Michela Ghitti

Use of Metadynamics in the Design of isoDGR‐Based αvβ3 Antagonists To Fine‐Tune the Conformational Ensemble

The integrin family of cell-adhesion receptors regulates cellular functions crucial to the initiation, progression, and metastasis of solid tumors. In particular, integrin avb3 plays a key role in endothelial cell survival and migration during tumor angiogenesis. It is therefore gaining increasing importance as a drug target in antiangiogenic cancer therapy. The sequence Arg-Gly-Asp (RGD), which is contained in natural avb3 interactors, such as vitronectin, fibronectin, fibrinogen, osteopontin, and tenascin, is by far the most prominent ligand to promote specific cell adhesion through stimulation. This sequence is therefore attractive as a lead for the development of different integrin antagonists. Recent biochemical studies showed that deamidation of the NGR sequence gives rise to isoDGR, a new avb3-binding motif. This sequence constitutes a novel class of peptidic integrin ligands and paves the way to drug-design studies with a focus on the synthesis and characterization of a new generation of isoDGR-based macrocycles. For the design of low-molecular-mass isoDGR-containing molecules, an accurate determination of their biologically active conformation is a prerequisite. The presence of the b bond induces high flexibility in isoDGR-containing macrocycles and thus augments the range of accessible interconverting conformations. However, the identification of relevant conformations that might affect binding affinity is challenging for standard spectroscopic and diffraction techniques. Atomistic simulations, such as molecular dynamics (MD), replica-exchange molecular dynamics (REMD), and Monte Carlo (MC) simulations, can complement experimental data. However, they often fail to generate reliable equilibrium conformations because of the rugged and complex nature of the free-energy surface (FES) that is accessible to the system. As a consequence, computational sampling is often relegated to some local, unrealistic minima, which compromise subsequent docking studies. As computational drug design becomes increasingly reliant on virtual screening and on high-throughput 3D modeling, the need for fast and accurate computational methods for sampling of the ensemble of energetically accessible conformations is warranted. In this context, several techniques, including the local-elevation method, taboo search, the Wang–Landau method, adaptive force bias, conformational flooding, umbrella sampling, weighted histogram techniques, transitionstate theory, and path sampling, have been developed to address the sampling problem, through either reconstruction of the free energy or the direct acceleration of events that might happen on a long timescale (“rare events”). Related to these methods, metadynamics (MetaD) has emerged as a powerful coarse-grained non-Markovian molecular-dynamics approach for the acceleration of rare events and the efficient and rapid computation of multidimensional free-energy surfaces as a function of a restricted number of degrees of freedom, named collective variables (CVs). If the CVs are appropriately chosen for the system under investigation, MetaD directly provides a good estimate of the free energy of the system projected into the CVs (see the Supporting Information for details). Notably, the free energy is not immediately deducible by other sampling methods, such as umbrella sampling, in which the free-energy profile is not obtained directly from the simulations and requires an additional computational step, such as the weighted histogram analysis method (WHAM). In this study, we developed a protocol based on the combination of MetaD and docking simulations to analyze the conformations and the avb3-binding properties of isoDGR-containing cyclopeptides and to predict the conformational effects of chemical modifications and discriminate binders from nonbinders in silico. To investigate the conformational equilibrium of RGD-, DGR-, and isoDGRcontaining cyclopeptides (cyclization mode involving cysteine side chains) and to exhaustively explore their FESs, we performed well-temperedMetaD simulations, for which we chose Gly f and y angles as CVs (Figure 1; for simulation details, see the Supporting Information). As it lacks a side chain, Gly has large conformational freedom around its backbone dihedral angles. Therefore, Gly can explore a considerably larger area in the Ramachandran energy diagram than any other amino acid, and occupies five [*] Dr. A. Spitaleri, Dr. M. Ghitti, Dr. S. Mari, Dr. G. Musco Dulbecco Telethon Institute, Biomolecular NMR Laboratory c/o Center of Genomic and Bioinformatics S. Raffaele Scientific Institute via Olgettina 58, 20132 Milan (Italy) Fax: (+39)02-2643-4153 E-mail: giovanna.musco@hsr.it

2D TR‐NOESY Experiments Interrogate and Rank Ligand–Receptor Interactions in Living Human Cancer Cells

Recent advances in cancer therapy include the design ofmolecules that interfer with tumor angiogenesis and moietiesthat recognize specific receptors expressed onto the tumorendothelium and/or onto tumor cells, thus allowing theligand-directed targeted delivery of various drugs and par-ticles to tumors. In this context, integrin avb3 and themembrane-spanning surface protein aminopeptidase N(CD13) play a pivotal role in tumor growth and metastaticspread, as they are important membrane-bound receptorshighly expressed during angiogenesis.

Metadynamics Simulations Rationalise the Conformational Effects Induced by N‐Methylation of RGD Cyclic Hexapeptides

We combined metadynamics, docking and molecular mechanics/generalised born surface area (MM/GBSA) re-scoring methods to investigate the impact of single and multiple N-methylation on a set of RGD cyclopeptides displaying different affinity for integrin αIIbβ3. We rationalised the conformational effects induced by N-methylation and its interplay with receptor affinity, obtaining good agreement with experimental data. This approach can be exploited before entering time-consuming and expensive synthesis and binding experiments.

