Identification and characterization of novel candidate compounds targeting 6- and 7-transmembrane µ-opioid receptor isoforms.

BACKGROUND AND PURPOSE: The µ-opioid receptor (µ receptor) is the primary target for opioid analgesics. The 7-transmembrane (TM) and 6TM µ receptor isoforms mediate inhibitory and excitatory cellular effects. Here, we developed compounds selective for 6TM- or 7TM-µ receptors to further our understanding of the pharmacodynamic properties of µ receptors. EXPERIMENTAL APPROACH: We performed virtual screening of the ZINC Drug Now library of compounds using in silico 7TM- and 6TM-µ receptor structural models and identified potential compounds that are selective for 6TM- and/or 7TM-µ receptors. Subsequently, we characterized the most promising candidate compounds in functional in vitro studies using Be2C neuroblastoma transfected cells, behavioural in vivo pain assays using various knockout mice and in ex vivo electrophysiology studies. KEY RESULTS: Our virtual screen identified 30 potential candidate compounds. Subsequent functional in vitro cellular assays shortlisted four compounds (#5, 10, 11 and 25) that demonstrated 6TM- or 7TM-µ receptor-dependent NO release. In in vivo pain assays these compounds also produced dose-dependent hyperalgesic responses. Studies using mice that lack specific opioid receptors further established the µ receptor-dependent nature of identified novel ligands. Ex vivo electrophysiological studies on spontaneous excitatory postsynaptic currents in isolated spinal cord slices also validated the hyperalgesic properties of the most potent 6TM- (#10) and 7TM-µ receptor (#5) ligands. CONCLUSION AND IMPLICATIONS: Our novel compounds represent a new class of ligands for µ receptors and will serve as valuable research tools to facilitate the development of opioids with significant analgesic efficacy and fewer side-effects.

Stabilization of µ-opioid receptor facilitates its cellular translocation and signaling.

The G protein-coupled µ-opioid receptor (µ-OR) mediates the majority of analgesia effects for morphine and other pain relievers. Despite extensive studies of its structure and activation mechanisms, the inherently low maturation efficiency of µ-OR represents a major hurdle to understanding its function. Here we computationally designed µ-OR mutants with altered stability to probe the relationship between cell-surface targeting, signal transduction, and agonist efficacy. The stabilizing mutation T315Y enhanced µ-OR trafficking to the plasma membrane and significantly promoted the morphine-mediated inhibition of downstream signaling. In contrast, the destabilizing mutation R165Y led to intracellular retention of µ-OR and reduced the response to morphine stimulation. These findings suggest that µ-OR stability is an important factor in regulating receptor signaling and provide a viable avenue to improve the efficacy of analgesics.

Reversible and Tunable Photoswitching of Protein Function through Genetic Encoding of Azobenzene Amino Acids in Mammalian Cells.

The genetic encoding of three different azobenzene phenylalanines with different photochemical properties was achieved in human cells by using an engineered pyrrolysyl tRNA/tRNA synthetase pair. In order to demonstrate reversible light control of protein function, azobenzenes were site-specifically introduced into firefly luciferase. Computational strategies were applied to guide the selection of potential photoswitchable sites that lead to a reversibly controlled luciferase enzyme. In addition, the new azobenzene analogues provide enhanced thermal stability, high photoconversion, and responsiveness to visible light. These small-molecule photoswitches can reversibly photocontrol protein function with excellent spatiotemporal resolution, and preferred sites for incorporation can be computationally determined, thus providing a new tool for investigating biological processes.

miRNA-711 Binds and Activates TRPA1 Extracellularly to Evoke Acute and Chronic Pruritus.

Increasing evidence suggests that extracellular miRNAs may serve as biomarkers of diseases, but the physiological relevance of extracellular miRNA is unclear. We find that intradermal cheek injection of miR-711 induces TRPA1-depedent itch (scratching) without pain (wiping) in naive mice. Extracellular perfusion of miR-711 induces TRPA1 currents in both Trpa1-expressing heterologous cells and native sensory neurons through the core sequence GGGACCC. Computer simulations reveal that the core sequence binds several residues at the extracellular S5-S6 loop of TRPA1, which are critical for TRPA1 activation by miR-711 but not allyl isothiocyanate. Intradermal inoculation of human Myla cells induces lymphoma and chronic itch in immune-deficient mice, associated with increased serum levels of miR-711, secreted from cancer cells. Lymphoma-induced chronic itch is suppressed by miR-711 inhibitor and a blocking peptide that disrupts the miR-711/TRPA1 interaction. Our findings demonstrated an unconventional physiological role of extracellular naked miRNAs as itch mediators and ion channel modulators.

