In particular, we analyzed conductance, permeability and/or structural differences of deafness-related mutations of Cx26 intercellular gap junction channels or unpaired hemichannels, using different molecular dynamics approaches and statistical mechanics. The first part of this thesis introduces the structure of gap junction channels and the connexin proteins that form them. Next, it dwells on the function of gap junction channels and connexin hemichannels, particularly on their conductance and permeability, two fundamental properties explored in the rest of the work. This introductory section contains also a brief overview of electrophysiology experiments performed on connexin channels. It ends with a section on gating properties that modify conductance state and a discussion of disease-related connexin mutations. The second part presents the key results obtained during this Ph.D. work, beginning with the full atom model on which the work was based, and a discussion of the improvements achieved during this project. The rest of the results are subdivided into three topics: (a) structural analysis, (b) conductance and (c) permeability. a) This section summarizes the results obtained for the mutant Cx26 C169Y, the analysis of which shows a large displacement of the extracellular loops and, consequently, of residues critically involved in hemichannel docking. b) This section introduces two methods to compute the ionic conductance of a connexin hemichannel and applies them to three different models: the mutant Cx26 M34T, the WT Cx26 hemichannel in a Coarse-Grained representation and the full-atom WT hemichannel model bound to an antibody. c) This section starts with the explanation of the method used to compute the potential of mean force (PMF) for the passage of a molecule through the channel pore and a possible interpretation of this potential. The PMF was then used to quantify permeation of different molecules (IP3, ATP) through Cx26 WT and V84L mutant hemichannels. The chapter ends with a structural analysis which accounts for the differences in the permeability of the two models. This thesis also contains an overall discussion of the results highlighted above and a methodological appendix.Questo progetto Ph.D. si è concentrato sullo studio dei canali di membrana composti dalla proteina connessina 26 (Cx26). In particolare, abbiamo analizzato la conduttanza, la permeabilità e/o differenze strutturali di mutazioni correlate a sordità di canali di giunzione Gap Cx26 o emicanali, utilizzando diversi approcci di dinamica molecolare e meccanica statistica. La prima parte di questa tesi presenta la struttura dei canali di giunzione gap e le ​​connessine che li formano. Successivamente, si sofferma sulla funzione dei canali giunzione gap e emicanali di connessine, in particolare sulla loro conduttanza e permeabilità, due proprietà fondamentali esplorate nel resto del lavoro. Questa sezione introduttiva contiene anche una breve panoramica di esperimenti di elettrofisiologia condotti sui canali connessina. Si conclude con una sezione sulle proprietà di gating che modificano lo stato di conduttanza e una discussione sulle mutazioni della connessina correlate a malattie. La seconda parte presenta i principali risultati ottenuti nel corso di questo dottorato, a partire dal modello full atom su cui si basa il lavoro, e una discussione dei miglioramenti ottenuti durante questo progetto. Il resto dei risultati sono suddivisi in tre temi: (a) analisi strutturale, (b) conduttanza e (c) permeabilità. a) Questa sezione riassume i risultati ottenuti per il mutante Cx26 C169Y, la cui analisi mostra un grande spostamento delle spire extracellulari e, conseguentemente, dei residui criticamente coinvolti nell-aggancio degli emicanali. b) In questa sezione introduce due metodi per calcolare la conduttanza ionica di un emicanale di connessina e li applica a tre diversi modelli: il mutante Cx26 M34T, l-emicanale WT Cx26 in una rappresentazione a grana grossa (coarse grained) e il modello di emicanale WT full-atomo legato ad un anticorpo. c) Questa sezione inizia con la spiegazione del metodo utilizzato per calcolare il potenziale della forza media (PMF) per il passaggio di una molecola attraverso il poro del canale e una possibile interpretazione di questo potenziale. Il PMF è stato poi utilizzato per quantificare la permeazione di molecole diverse (IP3, ATP e glucosio) attraverso Cx26 WT e l-emicanale mutante V84L. Il capitolo termina con un'analisi strutturale che rappresenta le differenze nella permeabilità dei due modelli. Questa tesi contiene anche una discussione generale dei risultati evidenziati sopra e una appendice metodologica

