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Protein-assembly defects due to an enrichment of aberrant conformational protein variants are emerging as a new frontier in therapeutics design. Understanding the structural elements that rewire the conformational dynamics of proteins and pathologically perturb functionally oriented ensembles is important for inhibitor development. Chaperones are hub proteins for the assembly of multiprotein complexes and an enrichment of aberrant conformers can affect the cellular proteome, and in turn, phenotypes. Here, we integrate computational and experimental tools to investigte how N-glycosylation of specific residues in glucose-regulated protein 94 (GRP94) modulates internal dynamics and alters the conformational fitness of regions fundamental for the interaction with ATP and synthetic ligands and impacts substructures important for the recognition of interacting proteins. N-glycosylation plays an active role in modulating the energy landscape of GRP94, and we provide support for leveraging the knowledge on distinct glycosylation variants to design molecules targeting GRP94 disease-associated conformational states and assemblies., Competing Interests: Declaration of interests G. Chiosis, A.R., C.S.D., and P.Y. are inventors on patents covering PU-WS13 and associated composition of matter. G. Chiosis is a founder of Samus Therapeutics., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

SARS-CoV-2 has evolved rapidly in the first 3 years of pandemic diffusion. The initial evolution of the virus appeared to proceed through big jumps in sequence changes rather than through the stepwise accumulation of point mutations on already established variants. Here, we examine whether this nonlinear mutational process reverberates in variations of the conformational dynamics of the SARS-CoV-2 Spike protein (S-protein), the first point of contact between the virus and the human host. We run extensive microsecond-scale molecular dynamics simulations of seven distinct variants of the protein in their fully glycosylated state and set out to elucidate possible links between the mutational spectrum of the S-protein and the structural dynamics of the respective variant, at global and local levels. The results reveal that mutation-dependent structural and dynamic modulations mostly consist of increased coordinated motions in variants that acquire stability and in an increased internal flexibility in variants that are less stable. Importantly, a limited number of functionally important substructures (the receptor binding domain, in particular) share the same time of movements in all variants, indicating efficient preorganization for functional regions dedicated to host interactions. Our results support a model in which the internal dynamics of the S-proteins from different strains varies in a way that reflects the observed random and non-stepwise jumps in sequence evolution, while conserving the functionally oriented traits of conformational dynamics necessary to support productive interactions with host receptors.

Enteric hyperoxaluria (EH) is a known complication of Roux-en-Y gastric bypass (RYGB) and can lead to nephrolithiasis, oxalate-induced nephropathy, and end-stage renal disease. Recurrent EH-induced renal impairment has been reported after kidney transplantation and may lead to allograft loss. EH occurs in up to one quarter of patients following malabsorption-based bariatric operations. We present a report of medically refractory EH in a renal transplant recipient with allograft dysfunction that was successfully managed with reversal of RYGB. The patient developed renal failure 7 years following gastric bypass requiring renal transplant. Following an uneventful living donor kidney transplant, the patient developed recurrent subacute allograft dysfunction. A diagnosis of oxalate nephropathy was made based on biopsy findings of renal tubular calcium oxalate deposition in conjunction with elevated serum oxalate levels and elevated 24-hr urinary oxalate excretion. Progressive renal failure ensued despite medical management. The patient underwent reversal of her RYGB, which resulted in recovery of allograft function. This report highlights an under-recognized, potentially treatable cause of renal allograft failure in patients with underlying gastrointestinal pathology or history of bariatric surgery and proposes a strategy for management of patients with persistent hyperoxaluria based on a review of the literature.

Introduction: Superobesity (SO) is defined as a BMI > 50 Kg/m 2 , and represents the extreme severity of the disease, resulting in a challenge for the surgeons., Methods: In this retrospective study we aimed to compare the outcomes of SO patients compared to morbidly obese (MO) patients., Results: We included in this study 154 MO patients, with a median preoperative BMI of 40.8 kg/m 2 , and 19 SO patients with median preoperative BMI of 54.9 kg/m 2 . The MO patients underwent sleeve gastrectomy (SG) in 62 (40.3%) cases, laparoscopic Roux-and-Y gastric bypass (LRYGBP) in 85 (55.2%) cases and One-Anastomosis Gastric Bypass (OAGB) in 7 (4.5%) cases. underwent OAGB. The patients in the SO group were submitted to SG in 11 (57.9%) cases, LRYGBP in 5 (26.3%) cases, and OAGB in 3 (15.8%). At 24-month follow-up, an excess weight loss (EWL) >50% was achieved in 129 (83.8%) patients in the MO group and in 15 (78.9%) in the SO group ( p  = 0.53). A BMI < 35 kg/m 2 was achieved in 137 (89%) patients in the MO group and from 8 (42.2%) patients in the SO group ( p  < 0.001). The total weight loss was significantly directly related to the initial BMI. Superobesity was identified as independent risk factor for surgical failure when considering the outcome of BMI < 35 kg/m 2 ., Discussion: Our study confirms that, although SO patients tend to gain a greater weight loss than MO patients, they less frequently achieve the desired BMI target. In this setting, it should be necessary to re-consider malabsorptive procedures as first choice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Medas, Moroni, Deidda, Zorcolo, Restivo, Canu, Cappellacci, Calò, Pintus and Fantola.)

Protein-protein interactions (PPIs) have emerged in the past years as significant pharmacological targets in the development of new therapeutics due to their key roles in determining pathological pathways. Herein, we present fragments on energy surfaces, a simple and general design strategy that integrates the analysis of the dynamic and energetic signatures of proteins to unveil the substructures involved in PPIs, with docking, selection, and combination of drug-like fragments to generate new PPI inhibitor candidates. Specifically, structural representatives of the target protein are used as inputs for the blind physics-based prediction of potential protein interaction surfaces using the matrix of low coupling energy decomposition method. The predicted interaction surfaces are subdivided into overlapping windows that are used as templates to direct the docking and combination of fragments representative of moieties typically found in active drugs. This protocol is then applied and validated using structurally diverse, important PPI targets as test systems. We demonstrate that our approach facilitates the exploration of the molecular diversity space of potential ligands, with no requirement of prior information on the location and properties of interaction surfaces or on the structures of potential lead compounds. Importantly, the hit molecules that emerge from our ab initio design share high chemical similarity with experimentally tested active PPI inhibitors. We propose that the protocol we describe here represents a valuable means of generating initial leads against difficult targets for further development and refinement.