Molecular Insights into hERG Potassium Channel Blockade by Lubeluzole.

BACKGROUND/AIMS: Lubeluzole is a benzothiazole derivative that has shown neuroprotective properties in preclinical models of ischemic stroke. However, clinical research on lubeluzole is now at a standstill, since lubeluzole seems to be associated with the acquired long QT syndrome and ventricular arrhythmias. Since the cardiac cellular effects of lubeluzole have not been described thus far, an explanation for the lubeluzole-induced QT interval prolongation is lacking. METHODS: We tested the affinity of lubeluzole, its enantiomer, and the racemate for hERG channel using the patch-clamp technique. We synthesized and tested two simplified model compounds corresponding to two moieties included in the lubeluzole structure. The obtained experimental results were rationalized by docking simulation on the recently reported cryo-electron microscopy (cryo-EM) structure of hERG. Group efficiency analysis was performed in order to individuate the fragment most contributing to binding. RESULTS: We found that lubeluzole and its R enantiomer are highly potent inhibitors of human ether-ago-go-related gene (hERG) channel with an IC50 value of 12.9 ± 0.7 nM and 11.3 ± 0.8 nM, respectively. In the presence of lubeluzole, steady-state activation and inactivation of hERG channel were shifted to more negative potentials and inactivation kinetics was accelerated. Mutations of aromatic residues (Y652A and F656A) in the channel inner cavity significantly reduced the inhibitory effect of lubeluzole. Molecular docking simulations performed on the near atomic resolution cryo-electron microscopy structures of hERG supported the role of Y652 and F656 as the main contributors to high affinity binding. Group efficiency analysis indicated that both 1,3-benzothiazol-2-amine and 3-aryloxy-2-propanolamine moieties contribute to drug binding with the former giving higher contribution. CONCLUSIONS: This study suggests the possibility to modulate lubeluzole hERG blockade by introducing suitable substituents onto one or both constituting portions of the parent compound in order to either reduce potency (i. e. torsadogenic potential) or potentiate affinity (useful for class III antiarrhythmic and anticancer agent development).

Discovery of Novel Adenosine Receptor Antagonists through a Combined Structure- and Ligand-Based Approach Followed by Molecular Dynamics Investigation of Ligand Binding Mode.

An intense effort is made by pharmaceutical and academic research laboratories to identify and develop selective antagonists for each adenosine receptor (AR) subtype as potential clinical candidates for "soft" treatment of various diseases. Crystal structures of subtypes A2A and A1ARs offer exciting opportunities for structure-based drug design. In the first part of the present work, Maybridge HitFinder library of 14400 compounds was utilized to apply a combination of structure-based against the crystal structure of A2AAR and ligand-based methodologies. The docking poses were rescored by CHARMM energy minimization and calculation of the desolvation energy using Poisson-Boltzmann equation electrostatics. Out of the eight selected and tested compounds, five were found positive hits (63% success). Although the project was initially focused on targeting A2AAR, the identified antagonists exhibited low micromolar or micromolar affinity against A2A/A3, ARs, or A3AR, respectively. Based on these results, 19 compounds characterized by novel chemotypes were purchased and tested. Sixteen of them were identified as AR antagonists with affinity toward combinations of the AR family isoforms (A2A/A3, A1/A3, A1/A2A/A3, and A3). The second part of this work involves the performance of hundreds of molecular dynamics (MD) simulations of complexes between the ARs and a total of 27 ligands to resolve the binding interactions of the active compounds, which were not achieved by docking calculations alone. This computational work allowed the prediction of stable and unstable complexes which agree with the experimental results of potent and inactive compounds, respectively. Of particular interest is that the 2-amino-thiophene-3-carboxamides, 3-acylamino-5-aryl-thiophene-2-carboxamides, and carbonyloxycarboximidamide derivatives were found to be selective and possess a micromolar to low micromolar affinity for the A3 receptor.

Molecular Mechanisms of the R61T Mutation in Apolipoprotein E4: A Dynamic Rescue.