Probing the electrostatic aggregation of nanoparticles with oppositely charged molecular ions

Abstract The co‐assembly of charged nanoparticles with oppositely charged molecular ions has emerged as a promising technique in the fabrication of nanoparticle superstructures. However, the underlying mechanism behind these molecular ions in mediating the repulsion between these charged nanoparticles remains elusive. Herein, coarse‐grained molecular dynamics simulations are used to elucidate the effects of valency, shape, and size of molecular anions on their co‐assembly with gold nanoparticles coated with positively charged ligands. The findings suggest that the valency, shape, and size of molecular anions significantly influence the repulsion and aggregating dynamics among these positively charged nanoparticles. Moreover, the free energy calculations reveal that ring‐shaped molecular anions with higher valences and larger sizes are more effective at reducing the repulsion between these gold nanoparticles and thus enhance the stability of the aggregate. This study contributes to a better understanding of the critical roles of valence, shape, and size of ions in mediating the electrostatic co‐assembly of nanoparticles with oppositely charged ions, and it also guides the future design of DNA templates and DNA origami in co‐assembly with oppositely charged nanoparticles

In Silico Maturation of a Nanomolar Antibody against the Human CXCR2

The steady increase in computational power in the last 50 years is opening unprecedented opportunities in biology, as computer simulations of biological systems have become more accessible and can reproduce experimental results more accurately. Here, we wanted to test the ability of computer simulations to replace experiments in the limited but practically useful scope of improving the biochemical characteristics of the abN48 antibody, a nanomolar antagonist of the CXC chemokine receptor 2 (CXCR2) that was initially selected from a combinatorial antibody library. Our results showed a good correlation between the computed binding energies of the antibody to the peptide target and the experimental binding affinities. Moreover, we showed that it is possible to design new antibody sequences in silico with a higher affinity to the desired target using a Monte Carlo Metropolis algorithm. The newly designed sequences had an affinity comparable to the best ones obtained using in vitro affinity maturation and could be obtained within a similar timeframe. The methodology proposed here could represent a valid alternative for improving antibodies in cases in which experiments are too expensive or technically tricky and could open an opportunity for designing antibodies for targets that have been elusive so far

Molecular dynamics simulations highlight structural and functional alterations in deafness-related M34T mutation of connexin 26

Mutations of the GJB2 gene encoding the connexin 26 (Cx26) gap junction protein, which is widely expressed in the inner ear, are the primary cause of hereditary non\u2013syndromic hearing loss in several populations. The deafness\u2013associated single amino acid substitution of methionine 34 (M34) in the first transmembrane helix (TM1) with a threonine (T) ensues in the production of mutant Cx26M34T channels that are correctly synthesize and assembled in the plasma membrane. However, mutant channels overexpressed in HeLa cells retain only 9% of the wild type unitary conductance. Here we extend and rationalize those findings by comparing wild type Cx26 (Cx26WT) and Cx26M34T mutant channels in silico, using molecular dynamics simulation. Our results indicate that the quaternary structure of the Cx26M34T hemichannel is altered at the level of the pore funnel due to the disruption of the hydrophobic interaction between M34 and tryptophan 3 (W3) in the N\u2013terminal helix (NTH). Our simulations also show that external force stimuli applied to the NTHs can detach them from the inner wall of the pore more readily in the mutant than in the wild type hemichannel. These structural alterations significantly increase the free energy barrier encountered by permeating ions, correspondingly decreasing the unitary conductance of the Cx26M34T hemichannel. Our results accord with the proposal that the mutant resides most of the time in a low conductance state. However, the small the displacement of the NTHs in our Cx26M34T hemichannel model is not compatible with the formation of a pore plug as in the related Cx26M34A mutant