Affitins constitute a class of small proteins belonging to Sul7d family, which, in microorganisms such as Sulfolobus acidocaldarius , bind DNA preventing its denaturation. Thanks to their stability and small size (60-66 residues in length) they have been considered as ideal candidates for engineering and have been used for more than 10 years now, for different applications. The individuation of a mutant able to recognize a specific target does not imply the knowledge of the binding geometry between the two proteins. However, its identification is of undoubted importance but not always experimentally accessible. For this reason, computational approaches such as protein-protein docking can be helpful for an initial structural characterization of the complex. This method, which produces tens of putative binding geometries ordered according to a binding score, needs to be followed by a further reranking procedure for finding the most plausible one. In the present paper, we use the server ClusPro for generating docking models of affitins with different protein partners whose experimental structures are available in the Protein Data Bank. Then, we apply two protocols for reranking the docking models. The first one investigates their stability by means of Molecular Dynamics simulations; the second one, instead, compares the docking models with the interacting residues predicted by the Matrix of Local Coupling Energies method. Results show that the more efficient way to deal with the reranking problem is to consider the information given by the two protocols together, i.e. employing a consensus approach., Competing Interests: Author AM is employed by Bracco S.p.A. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ranaudo, Cosentino, Greco, Moro, Bonardi, Maiocchi and Moroni.)

Cadherins are cell-surface proteins that mediate cell-cell adhesion. By regulating their grip formation and strength, cadherins play a pivotal role during normal tissue morphogenesis and homeostasis of multicellular organisms. However, their dysfunction is associated with cell migration and proliferation, cancer progression and metastasis. The conserved amino acid sequence His-Ala-Val (HAV) in the extracellular domain of cadherins is implicated in cadherin-mediated adhesion and migration. Antagonists of cadherin adhesion such as monoclonal antibodies and small molecule inhibitors based on HAV peptides, are of high therapeutic value in cancer treatment. However, antibodies are not stable outside their natural environment and are expensive to produce, while peptides have certain limitations as a drug as they are prone to proteolysis. Herein, we propose as alternative, a synthetic antibody based on molecularly imprinted polymer nanogels (MIP-NGs) to target the HAV domain. The MIP-NGs are biocompatible, have high affinity for N-cadherin and inhibit cell adhesion and migration of human cervical adenocarcinoma (HeLa) cells, as demonstrated by cell aggregation and Matrigel invasion assays, respectively. The emergence of MIPs as therapeutics for fighting cancer is still in its infancy and this novel demonstration reinforces the fact that they have a rightful place in cancer treatment.

Herein we examine the determinants of the allosteric inhibition of the mitochondrial chaperone TRAP1 by a small molecule ligand. The knowledge generated is harnessed into the design of novel derivatives with interesting biological properties. TRAP1 is a member of the Hsp90 family of proteins, which work through sequential steps of ATP processing coupled to client-protein remodeling. Isoform selective inhibition of TRAP1 can provide novel information on the biomolecular mechanisms of molecular chaperones, as well as new insights into the development of small molecules with therapeutic potential. Our analysis of the interactions between an active first-generation allosteric ligand and TRAP1 shows how the small molecule induces long-range perturbations that influence the attainment of reactive poses in the active site. At the same time, the dynamic adaptation of the allosteric binding pocket to the presence of the first-generation compound sets the stage for the design of a set of second-generation ligands: the characterization of the formation/disappearance of pockets around the allosteric site that is used to guide optimize the ligands' fit for the allosteric site and improve inhibitory activities. The effects of the newly designed molecules are validated experimentally in vitro and in vivo. We discuss the implications of our approach as a promising strategy towards understanding the molecular determinants of allosteric regulation in chemical and molecular biology, and towards speeding up the design of allosteric small molecule modulators., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The tiger mosquito ( Aedes albopictus) is one of the most invasive species in the world and a competent vector for numerous arboviruses, thus the study and monitoring of its fast worldwide spread is crucial for global public health. The small extra-nuclear and maternally-inherited mitochondrial DNA represents a key tool for reconstructing phylogenetic and phylogeographic relationships within a species, especially when analyzed at the mitogenome level. Here the mitogenome variation of 76 tiger mosquitoes, 37 of which new and collected from both wild adventive populations and laboratory strains, was investigated. This analysis significantly improved the global mtDNA phylogeny of Ae. albopictus , uncovering new branches and sub-branches within haplogroup A1, the one involved in its recent worldwide spread. Our phylogeographic approach shows that the current distribution of tiger mosquito mitogenome variation has been strongly affected by clonal and sub-clonal founder events, sometimes involving wide geographic areas, even across continents, thus shedding light on the Asian sources of worldwide adventive populations. In particular, different starting points for the two major clades within A1 are suggested, with A1a spreading mainly along temperate areas from Japanese and Chinese sources, and A1b arising and mainly diffusing in tropical areas from a South Asian source., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Battaglia, Agostini, Moroni, Colombo, Lombardo, Rambaldi Migliore, Gabrieli, Garofalo, Gagliardi, Gomulski, Ferretti, Semino, Malacrida, Gasperi, Achilli, Torroni and Olivieri.)

Multifunctional human collagen lysyl hydroxylase (LH/PLOD) enzymes catalyze post-translational hydroxylation and subsequent glycosylation of collagens, enabling their maturation and supramolecular organization in the extracellular matrix (ECM). Recently, the overexpression of LH/PLODs in the tumor microenvironment results in abnormal accumulation of these collagen post-translational modifications, which has been correlated with increased metastatic progression of a wide variety of solid tumors. These observations make LH/PLODs excellent candidates for prospective treatment of aggressive cancers. The recent years have witnessed significant research efforts to facilitate drug discovery on LH/PLODs, including molecular structure characterizations and development of reliable high-throughput enzymatic assays. Using a combination of biochemistry and in silico studies, we characterized the dual role of Fe 2+ as simultaneous cofactor and inhibitor of lysyl hydroxylase activity and studied the effect of a promiscuous Fe 2+ chelating agent, 2,2'-bipyridil, broadly considered a lysyl hydroxylase inhibitor. We found that at low concentrations, 2,2'-bipyridil unexpectedly enhances the LH enzymatic activity by reducing the inhibitory effect of excess Fe 2+ . Together, our results show a fine balance between Fe 2+ -dependent enzymatic activity and Fe 2+ -induced self-inhibited states, highlighting exquisite differences between LH/PLODs and related Fe 2+ , 2-oxoglutarate dioxygenases and suggesting that conventional structure-based approaches may not be suited for successful inhibitor development. These insights address outstanding questions regarding druggability of LH/PLOD lysyl hydroxylase catalytic site and provide a solid ground for upcoming drug discovery and screening campaigns., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Scietti, Moroni, Mattoteia, Fumagalli, De Marco, Negro, Chiapparino, Serapian, De Giorgi, Faravelli, Colombo and Forneris.)

Molecular dynamics (MD) simulations are coming of age in the study of nucleic acids, including specific tertiary structures such as G-quadruplexes. While being precious for providing structural and dynamic information inaccessible to experiments at the atomistic level of resolution, MD simulations in this field may still be limited by several factors. These include the force fields used, different models for ion parameters, ionic strengths, and water models. We address various aspects of this problem by analyzing and comparing microsecond-long atomistic simulations of the G-quadruplex structure formed by the human immunodeficiency virus long terminal repeat (HIV LTR)-III sequence for which nuclear magnetic resonance (NMR) structures are available. The system is studied in different conditions, systematically varying the ionic strengths, ion numbers, and water models. We comparatively analyze the dynamic behavior of the G-quadruplex motif in various conditions and assess the ability of each simulation to satisfy the nuclear magnetic resonance (NMR)-derived experimental constraints and structural parameters. The conditions taking into account K + -ions to neutralize the system charge, mimicking the intracellular ionic strength, and using the four-atom water model are found to be the best in reproducing the experimental NMR constraints and data. Our analysis also reveals that in all of the simulated environments residues belonging to the duplex moiety of HIV LTR-III exhibit the highest flexibility.