The apolipoprotein E4 (ApoE4) gene is the strongest genetic risk factor for Alzheimer's disease (AD). With respect to the other common isoforms of this protein (ApoE2 and ApoE3), ApoE4 is characterized by lower stability that underlies the formation of a stable interaction between the protein's N- and C-terminal domains. AD-related cellular dysfunctions have been linked to this ApoE4 misfolded state. In this regard, it has been reported that the mutation R61T is able to rescue the deleterious cellular effects of ApoE4 by preventing the formation of the misfolded intermediate state. However, a clear description of the structural features at the basis of the R61T-ApoE4 mutant's protective effect is still missing. Recently, using extensive molecular dynamics simulations, we have identified a structural model of an ApoE4 misfolded intermediate state. Building on our previous work, here we explore the dynamical changes induced by the R61T mutation in the ApoE4 native and misfolded states. Notably, we do not observe any local changes in the domains in the R61T-ApoE4 system, rather a general loss of correlated movements in the entire protein structure. More specifically, we detect increased dynamics in the hinge region, which is essential for ApoE4 domain-domain interaction. Consistent with previously reported data on altered phospholipid and receptor binding, we hypothesize that mutations destabilizing the ApoE4 intermediate state change hinge region dynamics, which propagates to distal functional regions of the protein and modifies ApoE4's functional properties. This unique behavior of the ApoE4 hinge region provides, to our knowledge, a novel understanding of ApoE4's role in AD.

Reducing the Flexibility of Type†II Dehydroquinase for Inhibition: A Fragment-Based Approach and Molecular Dynamics Study.

A multidisciplinary approach was used to identify and optimize a quinazolinedione-based ligand that would decrease the flexibility of the substrate-covering loop (catalytic loop) of the type II dehydroquinase from Helicobacter pylori. This enzyme, which is essential for the survival of this bacterium, is involved in the biosynthesis of aromatic amino acids. A computer-aided fragment-based protocol (ALTA) was first used to identify the aromatic fragments able to block the interface pocket that separates two neighboring enzyme subunits and is located at the active site entrance. Chemical modification of its non-aromatic moiety through an olefin cross-metathesis and Seebach's self-reproduction of chirality synthetic principle allowed the development of a quinazolinedione derivative that disables the catalytic loop plasticity, which is essential for the enzyme's catalytic cycle. Molecular dynamics simulations revealed that the ligand would force the catalytic loop into an inappropriate arrangement for catalysis by strong interactions with the catalytic tyrosine and by expelling the essential arginine out of the active site.

Computational Modeling of Small Molecule Ligand Binding Interactions and Affinities.

Understanding and controlling biological phenomena via structure-based drug screening efforts often critically rely on accurate description of protein-ligand interactions. However, most of the currently available computational techniques are affected by severe deficiencies in both protein and ligand conformational sampling as well as in the scoring of the obtained docking solutions. To overcome these limitations, we have recently developed MedusaDock, a novel docking methodology, which simultaneously models ligand and receptor flexibility. Coupled with MedusaScore, a physical force field-based scoring function that accounts for the protein-ligand interaction energy, MedusaDock, has reported the highest success rate in the CSAR 2011 exercise. Here, we present a standard computational protocol to evaluate the binding properties of the two enantiomers of the non-selective ß-blocker propanolol in the ß2 adrenergic receptor's binding site. We describe details of our protocol, which have been successfully applied to several other targets.

Docking and Scoring with Target-Specific Pose Classifier Succeeds in Native-Like Pose Identification But Not Binding Affinity Prediction in the CSAR 2014 Benchmark Exercise.