Identification of TMPRSS6 cleavage sites of hemojuvelin

Hemojuvelin (HJV), the coreceptor of the BMP‐SMAD pathway that up‐regulates hepcidin transcription, is a repulsive guidance molecule (RGMc) which undergoes a complex intracellular processing. Following autoproteolysis, it is exported to the cell surface both as a full‐length and a heterodimeric protein. In vitro membrane HJV (m‐HJV) is cleaved by the transmembrane serine protease TMPRSS6 to attenuate signalling and to inhibit hepcidin expression. In this study, we investigated the number and position of HJV cleavage sites by mutagenizing arginine residues (R), potential TMPRSS6 targets, to alanine (A). We analysed translation and membrane expression of HJV R mutants and the pattern of fragments they release in the culture media in the presence of TMPRSS6. Abnormal fragments were observed for mutants at arginine 121, 176, 218, 288 and 326. Considering that all variants, except HJVR121A, lack autoproteolytic activity and some (HJVR176A and HJVR288A) are expressed at reduced levels on cell surface, we identified the fragments originating from either full‐length or heterodimeric proteins and defined the residues 121 and 326 as the TMPRSS6 cleavage sites in both isoforms. Using the N‐terminal FLAG‐tagged HJV, we showed that residue 121 is critical also in the rearrangement of the N‐terminal heterodimeric HJV. Exploiting the recently reported RGMb crystallographic structure, we generated a model of HJV that was used as input structure for all‐atoms molecular dynamics simulation in explicit solvent. As assessed by in silico studies, we concluded that some arginines in the von Willebrand domain appear TMPRSS6 insensitive, likely because of partial protein structure destabilization.

NMR and Computational Methods in the Structural and Dynamic Characterization of Ligand-Receptor Interactions

The recurrent failures in drug discovery campaigns, the asymmetry between the enormous financial investments and the relatively scarce results have fostered the development of strategies based on complementary methods. In this context in recent years the rigid lock-and-key binding concept had to be revisited in favour of a dynamic model of molecular recognition accounting for conformational changes of both the ligand and the receptor. The high level of complexity required by a dynamic description of the processes underlying molecular recognition requires a multidisciplinary investigation approach. In this perspective, the combination of nuclear magnetic resonance spectroscopy with molecular docking, conformational searches along with molecular dynamics simulations has given new insights into the dynamic mechanisms governing ligand receptor interactions, thus giving an enormous contribution to the identification and design of new and effective drugs. Herein a succinct overview on the applications of both NMR and computational methods to the structural and dynamic characterization of ligand-receptor interactions will be presented.

Glycine N-Methylation in NGR-Tagged Nanocarriers Prevents Isoaspartate Formation and Integrin Binding without Impairing CD13 Recognition and Tumor Homing

NGR (asparagine-glycine-arginine) is a tumor vasculature-homing peptide motif widely used for the functionalization of drugs, nanomaterials and imaging compounds for cancer treatment and diagnosis. Unfortunately, this motif has a strong propensity to undergo rapid deamidation. This reaction, which converts NGR into isoDGR, is associated with receptor switching from CD13 to integrins, with potentially important manufacturing, pharmacological and toxicological implications. It is found that glycine N-methylation of NGR-tagged nanocarriers completely prevents asparagine deamidation without impairing CD13 recognition. Studies in animal models have shown that the methylated NGR motif can be exploited for delivering radiolabeled compounds and nanocarriers, such as tumor necrosis factor-α (TNF)-bearing nanogold and liposomal doxorubicin, to tumors with improved selectivity. These findings suggest that this NGR derivative is a stable and efficient tumor-homing ligand that can be used for delivering functional nanomaterials to tumor vasculature.

Structural basis for PHDVC5HCHNSD1-C2HRNizp1 interaction: implications for Sotos syndrome.

Sotos syndrome is an overgrowth syndrome caused by mutations within the functional domains of NSD1 gene coding for NSD1, a multidomain protein regulating chromatin structure and gene expression. In particular, PHDVC5HCHNSD1 tandem domain, composed by a classical (PHDV) and an atypical (C5HCH) plant homeo-domain (PHD) finger, is target of several pathological missense-mutations. PHDVC5HCHNSD1 is also crucial for NSD1-dependent transcriptional regulation and interacts with the C2HR domain of transcriptional repressor Nizp1 (C2HRNizp1) in vitro. To get molecular insights into the mechanisms dictating the patho-physiological relevance of the PHD finger tandem domain, we solved its solution structure and provided a structural rationale for the effects of seven Sotos syndrome point-mutations. To investigate PHDVC5HCHNSD1 role as structural platform for multiple interactions, we characterized its binding to histone H3 peptides and to C2HRNizp1 by ITC and NMR. We observed only very weak electrostatic interactions with histone H3 N-terminal tails, conversely we proved specific binding to C2HRNizp1. We solved C2HRNizp1 solution structure and generated a 3D model of the complex, corroborated by site-directed mutagenesis. We suggest a mechanistic scenario where NSD1 interactions with cofactors such as Nizp1 are impaired by PHDVC5HCHNSD1 pathological mutations, thus impacting on the repression of growth-promoting genes, leading to overgrowth conditions.