The barn swallow (Hirundo rustica) poses a number of fascinating scientific questions, including the taxonomic status of postulated subspecies. Here, we obtained and assessed the sequence variation of 411 complete mitogenomes, mainly from the European H. r. rustica, but other subspecies as well. In almost every case, we observed subspecies-specific haplogroups, which we employed together with estimated radiation times to postulate a model for the geographical and temporal worldwide spread of the species. The female barn swallow carrying the Hirundo rustica ancestral mitogenome left Africa (or its vicinity) around 280 thousand years ago (kya), and her descendants expanded first into Eurasia and then, at least 51 kya, into the Americas, from where a relatively recent (<20 kya) back migration to Asia took place. The exception to the haplogroup subspecies specificity is represented by the sedentary Levantine H. r. transitiva that extensively shares haplogroup A with the migratory European H. r. rustica and, to a lesser extent, haplogroup B with the Egyptian H. r. savignii. Our data indicate that rustica and transitiva most likely derive from a sedentary Levantine population source that split at the end of the Younger Dryas (YD) (11.7 kya). Since then, however, transitiva received genetic inputs from and admixed with both the closely related rustica and the adjacent savignii. Demographic analyses confirm this species' strong link with climate fluctuations and human activities making it an excellent indicator for monitoring and assessing the impact of current global changes on wildlife., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Laparoscopy is the surgical standard of care for bariatric procedures; however, during the last two decades, the robotic approach has gained increasing interest. It is currently considered a safe and effective alternative to laparoscopy. This literature review investigates the role of the robotic approach for primary and revisional bariatric procedures, with the particular aim of comparing this technique with the standard-of-care laparoscopic approach. The feasibility of robotic dissection and suturing could have potential advantages: robotics may prevent the risk of leak and bleeding and other surgical complications, determining potential benefits in terms of operative time, length of hospital stay, and learning curve. Considering primary procedures, the literature reveals no advantages in robotic versus the laparoscopic approach for adjustable gastric banding and sleeve gastrectomy. Robotic Roux-en-Y gastric bypass is associated with a longer operative time and a shorter hospital length of stay than laparoscopy. The robotic approach in revisional surgery has been proven to be safe and effective. Despite the longer operative time, the robotic platform could achieve a lower bleeding rate compared with laparoscopy. The surgeon's selection criteria related to referrals to the robotic approach of difficult-perceived cases could represent a bias. In conclusion, robotic surgery can be considered a safe and effective approach in both primary and revisional bariatric surgery, despite the lack of evidence to support its routine use in primary bariatric surgery. However, in revisional bariatric surgery and in surgical complex procedures, the robotic approach could have potential benefits in terms of surgical complications and learning curves., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fantola, Moroni, Runfola, Lai, Pintus, Gallucci, Pennestrì and Raffaelli.)

Congenital heart disease (CHD) is present in 1% of live births, yet identification of causal mutations remains challenging. We hypothesized that genetic determinants for CHDs may lie in the protein interactomes of transcription factors whose mutations cause CHDs. Defining the interactomes of two transcription factors haplo-insufficient in CHD, GATA4 and TBX5, within human cardiac progenitors, and integrating the results with nearly 9,000 exomes from proband-parent trios revealed an enrichment of de novo missense variants associated with CHD within the interactomes. Scoring variants of interactome members based on residue, gene, and proband features identified likely CHD-causing genes, including the epigenetic reader GLYR1. GLYR1 and GATA4 widely co-occupied and co-activated cardiac developmental genes, and the identified GLYR1 missense variant disrupted interaction with GATA4, impairing in vitro and in vivo function in mice. This integrative proteomic and genetic approach provides a framework for prioritizing and interrogating genetic variants in heart disease., Competing Interests: Declaration of interests D.S. is scientific co-founder, shareholder, and director of Tenaya Therapeutics. B.G.B. and B.R.C. are scientific co-founders and shareholders of Tenaya Therapeutics. K.S.P. is a shareholder of Tenaya Therapeutics. N.J.K. has received research support from Vir Biotechnology and F. Hoffmann-La Roche. N.J.K. has consulting agreements with the Icahn School of Medicine at Mount Sinai, New York, Maze Therapeutics, and Interline Therapeutics; is a shareholder of Tenaya Therapeutics, Maze Therapeutics, and Interline Therapeutics; and is financially compensated by GEn1E Lifesciences, Inc. and Twist Bioscience Corp., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Bariatric surger (BS) is characterized by lipid metabolic changes as a response to the massive release of non-esterified fatty acids (NEFA) from adipose depots. The study aimed at evaluating changes in polyunsaturated fatty acids (PUFA) metabolism and biosynthesis of the lipid mediators N -acylethanolamines (NAE), as indices of nuclear peroxisome proliferator-activated receptor (PPAR)-α activation. The observational study was performed on 35 subjects (27 female, 8 male) with obesity, undergoing bariatric surgery. We assessed plasma FA and NAE profiles by LC-MS/MS, clinical parameters and anthropometric measures before and 1 and 6 months after bariatric surgery. One month after bariatric surgery, as body weight and clinical parameters improved significantly, we found higher plasma levels of N -oleoylethanolamine, arachidonic and a 22:6-n3/20:5-n3 ratio as evidence of PPAR-α activation. These changes corresponded to higher circulating levels of NEFA and a steep reduction of the fat mass. After 6 months 22:6-n3/20:5-n3 remained elevated and fat mass was further reduced. Our data suggest that the massive release of NEFA from adipose tissue at 1-Post, possibly by inducing PPAR-α, may enhance FA metabolism contributing to fat depot reduction and improved metabolic parameters in the early stage. However, PUFA metabolic changes favor n6 PUFA biosynthesis, requiring a nutritional strategy aimed at reducing the n6/n3 PUFA ratio.

The SARS-CoV-2 spike (S) protein is exposed on the viral surface and is the first point of contact between the virus and the host. For these reasons it represents the prime target for Covid-19 vaccines. In recent months, variants of this protein have started to emerge. Their ability to reduce or evade recognition by S-targeting antibodies poses a threat to immunological treatments and raises concerns for their consequences on vaccine efficacy. To develop a model able to predict the potential impact of S-protein mutations on antibody binding sites, we performed unbiased multi-microsecond molecular dynamics of several glycosylated S-protein variants and applied a straightforward structure-dynamics-energy based strategy to predict potential changes in immunogenic regions on each variant. We recover known epitopes on the reference D614G sequence. By comparing our results, obtained on isolated S-proteins in solution, to recently published data on antibody binding and reactivity in new S variants, we directly show that modifications in the S-protein consistently translate into the loss of potentially immunoreactive regions. Our findings can thus be qualitatively reconnected to the experimentally characterized decreased ability of some of the Abs elicited against the dominant S-sequence to recognize variants. While based on the study of SARS-CoV-2 spike variants, our computational epitope-prediction strategy is portable and could be applied to study immunoreactivity in mutants of proteins of interest whose structures have been characterized, helping the development/selection of vaccines and antibodies able to control emerging variants.