The CSAR 2014 exercise provided an important benchmark for testing current approaches for pose identification and ligand ranking using three X-ray characterized proteins: Factor Xa (FXa), Spleen Tyrosine Kinase (SYK), and tRNA Methyltransferase (TRMD). In Phase 1 of the exercise, we employed Glide and MedusaDock docking software, both individually and in combination, with the special target-specific pose classifier trained to discriminate native-like from decoy poses. All approaches succeeded in the accurate detection of native and native-like poses. We then used Glide SP and MedusaScore scoring functions individually and in combination with the pose-scoring approach to predict relative binding affinities of the congeneric series of ligands in Phase 2 of the exercise. Similar to other participants in the CSAR 2014 exercise, we found that our models showed modest prediction accuracy. Quantitative structure-activity relationship (QSAR) models developed for the FXa ligands using available bioactivity data from ChEMBL showed relatively low prediction accuracy for the CSAR 2014 ligands of the same target. Interestingly, QSAR models built with CSAR data only yielded Spearman correlation coefficients as high as ρ = 0.69 for FXa and ρ = 0.79 for SYK based on 5-fold cross-validation. Virtual screening of the DUD library using the FXa structure was successful in discriminating between active compounds and decoys in spite of poor ranking accuracy of the underlying scoring functions. Our results suggest that two of the three common tasks associated with molecular docking, i.e., native-like pose identification and virtual screening, but not binding affinity prediction, could be accomplished successfully for the CSAR 2014 challenge data set.

Pharmacological Chaperones: Design and Development of New Therapeutic Strategies for the Treatment of Conformational Diseases.

Errors in protein folding may result in premature clearance of structurally aberrant proteins, or in the accumulation of toxic misfolded species or protein aggregates. These pathological events lead to a large range of conditions known as conformational diseases. Several research groups have presented possible therapeutic solutions for their treatment by developing novel compounds, known as pharmacological chaperones. These cell-permeable molecules selectively provide a molecular scaffold around which misfolded proteins can recover their native folding and, thus, their biological activities. Here, we review therapeutic strategies, clinical potentials, and cost-benefit impacts of several classes of pharmacological chaperones for the treatment of a series of conformational diseases.

The Molecular Basis for Dual Fatty Acid Amide Hydrolase (FAAH)/Cyclooxygenase (COX) Inhibition.

The design of multitarget-directed ligands is a promising strategy for discovering innovative drugs. Here, we report a mechanistic study that clarifies key aspects of the dual inhibition of the fatty acid amide hydrolase (FAAH) and the cyclooxygenase (COX) enzymes by a new multitarget-directed ligand named ARN2508 (2-[3-fluoro-4-[3-(hexylcarbamoyloxy)phenyl]phenyl]propanoic acid). This potent dual inhibitor combines, in a single scaffold, the pharmacophoric elements often needed to block FAAH and COX, that is, a carbamate moiety and the 2-arylpropionic acid functionality, respectively. Molecular modeling and molecular dynamics simulations suggest that ARN2508 uses a noncovalent mechanism of inhibition to block COXs, while inhibiting FAAH via the acetylation of the catalytic Ser241, in line with previous experimental evidence for covalent FAAH inhibition. This study proposes the molecular basis for the dual FAAH/COX inhibition by this novel hybrid scaffold, stimulating further experimental studies and offering new insights for the rational design of novel anti-inflammatory agents that simultaneously act on FAAH and COX.

Structural and functional interactions between six-transmembrane µ-opioid receptors and ß2-adrenoreceptors modulate opioid signaling.

The primary molecular target for clinically used opioids is the µ-opioid receptor (MOR). Besides the major seven-transmembrane (7TM) receptors, the MOR gene codes for alternatively spliced six-transmembrane (6TM) isoforms, the biological and clinical significance of which remains unclear. Here, we show that the otherwise exclusively intracellular localized 6TM-MOR translocates to the plasma membrane upon coexpression with ß2-adrenergic receptors (ß2-ARs) through an interaction with the fifth and sixth helices of ß2-AR. Coexpression of the two receptors in BE(2)-C neuroblastoma cells potentiates calcium responses to a 6TM-MOR ligand, and this calcium response is completely blocked by a selective ß2-antagonist in BE(2)-C cells, and in trigeminal and dorsal root ganglia. Co-administration of 6TM-MOR and ß2-AR ligands leads to substantial analgesic synergy and completely reverses opioid-induced hyperalgesia in rodent behavioral models. Together, our results provide evidence that the heterodimerization of 6TM-MOR with ß2-AR underlies a molecular mechanism for 6TM cellular signaling, presenting a unique functional responses to opioids. This signaling pathway may contribute to the hyperalgesic effects of opioids that can be efficiently blocked by ß2-AR antagonists, providing a new avenue for opioid therapy.

Differential Regulation of 6- and 7-Transmembrane Helix Variants of µ-Opioid Receptor in Response to Morphine Stimulation.