Dissecting intrinsic and ligand-induced structural communication in the β3 headpiece of integrins

BACKGROUND Graph theory is widely used to dissect structural communication in biomolecular systems. Here, graph theory-based approaches were applied to the headpiece of integrins, adhesion cell-surface receptors that transmit signals across the plasma membranes. METHODS Protein Structure Network (PSN) analysis incorporating dynamic information either from molecular dynamics simulations or from Elastic Network Models was applied to the β3 domains from integrins αVβ3 and αIIbβ3 in their apo and ligand-bound states. RESULTS Closed and open states of the β headpiece are characterized by distinct allosteric communication pathways involving highly conserved amino acids at the two different α/β interfaces in the βI domain, the closed state being prompted to the closed-to-open transition. In the closed state, pure antagonism is associated with the establishment of communication pathways that start from the ligand, pass through the β1/α3,α4 interface, and end up in the hybrid domain by involving the Y110-Q82 link, which is weakened in the agonist-bound states. CONCLUSIONS Allosteric communication in integrins relies on highly conserved and functionally relevant amino acid residues. The αβα-sandwich architecture of integrin βI domain dictates the structural communication between ligand binding site and hybrid domain. Differently from agonists, pure antagonists are directly involved in allosteric communication pathways and exert long-distance strengthening of the βI/hybrid interface. Release of the structure network in the ligand binding site is associated with the close-to-open transition accompanying the activation process. GENERAL SIGNIFICANCE The study strengthens the power of graph-based analyses to decipher allosteric communication intrinsic to protein folds and modified by functionally different ligands.

A rationally designed NRP1-independent superagonist SEMA3A mutant is an effective anticancer agent

An NRP1-independent, non-natural SEMA3A mutant isoform is a parenterally deliverable anticancer drug that normalizes the vasculature. Semaphoring to tumor vasculature Solid tumors typically have blood vessels that are not only increased in number but also exhibit various structural and functional abnormalities. Thus, vascular normalization is frequently proposed as an antitumor strategy, with the goal of improving intratumoral oxygenation and drug delivery. SEMA3A, a protein from the semaphorin family, is a known vascular normalizing agent but not a good therapeutic candidate due to adverse effects. To address this concern, Gioelli et al. engineered a mutant version of SEMA3A that retained its vascular normalizing function but did not activate the pathway responsible for the main side effects. The authors then confirmed the ability of mutant SEMA3A to normalize tumor vasculature and demonstrated its anticancer effects alone and combined with chemotherapy. Vascular normalizing strategies, aimed at ameliorating blood vessel perfusion and lessening tissue hypoxia, are treatments that may improve the outcome of cancer patients. Secreted class 3 semaphorins (SEMA3), which are thought to directly bind neuropilin (NRP) co-receptors that, in turn, associate with and elicit plexin (PLXN) receptor signaling, are effective normalizing agents of the cancer vasculature. Yet, SEMA3A was also reported to trigger adverse side effects via NRP1. We rationally designed and generated a safe, parenterally deliverable, and NRP1-independent SEMA3A point mutant isoform that, unlike its wild-type counterpart, binds PLXNA4 with nanomolar affinity and has much greater biochemical and biological activities in cultured endothelial cells. In vivo, when parenterally administered in mouse models of pancreatic cancer, the NRP1-independent SEMA3A point mutant successfully normalized the vasculature, inhibited tumor growth, curbed metastatic dissemination, and effectively improved the supply and anticancer activity of chemotherapy. Mutant SEMA3A also inhibited retinal neovascularization in a mouse model of age-related macular degeneration. In summary, mutant SEMA3A is a vascular normalizing agent that can be exploited to treat cancer and, potentially, other diseases characterized by pathological angiogenesis.

A persulfidation-based mechanism controls aquaporin-8 conductance

A two-step posttranslational modification of AQP8 provides a mechanism regulating plasma membrane H2O2 conductance. Upon engagement of tyrosine kinase receptors, nicotinamide adenine dinucleotide phosphate (NADPH)–oxidases release H2O2 in the extracellular space. We reported previously that aquaporin-8 (AQP8) transports H2O2 across the plasma membrane and is reversibly gated during cell stress, modulating signal strength and duration. We show that AQP8 gating is mediated by persulfidation of cysteine 53 (C53). Treatment with H2S is sufficient to block H2O2 entry in unstressed cells. Silencing cystathionine β-synthase (CBS) prevents closure, suggesting that this enzyme is the main source of H2S. Molecular modeling indicates that C53 persulfidation displaces a nearby histidine located in the narrowest part of the channel. We propose that H2O2 molecules transported through AQP8 sulfenylate C53, making it susceptible to H2S produced by CBS. This mechanism tunes H2O2 transport and may control signaling and limit oxidative stress.