TRAP1, the mitochondrial isoform of heat shock protein (Hsp)90 chaperones, is a key regulator of metabolism and organelle homeostasis in diverse pathological states. While selective TRAP1 targeting is an attractive goal, classical active-site-directed strategies have proved difficult, due to high active site conservation among Hsp90 paralogs. Here, we discuss advances in developing TRAP1-directed strategies, from lead modification with mitochondria delivery groups to the computational discovery of allosteric sites and ligands. Specifically, we address the unique opportunities that targeting TRAP1 opens up in tackling fundamental questions on its biology and in unveiling new therapeutic approaches. Finally, we show how crucial to this endeavor is our ability to predict the activities of TRAP1-selective allosteric ligands and to optimize target engagement to avoid side effects., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The mitochondrial paralog of the Hsp90 chaperone family TRAP1 is often induced in tumors, but the mechanisms controlling its expression, as well as its physiological functions remain poorly understood. Here, we find that TRAP1 is highly expressed in the early stages of Zebrafish development, and its ablation delays embryogenesis while increasing mitochondrial respiration of fish larvae. TRAP1 expression is enhanced by hypoxic conditions both in developing embryos and in cancer models of Zebrafish and mammals. The TRAP1 promoter contains evolutionary conserved hypoxic responsive elements, and HIF1α stabilization increases TRAP1 levels. TRAP1 inhibition by selective compounds or by genetic knock-out maintains a high level of respiration in Zebrafish embryos after exposure to hypoxia. Our data identify TRAP1 as a primary regulator of mitochondrial bioenergetics in highly proliferating cells following reduction in oxygen tension and HIF1α stabilization.

Allosteric drugs have been attracting increasing interest over the past few years. In this context, it is common practice to use high-throughput screening for the discovery of non-natural allosteric drugs. While the discovery stage is supported by a growing amount of biological information and increasing computing power, major challenges still remain in selecting allosteric ligands and predicting their effect on the target protein's function. Indeed, allosteric compounds can act both as inhibitors and activators of biological responses. Computational approaches to the problem have focused on variations on the theme of molecular docking coupled to molecular dynamics with the aim of recovering information on the (long-range) modulation typical of allosteric proteins.

Aims: TNF receptor-associated protein 1 (TRAP1), the mitochondrial paralog of the heat shock protein 90 (Hsp90) family of molecular chaperones, is required for neoplastic growth in several tumor cell models, where it inhibits succinate dehydrogenase (SDH) activity, thus favoring bioenergetic rewiring, maintenance of redox homeostasis, and orchestration of a hypoxia-inducible factor 1-alpha (HIF1α)-mediated pseudohypoxic program. Development of selective TRAP1 inhibitors is instrumental for targeted development of antineoplastic drugs, but it has been hampered up to now by the high degree of homology among catalytic pockets of Hsp90 family members. The vegetal derivative honokiol and its lipophilic bis-dichloroacetate ester, honokiol DCA (HDCA), are small-molecule compounds with antineoplastic activity. HDCA leads to oxidative stress and apoptosis in in vivo tumor models and displays an action that is functionally opposed to that of TRAP1, as it induces both SDH and the mitochondrial deacetylase sirtuin-3 (SIRT3), which further enhances SDH activity. We investigated whether HDCA could interact with TRAP1, inhibiting its chaperone function, and the effects of HDCA on tumor cells harboring TRAP1. Results: An allosteric binding site in TRAP1 is able to host HDCA, which inhibits TRAP1 but not Hsp90 ATPase activity. In neoplastic cells, HDCA reverts TRAP1-dependent downregulation of SDH, decreases proliferation rate, increases mitochondrial superoxide levels, and abolishes tumorigenic growth. Innovation: HDCA is a potential lead compound for the generation of antineoplastic approaches based on the allosteric inhibition of TRAP1 chaperone activity. Conclusions: We have identified a selective TRAP1 inhibitor that can be used to better dissect TRAP1 biochemical functions and to tailor novel tumor-targeting strategies.

The Hsp90 chaperone system interacts with a wide spectrum of client proteins, forming variable and dynamic multiprotein complexes that involve the intervention of cochaperone partners. Recent results suggest that the role of Hsp90 complexes is to establish interactions that suppress unwanted client activities, allow clients to be protected from degradation and respond to biochemical signals. Cryo-electron microscopy (cryoEM) provided the first key molecular picture of Hsp90 in complex with a kinase, Cdk4, and a cochaperone, Cdc37. Here, we use a combination of molecular dynamics (MD) simulations and advanced comparative analysis methods to elucidate key aspects of the functional dynamics of the complex, with different nucleotides bound at the N-terminal Domain of Hsp90. The results reveal that nucleotide-dependent structural modulations reverberate in a striking asymmetry of the dynamics of Hsp90 and identify specific patterns of long-range coordination between the nucleotide binding site, the client binding pocket, the cochaperone and the client. Our model establishes a direct atomic-resolution cross-talk between the ATP-binding site, the client region that is to be remodeled and the surfaces of the Cdc37-cochaperone., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Allosteric molecules provide a powerful means to modulate protein function. However, the effect of such ligands on distal orthosteric sites cannot be easily described by classical docking methods. Here, we applied machine learning (ML) approaches to expose the links between local dynamic patterns and different degrees of allosteric inhibition of the ATPase function in the molecular chaperone TRAP1. We focused on 11 novel allosteric modulators with similar affinities to the target but with inhibitory efficacy between the 26.3 and 76%. Using a set of experimentally related local descriptors, ML enabled us to connect the molecular dynamics (MD) accessible to ligand-bound (perturbed) and unbound (unperturbed) systems to the degree of ATPase allosteric inhibition. The ML analysis of the comparative perturbed ensembles revealed a redistribution of dynamic states in the inhibitor-bound versus inhibitor-free systems following allosteric binding. Linear regression models were built to quantify the percentage of experimental variance explained by the predicted inhibitor-bound TRAP1 states. Our strategy provides a comparative MD-ML framework to infer allosteric ligand functionality. Alleviating the time scale issues which prevent the routine use of MD, a combination of MD and ML represents a promising strategy to support in silico mechanistic studies and drug design.