The pharmacological effect of opioids originates, at the cellular level, by their interaction with the µ-opioid receptor (mOR) resulting in the regulation of voltage-gated Ca2+ channels and inwardly rectifying K+ channels that ultimately modulate the synaptic transmission. Recently, an alternative six trans-membrane helix isoform of mOR, (6TM-mOR) has been identified, but its function and signaling are still largely unknown. Here, we present the structural and functional mechanisms of 6TM-mOR signaling activity upon binding to morphine. Our data suggest that despite the similarity of binding modes of the alternative 6TM-mOR and the dominant seven trans-membrane helix variant (7TM-mOR), the interaction with morphine generates different dynamic responses in the two receptors, thus, promoting the activation of different mOR-specific signaling pathways. We characterize a series of 6TM-mOR-specific cellular responses, and observed that they are significantly different from those for 7TM-mOR. Morphine stimulation of 6TM-mOR does not promote a cellular cAMP response, while it increases the intracellular Ca2+ concentration and reduces the cellular K+ conductance. Our findings indicate that 6TM-mOR has a unique contribution to the cellular opioid responses. Therefore, it should be considered as a relevant target for the development of novel pharmacological tools and medical protocols involving the use of opioids.

ApoE4-specific Misfolded Intermediate Identified by Molecular Dynamics Simulations.

The increased risk of developing Alzheimer's disease (AD) is associated with the APOE gene, which encodes for three variants of Apolipoprotein E, namely E2, E3, E4, differing only by two amino acids at positions 112 and 158. ApoE4 is known to be the strongest risk factor for AD onset, while ApoE3 and ApoE2 are considered to be the AD-neutral and AD-protective isoforms, respectively. It has been hypothesized that the ApoE isoforms may contribute to the development of AD by modifying the homeostasis of ApoE physiological partners and AD-related proteins in an isoform-specific fashion. Here we find that, despite the high sequence similarity among the three ApoE variants, only ApoE4 exhibits a misfolded intermediate state characterized by isoform-specific domain-domain interactions in molecular dynamics simulations. The existence of an ApoE4-specific intermediate state can contribute to the onset of AD by altering multiple cellular pathways involved in ApoE-dependent lipid transport efficiency or in AD-related protein aggregation and clearance. We present what we believe to be the first structural model of an ApoE4 misfolded intermediate state, which may serve to elucidate the molecular mechanism underlying the role of ApoE4 in AD pathogenesis. The knowledge of the structure for the ApoE4 folding intermediate provides a new platform for the rational design of alternative therapeutic strategies to fight AD.

Differences in the Antinociceptive Effects and Binding Properties of Propranolol and Bupranolol Enantiomers.

UNLABELLED: Recent efforts have suggested that the ß-adrenergic receptor (ß-AR) system may be a novel and viable therapeutic target for pain reduction; however, most of the work to date has focused on the ß(2)-adrenergic receptor (AR). Here, we compared the antinociceptive effects of enantiomeric configurations of propranolol and bupranolol, two structurally similar nonselective ß-blocking drugs, against mouse models of inflammatory and chronic pain. In addition, we calculated in silico docking and measured the binding properties of propranolol and bupranolol for all 3 ß-ARs. Of the agents examined, S-bupranolol is superior in terms of its antinociceptive effect and exhibited fewer side effects than propranolol or its associated enantiomers. In contrast to propranolol, S-bupranolol exhibited negligible ß-AR intrinsic agonist activity and displayed a full competitive antagonist profile at ß(1)/ß(2)/ß(3)-ARs, producing a unique blockade of ß(3)-ARs. We have shown that S-bupranolol is an effective antinociceptive agent in mice without negative side effects. The distinctive profile of S-bupranolol is most likely mediated by its negligible ß-AR intrinsic agonist activity and unique blockade of ß(3)-AR. These findings suggest that S-bupranolol instead of propranolol may represent a new and effective treatment for a variety of painful conditions. PERSPECTIVE: The S enantiomer of bupranolol, a ß-receptor antagonist, shows greater antinociceptive efficacy and a superior preclinical safety profile and it should be considered as a unique ß-adrenergic receptor compound to advance future clinical pain studies.

An optimized polyamine moiety boosts the potency of human type II topoisomerase poisons as quantified by comparative analysis centered on the clinical candidate F14512.