The chick embryo chorioallantoic membrane (CAM) represents a powerful in vivo model to study several physiological and pathological processes including inflammation and tumor progression. Nevertheless, the possibility of deepening the molecular processes in the CAM system is biased by the absence/scarcity of chemical and biological reagents, designed explicitly for avian species. This is particularly true for transcriptional factors, proteinaceous molecules that regulate various cellular responses, including proliferation, survival, and differentiation. Here, we propose a detailed antibody-independent protocol to visualize the activation and nuclear translocation of transcriptional factors in cells or in tissues of different animal species. As a proof of concept, DNA/cAMP response element-binding protein (CREB) interaction was characterized on the CAM tissue using oligonucleotides containing the palindromic binding sequence of CREB. Scrambled oligonucleotides were used as controls. In situ DNA/protein interaction protocol is a versatile method that is useful for the study of transcription factors in the cell and tissue of different origins.

SARS-CoV-2 is a health threat with dire socioeconomical consequences. As the crucial mediator of infection, the viral glycosylated spike protein (S) has attracted the most attention and is at the center of efforts to develop therapeutics and diagnostics. Herein, we use an original decomposition approach to identify energetically uncoupled substructures as antibody binding sites on the fully glycosylated S. Crucially, all that is required are unbiased MD simulations; no prior knowledge of binding properties or ad hoc parameter combinations is needed. Our results are validated by experimentally confirmed structures of S in complex with anti- or nanobodies. We identify poorly coupled subdomains that are poised to host (several) epitopes and potentially involved in large functional conformational transitions. Moreover, we detect two distinct behaviors for glycans: those with stronger energetic coupling are structurally relevant and protect underlying peptidic epitopes, and those with weaker coupling could themselves be prone to antibody recognition.

TRAP1 is the mitochondrial paralog of the heat shock protein 90 (HSP90) chaperone family. Its activity as an energy metabolism regulator has important implications in cancer, neurodegeneration, and ischemia. Selective inhibitors of TRAP1 could inform on its mechanisms of action and set the stage for targeted drug development, but their identification was hampered by the similarity among active sites in HSP90 homologs. We use a dynamics-based approach to identify a TRAP1 allosteric pocket distal to its active site that can host drug-like molecules, and we select small molecules with optimal stereochemical features to target the pocket. These leads inhibit TRAP1, but not HSP90, ATPase activity and revert TRAP1-dependent downregulation of succinate dehydrogenase activity in cancer cells and in zebrafish larvae. TRAP1 inhibitors are not toxic per se, but they abolish tumorigenic growth of neoplastic cells. Our results indicate that exploiting conformational dynamics can expand the chemical space of chaperone antagonists to TRAP1-specific inhibitors with wide therapeutic opportunities., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

One of the most promising strategies to treat cancer is the use of therapeutic antibodies that disrupt cell-cell adhesion mediated by dysregulated cadherins. The principal site where cell-cell adhesion occurs encompasses Trp2 found at the N-terminal region of the protein. Herein, we employed the naturally exposed highly conserved peptide Asp1-Trp2-Val3-Ile4-Pro5-Pro6-Ile7, as epitope to prepare molecularly imprinted polymer nanoparticles (MIP-NPs) to recognize cadherins. Since MIP-NPs target the site responsible for adhesion, they were more potent than commercially available therapeutic antibodies for inhibiting cell-cell adhesion in cell aggregation assays, and for completely disrupting three-dimensional tumor spheroids as well as inhibiting invasion of HeLa cells. These biocompatible supramolecular anti-adhesives may potentially be used as immunotherapeutic or sensitizing agents to enhance antitumor effects of chemotherapy., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

NPAC is a cytokine-like nuclear factor involved in chromatin modification and regulation of gene expression. In humans, the C-terminal domain of NPAC has the conserved structure of the β-hydroxyacid dehydrogenases (β-HAD) protein superfamily, which forms a stable tetrameric core scaffold for demethylase enzymes and organizes multiple sites for chromatin interactions. In spite of the close structural resemblance to other β-HAD family members, the human NPAC dehydrogenase domain lacks a highly conserved catalytic lysine, substituted by a methionine. The reintroduction of the catalytic lysine by M437 K mutation results in a significant decrease of stability of the tetramer. Here, we have computationally investigated the molecular determinants of the functional differences between methionine and lysine-containing NPAC proteins. We find that the single mutation can determine strong consequences in terms of dynamics, stability, and ultimately ability to assemble in supramolecular complexes: the higher stability and lower flexibility of the methionine variant structurally preorganizes the monomer for tetramerization, whereas lysine increases flexibility and favors conformations that, while catalytically active, are not optimal for tetrameric assembly. We combine structure-dynamics analysis to an evolutionary study of NPAC sequences, showing that the methionine mutation occurs in a specifically flexible region of the lysine-containing protein, flanked by two domains that concentrate most of the stabilizing interactions. In our model, such separation of stability nuclei and flexible regions appears to favor the functional innovability of the protein.

Thrombospondin (TSP)-1 and TSP-2 share similar structures and functions, including a remarkable antiangiogenic activity. We have previously demonstrated that a mechanism of the antiangiogenic activity of TSP-1 is the interaction of its type III repeats domain with fibroblast growth factor-2 (FGF2), affecting the growth factor bioavailability and angiogenic activity. Since the type III repeats domain is conserved in TSP-2, this study aimed at investigating whether also TSP-2 retained the ability to interact with FGF2. The FGF2 binding properties of TSP-1 and TSP-2 and their recombinant domains were analyzed by solid-phase binding and surface plasmon resonance assays. TSP-2 bound FGF2 with high affinity (Kd = 1.3 nM). TSP-2/FGF2 binding was inhibited by calcium and heparin. The FGF2-binding domain of TSP-2 was located in the type III repeats and the minimal interacting sequence was identified as the GVTDEKD peptide in repeat 3C, corresponding to KIPDDRD, the active sequence of TSP-1. A second putative FGF2 binding sequence was also identified in repeat 11C of both TSPs. Computational docking analysis predicted that both the TSP-2 and TSP-1-derived heptapeptides interacted with FGF2 with comparable binding properties. Accordingly, small molecules based on the TSP-1 active sequence blocked TSP-2/FGF2 interaction. Binding of TSP-2 to FGF2 impaired the growth factor ability to interact with its cellular receptors, since TSP-2-derived fragments prevented the binding of FGF2 to both heparin (used as a structural analog of heparan sulfate proteoglycans) and FGFR-1. These findings identify TSP-2 as a new FGF2 ligand that shares with TSP-1 the same molecular requirements for interaction with the growth factor and a comparable capacity to block FGF2 interaction with proangiogenic receptors. These features likely contribute to TSP-2 antiangiogenic and antineoplastic activity, providing the rationale for future therapeutic applications.

Molecular chaperones HSP90 and HSP70 are essential regulators of the folding and activation of a disparate ensemble of client proteins. They function through ATP hydrolysis and the assembly of multiprotein complexes with cochaperones and clients. While their therapeutic relevance is recognized, important details underlying the links between ATP-dependent conformational dynamics and clients/cochaperones recruitment remain elusive. Allosteric modulators represent fundamental tools to obtain molecular insights into functional regulation. By selective perturbation of different aspects of HSP90/HSP70 activities, allosteric drugs can tune rather than completely inhibit signaling cascades, providing information on the relationships between structure-dynamics and function. Herein, we review advances in the design of HSP90 and HSP70 allosteric modulators. We consider inhibitors and activators in different biochemical and disease models. We discuss these compounds as probes to decipher the complexity of the chaperone machinery and that at the same time represent starting leads for the development of drugs against cancer and neurodegeneration.

Bioactive 2-benzazepines were accessed in an atom- and step-economical manner through a versatile palladium-catalyzed C-H activation strategy. The C-H arylation required low catalyst loading and a mild base, which was reflected by a broad scope and high functional-group tolerance. The benzotriazolodiazepinones were identified as new heat shock protein 90 (Hsp90) inhibiting lead compounds, with considerable potential for anti-cancer applications., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The Hsp90 family of molecular chaperones oversees the folding of a wide range of client proteins. Hsp90 is a homodimer, whose conformational states and functions are regulated by ATP-binding and hydrolysis. The crystal structure of mitochondrial Hsp90 (Trap1) showed that one of the two protomers in the active state is buckled, resulting in an asymmetric conformation. The asymmetry between the two protomers corresponds to the broadly conserved region responsible for client binding. Moreover, asymmetry determines differential hydrolysis for each protomer, with the buckled conformation favoring ATP processing. Experimental results show that after the first hydrolysis the dimer flips to a different asymmetric state while remaining in a closed conformation for the second hydrolysis. In this model, asymmetry plays a key role in the mechanism that drives chaperone function. Herein, we investigate the nucleotide-dependent internal dynamics of Trap1 with computational approaches. Our results shed light on the relationship between the nucleotide state in the N-terminal domain and the asymmetric modulation of the dynamic and structural properties of the client-binding region in the Middle domain. According to our analysis, this is the region that undergoes the most intense dynamic modulation upon nucleotide exchange. This result provides molecular insights into the roles of structural asymmetry in the regulation of Trap1 and suggests that this substructure is a promising target to modulate the functionally oriented aspects of Trap1 dynamics, therefore opening fresh opportunities for the design of selective therapeutics for Trap1-dependent diseases.

Introduction: The New York City (NYC) Macroscope is an electronic health record (EHR) surveillance system based on a distributed network of primary care records from the Hub Population Health System. In a previous 3-part series published in eGEMS , we reported the validity of health indicators from the NYC Macroscope; however, questions remained regarding their generalizability to other EHR surveillance systems., Methods: We abstracted primary care chart data from more than 20 EHR software systems for 142 participants of the 2013-14 NYC Health and Nutrition Examination Survey who did not contribute data to the NYC Macroscope. We then computed the sensitivity and specificity for indicators, comparing data abstracted from EHRs with survey data., Results: Obesity and diabetes indicators had moderate to high sensitivity (0.81-0.96) and high specificity (0.94-0.98). Smoking status and hypertension indicators had moderate sensitivity (0.78-0.90) and moderate to high specificity (0.88-0.98); sensitivity improved when the sample was restricted to records from providers who attested to Stage 1 Meaningful Use. Hyperlipidemia indicators had moderate sensitivity (≥0.72) and low specificity (≤0.59), with minimal changes when restricting to Stage 1 Meaningful Use., Discussion: Indicators for obesity and diabetes used in the NYC Macroscope can be adapted to other EHR surveillance systems with minimal validation. However, additional validation of smoking status and hypertension indicators is recommended and further development of hyperlipidemia indicators is needed., Conclusion: Our findings suggest that many of the EHR-based surveillance indicators developed and validated for the NYC Macroscope are generalizable for use in other EHR surveillance systems.

Introduction: Electronic health record (EHR) systems provide an opportunity to use a novel data source for population health surveillance. Validation studies that compare prevalence estimates from EHRs and surveys most often use difference testing, which can, because of large sample sizes, lead to detection of significant differences that are not meaningful. We explored a novel application of the two one-sided t test (TOST) to assess the equivalence of prevalence estimates in 2 population-based surveys to inform margin selection for validating EHR-based surveillance prevalence estimates derived from large samples., Methods: We compared prevalence estimates of health indicators in the 2013 Community Health Survey (CHS) and the 2013-2014 New York City Health and Nutrition Examination Survey (NYC HANES) by using TOST, a 2-tailed t test, and other goodness-of-fit measures., Results: A ±5 percentage-point equivalence margin for a TOST performed well for most health indicators. For health indicators with a prevalence estimate of less than 10% (extreme obesity [CHS, 3.5%; NYC HANES, 5.1%] and serious psychological distress [CHS, 5.2%; NYC HANES, 4.8%]), a ±2.5 percentage-point margin was more consistent with other goodness-of-fit measures than the larger percentage-point margins., Conclusion: A TOST with a ±5 percentage-point margin was useful in establishing equivalence, but a ±2.5 percentage-point margin may be appropriate for health indicators with a prevalence estimate of less than 10%. Equivalence testing can guide future efforts to validate EHR data.

Allosteric compounds that stimulate Hsp90 adenosine triphosphatase (ATPase) activity were rationally designed, showing anticancer potencies in the low micromolar to nanomolar range. In parallel, the mode of action of these compounds was clarified and a quantitative model that links the dynamic ligand-protein cross-talk to observed cellular and in vitro activities was developed. The results support the potential of using dynamics-based approaches to develop original mechanism-based cancer therapeutics., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

PARP-1 inhibition has been studied over the last decades for the treatment of various diseases. Despite the fact that several molecules act as PARP-1 inhibitors, a reduced number of compounds are used in clinical practice. To identify new compounds with a discriminatory PARP-1 inhibitory function, explicit-solvent molecular dynamics simulations using different inhibitors bound to the PARP-1 catalytic domain were performed. The representative structures obtained were used to generate structure-based pharmacophores, taking into account the dynamic features of receptor-ligand interactions. Thereafter, a virtual screening of compound databases using the pharmacophore models obtained was performed and the hits retrieved were subjected to molecular docking-based scoring. The drug-like molecules featuring the best ranking were evaluated for their PARP-1 inhibitory activity and IC50 values were calculated for the top scoring docked compounds. Altogether, three new PARP-1 inhibitor chemotypes were identified., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Electronic health records (EHRs) have the potential to offer real-time, inexpensive standardized health data about chronic health conditions. Despite rapid expansion, EHR data evaluations for chronic disease surveillance have been limited. We present design and methods for the New York City (NYC) Macroscope, an EHR-based chronic disease surveillance system. This methods report is the first in a three part series describing the development and validation of the NYC Macroscope. This report describes in detail the infrastructure underlying the NYC Macroscope; indicator definitions; design decisions that were made to maximize data quality; characteristics of the population sampled; completeness of data collected; and lessons learned from doing this work. The second report describes the methods used to evaluate the validity and robustness of NYC Macroscope prevalence estimates; presents validation results for estimates of obesity, smoking, depression and influenza vaccination; and discusses the implications of our findings for NYC and for other jurisdictions embarking on similar work. The third report applies the same validation methods to metabolic outcomes, including the prevalence, treatment and control of diabetes, hypertension and hyperlipidemia., Methods: We designed the NYC Macroscope for comparison to a local "gold standard," the 2013-14 NYC Health and Nutrition Examination Survey, and the telephonic 2013 Community Health Survey. NYC Macroscope indicators covered prevalence, treatment, and control of diabetes, hypertension, and hyperlipidemia; and prevalence of influenza vaccination, obesity, depression and smoking. Indicators were stratified by age, sex, and neighborhood poverty, and weighted to the in-care NYC population and limited to primary care patients. Indicator queries were distributed to a virtual network of primary care practices; 392 practices and 716,076 adult patients were retained in the final sample., Findings: The NYC Macroscope covered 10% of primary care providers and 15% of all adult patients in NYC in 2013 (8-47% of patients by neighborhood). Data completeness varied by domain from 98% for blood pressure among patients with hypertension to 33% for depression screening., Discussion: Design and validation efforts undertaken by NYC are described here to provide one potential blueprint for leveraging EHRs for population health monitoring. To replicate a model like NYC Macroscope, jurisdictions should establish buy-in; build informatics capacity; use standard, simple case defnitions; establish documentation quality thresholds; restrict to primary care providers; and weight the sample to a target population.