Combined computational-experimental analyses explain and quantify the spermine-vectorized F14512's boosted potency as a topoII poison. We found that an optimized polyamine moiety boosts drug binding to the topoII/DNA cleavage complex, rather than to the DNA alone. These results provide new structural bases and key reference data for designing new human topoII poisons.

COMT gene locus: new functional variants.

Catechol-O-methyltransferase (COMT) metabolizes catecholaminergic neurotransmitters. Numerous studies have linked COMT to pivotal brain functions such as mood, cognition, response to stress, and pain. Both nociception and risk of clinical pain have been associated with COMT genetic variants, and this association was shown to be mediated through adrenergic pathways. Here, we show that association studies between COMT polymorphic markers and pain phenotypes in 2 independent cohorts identified a functional marker, rs165774, situated in the 3' untranslated region of a newfound splice variant, (a)-COMT. Sequence comparisons showed that the (a)-COMT transcript is highly conserved in primates, and deep sequencing data demonstrated that (a)-COMT is expressed across several human tissues, including the brain. In silico analyses showed that the (a)-COMT enzyme features a distinct C-terminus structure, capable of stabilizing substrates in its active site. In vitro experiments demonstrated not only that (a)-COMT is catalytically active but also that it displays unique substrate specificity, exhibiting enzymatic activity with dopamine but not epinephrine. They also established that the pain-protective A allele of rs165774 coincides with lower COMT activity, suggesting contribution to decreased pain sensitivity through increased dopaminergic rather than decreased adrenergic tone, characteristic of reference isoforms. Our results provide evidence for an essential role of the (a)-COMT isoform in nociceptive signaling and suggest that genetic variations in (a)-COMT isoforms may contribute to individual variability in pain phenotypes.

µ-Opioid receptor 6-transmembrane isoform: A potential therapeutic target for new effective opioids.

The µ-opioid receptor (MOR) is the primary target for opioid analgesics. MOR induces analgesia through the inhibition of second messenger pathways and the modulation of ion channels activity. Nevertheless, cellular excitation has also been demonstrated, and proposed to mediate reduction of therapeutic efficacy and opioid-induced hyperalgesia upon prolonged exposure to opioids. In this mini-perspective, we review the recently identified, functional MOR isoform subclass, which consists of six transmembrane helices (6 TM) and may play an important role in MOR signaling. There is evidence that 6 TM MOR signals through very different cellular pathways and may mediate excitatory cellular effects rather than the classic inhibitory effects produced by the stimulation of the major (7 TM) isoform. Therefore, the development of 6 TM and 7 TM MOR selective compounds represents a new and exciting opportunity to better understand the mechanisms of action and the pharmacodynamic properties of a new class of opioids.

Carnosine inhibits Aß(42) aggregation by perturbing the H-bond network in and around the central hydrophobic cluster.

Aggregation of the amyloid-ß peptide (Aß) into fibrillar structures is a hallmark of Alzheimer's disease. Thus, preventing self-assembly of the Aß peptide is an attractive therapeutic strategy. Here, we used experimental techniques and atomistic simulations to investigate the influence of carnosine, a dipeptide naturally occurring in the brain, on Aß aggregation. Scanning force microscopy, circular dichroism and thioflavin T fluorescence experiments showed that carnosine does not modify the conformational features of Aß42 but nonetheless inhibits amyloid growth. Molecular dynamics (MD) simulations indicated that carnosine interacts transiently with monomeric Aß42 by salt bridges with charged side chains, and van der Waals contacts with residues in and around the central hydrophobic cluster ((17)LVFFA(21)). NMR experiments on the nonaggregative fragment Aß12-28 did not evidence specific intermolecular interactions between the peptide and carnosine, in agreement with MD simulations. However, a close inspection of the spectra revealed that carnosine interferes with the local propensity of the peptide to form backbone hydrogen bonds close to the central hydrophobic cluster (residues E22, S26 and N27). Finally, MD simulations of aggregation-prone Aß heptapeptide segments show that carnosine reduces the propensity to form intermolecular backbone hydrogen bonds in the region 18-24. Taken together, the experimental and simulation results (cumulative MD sampling of 0.2 ms) suggest that, despite the inability of carnosine to form stable contacts with Aß, it might block the pathway toward toxic aggregates by perturbing the hydrogen bond network near residues with key roles in fibrillogenesis.