Introduction: Electronic health records (EHRs) offer potential for population health surveillance but EHR-based surveillance measures require validation prior to use. We assessed the validity of obesity, smoking, depression, and influenza vaccination indicators from a new EHR surveillance system, the New York City (NYC) Macroscope. This report is the second in a 3-part series describing the development and validation of the NYC Macroscope. The first report describes in detail the infrastructure underlying the NYC Macroscope; design decisions that were made to maximize data quality; characteristics of the population sampled; completeness of data collected; and lessons learned from doing this work. This second report, which addresses concerns related to sampling bias and data quality, describes the methods used to evaluate the validity and robustness of NYC Macroscope prevalence estimates; presents validation results for estimates of obesity, smoking, depression and influenza vaccination; and discusses the implications of our findings for NYC and for other jurisdictions embarking on similar work. The third report applies the same validation methods described in this report to metabolic outcomes, including the prevalence, treatment and control of diabetes, hypertension and hyperlipidemia., Methods: NYC Macroscope prevalence estimates, overall and stratified by sex and age group, were compared to reference survey estimates for adult New Yorkers who reported visiting a doctor in the past year. Agreement was evaluated against 5 a priori criteria. Sensitivity and specificity were assessed by examining individual EHR records in a subsample of 48 survey participants., Results: Among adult New Yorkers in care, the NYC Macroscope prevalence estimate for smoking (15.2%) fell between estimates from NYC HANES (17.7 %) and CHS (14.9%) and met all 5 a priori criteria. The NYC Macroscope obesity prevalence estimate (27.8%) also fell between the NYC HANES (31.3%) and CHS (24.7%) estimates, but met only 3 a priori criteria. Sensitivity and specificity exceeded 0.90 for both the smoking and obesity indicators. The NYC Macroscope estimates of depression and influenza vaccination prevalence were more than 10 percentage points lower than the estimates from either reference survey. While specificity was > 0.90 for both of these indicators, sensitivity was < 0.70., Discussion: Through this work we have demonstrated that EHR data from a convenience sample of providers can produce acceptable estimates of smoking and obesity prevalence among adult New Yorkers in care; gained a better understanding of the challenges involved in estimating depression prevalence from EHRs; and identified areas for additional research regarding estimation of influenza vaccination prevalence. We have also shared lessons learned about how EHR indicators should be constructed and offer methodologic suggestions for validating them., Conclusions: This work adds to a rapidly emerging body of literature about how to define, collect and interpret EHR-based surveillance measures and may help guide other jurisdictions.

Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone responsible for the activation, maturation, and trafficking of several hundred client proteins in the cell. It is well known that (but not understood how) residues far away from Hsp90's nucleotide binding pocket can regulate its ATPase activity, a phenomenon called allosteric regulation. Here, the computational design of allosteric mutations was combined with in vitro and in vivo experiments to unravel nucleotide-responsive hot spots in the regulation of Hsp90. With this approach, we identified both activating and inhibiting regulation points and show that changes in those amino acids affect the conformational dynamics and ATPase activity of Hsp90 in vitro. Our observations that activating mutations loosen and inhibiting mutations rigidify the protein explain for the first time how Hsp90 changes in response to allosteric mutations. Additionally, mutations of these allosteric regulation points can be controlled by the interplay with Hsp90 co-chaperones, thus providing cells with an efficient mechanism of modifying Hsp90's intrinsic properties via different layers of regulation. Altogether, our results show that a framework for transmitting conformational information exists in the Hsp90 structure., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Introduction: Electronic health records (EHRs) from primary care providers can be used for chronic disease surveillance; however, EHR-based prevalence estimates may be biased toward people who seek care. This study sought to describe the characteristics of an in-care population and compare them with those of a not-in-care population to inform interpretation of EHR data., Methods: We used data from the 2013-2014 New York City Health and Nutrition Examination Survey (NYC HANES), considered the gold standard for estimating disease prevalence, and the 2013 Community Health Survey, and classified participants as in care or not in care, on the basis of their report of seeing a health care provider in the previous year. We used χ(2) tests to compare the distribution of demographic characteristics, health care coverage and access, and chronic conditions between the 2 populations., Results: According to the Community Health Survey, approximately 4.1 million (71.7%) adults aged 20 or older had seen a health care provider in the previous year; according to NYC HANES, approximately 4.7 million (75.1%) had. In both surveys, the in-care population was more likely to be older, female, non-Hispanic, and insured compared with the not-in-care population. The in-care population from the NYC HANES also had a higher prevalence of diabetes (16.7% vs 6.9%; P < .001), hypercholesterolemia (35.7% vs 22.3%; P < .001), and hypertension (35.5% vs 26.4%; P < .001) than the not-in-care population., Conclusion: Systematic differences between in-care and not-in-care populations warrant caution in using primary care data to generalize to the population at large. Future efforts to use primary care data for chronic disease surveillance need to consider the intended purpose of data collected in these systems as well as the characteristics of the population using primary care.

Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone's active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators.

The FGFs/FGFRs system is a recognized actionable target for therapeutic approaches aimed at inhibiting tumor growth, angiogenesis, metastasis, and resistance to therapy. We previously identified a non-peptidic compound (SM27) that retains the structural and functional properties of the FGF2-binding sequence of thrombospondin-1 (TSP-1), a major endogenous inhibitor of angiogenesis. Here we identified new small molecule inhibitors of FGF2 based on the initial lead. A similarity-based screening of small molecule libraries, followed by docking calculations and experimental studies, allowed selecting 7 bi-naphthalenic compounds that bound FGF2 inhibiting its binding to both heparan sulfate proteoglycans and FGFR-1. The compounds inhibit FGF2 activity in in vitro and ex vivo models of angiogenesis, with improved potency over SM27. Comparative analysis of the selected hits, complemented by NMR and biochemical analysis of 4 newly synthesized functionalized phenylamino-substituted naphthalenes, allowed identifying the minimal stereochemical requirements to improve the design of naphthalene sulfonates as FGF2 inhibitors.

The evaluation of free energy differences between specific states of a system is of fundamental interest in the study of (bio)chemical systems. Herein, we examine the use of the recently introduced confinement method (CM) to evaluate relative free energy changes upon protein/peptide mutations. CM is a path-independent technique that involves the transformation of a configurational state of the system into an ideal crystal permitting the direct computation of free energy differences. We illustrate the method by evaluating the differential stabilities between native and mutant sequences of a model peptide that has been extensively characterized by experimental approaches, the GB1 hairpin. We show a good correlation between calculated and experimental relative stabilities and discuss other possible applications of this method in the context of complex molecular conversions.

Hsp90 is a molecular chaperone of pivotal importance for multiple cell pathways. ATP-regulated internal dynamics are critical for its function and current pharmacological approaches block the chaperone with ATP-competitive inhibitors. Herein, a general approach to perturb Hsp90 through design of new allosteric ligands aimed at modulating its functional dynamics is proposed. Based on the characterization of a first set of 2-phenylbenzofurans showing stimulatory effects on Hsp90 ATPase and conformational dynamics, new ligands were developed that activate Hsp90 by targeting an allosteric site, located 65 Å from the active site. Specifically, analysis of protein responses to first-generation activators was exploited to guide the design of novel derivatives with improved ability to stimulate ATP hydrolysis. The molecules' effects on Hsp90 enzymatic, conformational, co-chaperone and client-binding properties were characterized through biochemical, biophysical and cellular approaches. These designed probes act as allosteric activators of the chaperone and affect the viability of cancer cell lines for which proper functioning of Hsp90 is necessary., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The fibroblast growth factor (FGF)/FGF receptor (FGFR) system plays a crucial role in cancer by affecting tumor growth, angiogenesis, drug resistance, and escape from anti-angiogenic anti-vascular endothelial growth factor therapy. The soluble pattern recognition receptor long-pentraxin 3 (PTX3) acts as a multi-FGF antagonist. Here we demonstrate that human PTX3 overexpression in transgenic mice driven by the Tie2 promoter inhibits tumor growth, angiogenesis, and metastasis in heterotopic, orthotopic, and autochthonous FGF-dependent tumor models. Using pharmacophore modeling of the interaction of a minimal PTX3-derived FGF-binding pentapeptide with FGF2, we identified a small-molecule chemical (NSC12) that acts as an extracellular FGF trap with significant implications in cancer therapy., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Proteins are not static objects. To carry out their functions in the cells and participate in biochemical interaction networks, proteins have to explore different conformational substates, which favor the adaptation to different partners and ultimately allow them to respond to changes in the environment. In this paper we discuss the implications of including the atomistic description of protein dynamics and flexibility in the context of drug discovery and design. The underlying idea is that a better understanding of the atomistic details of molecular recognition phenomena and conformational cross-talk between a ligand and a receptor can in fact translate in unexplored opportunities for the discovery of new drug like molecules. We will illustrate and discuss dynamics-based pharmacophores, the discovery of cryptic binding sites, the characterization and exploitation of allosteric regulation mechanisms and the definition of potential protein-protein interaction sites as potential sources of new bases for the rational design of small molecules endowed with specific biological functions. Overall, the inclusion of protein flexibility in the drug discovery process is starting to attract attention not only in the academic but also in the industrial community. This is supported by experimental tests that prove the actual feasibility of considering the explicit dynamics of drug-protein interactions at all relevant levels of resolution and the use of multiple receptor conformations in drug discovery, as affordable complements (if not an alternative) to classical High Throughput Screening (HTS) efforts based on static structures.

Modulation of Hsp90 C-terminal function represents a promising therapeutic approach for the treatment of cancer and neurodegenerative diseases. Current drug discovery efforts toward Hsp90 C-terminal inhibition focus on novobiocin, an antibiotic that was transformed into an Hsp90 inhibitor. Based on structural information obtained during the development of novobiocin derivatives and molecular docking studies, scaffolds containing a biphenyl moiety in lieu of the coumarin ring present in novobiocin were identified as new Hsp90 C-terminal inhibitors. Structure-activity relationship studies produced new derivatives that inhibit the proliferation of breast cancer cell lines at nanomolar concentrations, which corresponded directly with Hsp90 inhibition., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Inflammatory bowel disease (IBD) comprises several conditions with chronic or recurring immune response and inflammation of the gastrointestinal apparatus, of which ulcerative colitis and Crohn's disease are the commonest forms. This disease has a significant prevalence and it is of an unknown aethiology. Five-aminosalicylic acid (5-ASA) and its derivatives are among the oldest drugs approved for the treatment of the IBD. In this review we reapprise aspects of 5-ASA mechanism of action, safety, and efficacy that in our opinion make it a valuable drug that can be fruitfully tailored in personalised treatments as a therapeutic option alongside other immune-modifying agents.

Fibroblast growth factor-2 (FGF2) is a pleiotropic growth factor exhibiting a variety of biological activities. In this article, we studied the capacity of FGF2 purified with or without heparin affinity chromatography to self-associate. Analyzing the NMR HSQC spectra for different FGF2 concentrations, heparin-affinity purified FGF2 showed perturbations that indicate dimerization and are a higher-order oligomerization state. HSQC perturbation observed with different FGF2 concentrations revealed a heparin-binding site and two dimer interfaces. Thus, with increasing protein concentrations, FGF2 monomers make contacts with each other and form dimers or higher order oligomers. On the contrary, FGF2 purified with ion-exchange chromatography did not show similar perturbation indicating that self-association of FGF2 is eliminated if purification is done without heparin-affinity chromatography. The HSQC spectra of heparin-affinity purified FGF2 can be reproduced to some extent by adding heparin tetra-saccharide to ion exchange chromatography purified FGF2. Heparin-affinity purified FGF2 bound to acceptor and donor beads in a tagged form using His-tagged or GST-tagged proteins, also dimerized in the AlphaScreen™ assay. This assay was further validated using different experimental conditions and competitors. The assay constitutes an interesting tool to study dimerization of other FGF forms as well.

The interaction that occurs between molecules is a dynamic process that impacts both structural and conformational properties of the ligand and the ligand binding site. Herein, we investigate the dynamic cross-talk between a protein and the ligand as a source for new opportunities in ligand design. Analysis of the formation/disappearance of protein pockets produced in response to a first-generation inhibitor assisted in the identification of functional groups that could be introduced onto scaffolds to facilitate optimal binding, which allowed for increased binding with previously uncharacterized regions. MD simulations were used to elucidate primary changes that occur in the Hsp90 C-terminal binding pocket in the presence of first-generation ligands. This data was then used to design ligands that adapt to these receptor conformations, which provides access to an energy landscape that is not visible in a static model. The newly synthesized compounds demonstrated antiproliferative activity at ∼150 nM concentration. The method identified herein may be used to design chemical probes that provide additional information on structural variations of Hsp90 C-terminal binding site.