GPR101: Modeling a constitutively active receptor linked to X-linked acrogigantism.

GPR101 is a G protein-coupled receptor (GPCR) implicated in a rare form of genetic gigantism known as X-linked acrogigantism, or X-LAG. In particular, X-LAG patients harbor microduplications in the long arm of the X-chromosome that invariably include the GPR101 gene. Duplications of the GPR101 gene lead to the formation of a new chromatin domain that causes over-expression of the receptor in the pituitary tumors of the patients. Notably, GPR101 is a constitutively active receptor, which stimulates cells to produce the second messenger cyclic AMP (cAMP) in the absence of ligands. Moreover, GPR101 was recently reported to constitutively activate not only the cAMP pathway via Gs, but also other G protein subunits (Gq/11 and G12/13). Hence, chemicals that block the constitutive activity of GPR101, known as inverse agonists, have the potential to be useful for the development of pharmacological tools for the treatment of X-LAG. In this study, we provide structural insights into the putative structure of GPR101 based on in-house built homology models, as well as third party models based on the machine learning methods AlphaFold and AlphaFold-Multistate. Moreover, we report a molecular dynamics study, meant to further probe the constitutive activity of GPR101. Finally, we provide a structural comparison with the closest GPCRs, which suggests that GPR101 does not share their natural ligands. While this manuscript was under review, cryo-electron microscopy structures of GPR101 were reported. These structures are expected to enable computer-aided ligand discovery efforts targeting GPR101.

Rituximab Followed by Belimumab Controls Severe Lupus Nephritis and Bullous Pemphigoid in Systemic Lupus Erythematosus Refractory to Several Combination Therapies.

Systemic lupus erythematosus (SLE) and bullous pemphigoid (BP) are chronic autoimmune diseases in which B cells play an important pathogenic role in the different stages of the disease. B cell-targeted therapies have been suggested as a new rational approach for treating SLE. Rituximab (RTX), an anti-CD20 chimeric monoclonal antibody, failed to achieve primary endpoints in two clinical trials (EXPLORER and LUNAR) despite multiple observational and retrospective studies showing its beneficial effect on SLE. Moreover, RTX is recommended in cases of BP that is unresponsive to conventional treatments. Belimumab (BLM), a human immunoglobulin G1 λ monoclonal antibody that inhibits soluble B-lymphocyte stimulator (BlyS)/B-cell activating factor (BAFF), is the only biological treatment approved for standard therapy of refractory autoantibody-positive active SLE. Animal models and a few case reports have supported the efficacy of the combined use of RTX followed by BLM as maintenance therapy in severe lupus nephritis (LN), suggesting that their combined use may be more effective than their single use, without compromising safety. In this study, we describe the clinical case of a SLE patient with predominant renal involvement in overlap with BP, refractory to conventional therapy including RTX alone, achieving significant steroid sparing and clinical remission under sequential treatment of RTX-BLM. Moreover, we describe the first case of BP successfully treated with BLM. This case report may encourage further clinical research studies in B lymphocyte targeted combination therapy in patients affected by SLE with major organ involvement or with refractory disease, suggesting that RTX and BLM sequential therapy may be a valid option for the treatment of SLE manifestations, including conventional therapy and RTX-resistant LN.

Lists of Chemical Warfare Agents and Precursors from International Nonproliferation Frameworks: Structural Annotation and Chemical Fingerprint Analysis.

To support efforts to stem the proliferation of chemical weapons (CWs), we have curated and structurally annotated CW-control lists from three key international nonproliferation frameworks: the Chemical Weapons Convention (CWC), the Australia Group (AG), and the Wassenaar Arrangement. The curated lists are available as web tables at the Costanzi Research website (https://costanziresearch.com/cw-control-lists/). The annotations include manually curated 2D structural images, which provide a means to appreciate at a glance the similarities and differences between different entries, as well as downloadable 2D structures, in two different formats and three different structural identifiers, namely, simplified molecular-input line-entry system, standard InChI, and standard InChIKey, which are intended to provide a platform for cheminformatics analyses. The tables also include links to National Center for Biotechnology Information's PubChem and National Institute of Standards and Technology's Chemistry WebBook cards, hence providing prompt access to a wealth of physicochemical, analytical chemistry, and toxicological information. To showcase the importance of structural annotations, we discuss a discrepancy in a CW-control list covering the defoliant Agent Orange, which we identified through our curation process, and propose a solution to address it. Moreover, we present the results of chemical fingerprinting analyses, through which we clustered the entries of the three CW-control lists under study into structurally related groups and studied the overlaps between the three lists. As an application of this study, we examine the recent updates of CWC Schedule 1 and the AG precursors list, highlighting the relationships between the two amendments and proposing the possible addition of further chemicals. Our research is intended to facilitate the communication between scientific advisors and policymakers as well as the work of chemists and cheminformaticians involved in the CW nonproliferation field. Ultimately, we seek to provide tools to bolster the control of CWs and support the global efforts to rid the world of this category of weapons.

Deterioration in Clinical Status Is Not Enough to Suspend Eculizumab: A Genetic Complement-Mediated Atypical Hemolytic Uremic Syndrome Case Report.

BACKGROUND: Atypical hemolytic uremic syndrome (aHUS) is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. Mutations in CFI gene coding for complement regulation factors and in THBD gene coding for endothelial cell receptor thrombomodulin could predispose to the disease and hypertension can trigger the onset. CASE PRESENTATION: A 51-year-old female patient who had received kidney transplant eighteen years ago presented with hypertensive peak and hemolysis pattern. Normal ADAMTS13 levels as well as negative culture and serology for Shiga-toxin excluded, respectively, thrombotic thrombocytopenic purpura (TTP) and typical HUS caused by Shiga toxin-producing Escherichia coli (STEC-HUS). In suspicion of aHUS, we administered eculizumab and hemodialysis sessions were started as the patient showed severe renal failure. After an initial response, the patient developed cerebral hemorrhage. After last eculizumab administration, according to hematological parameters, an unsatisfactory response was observed: given the worsening clinical scenario, we withdrew eculizumab. Pathogenic mutations in CFI and THBD genes were found. After eculizumab reinitiation, looking at hemolysis indexes, we observed a suboptimal response as well as an otherwise adequate renal one: renal graft function was recovered despite persistence of hemolysis signs, after 6 months on regular dialysis. CONCLUSION: For the first time, we report an aHUS case in which a peculiar combination of mutations in CFI and THBD is found. We describe the importance of continuing eculizumab despite deterioration of patient's clinical conditions.

Influence of the Structural Accuracy of Homology Models on Their Applicability to Docking-Based Virtual Screening: The ß2 Adrenergic Receptor as a Case Study.

How accurate do structures of the ß2 adrenergic receptor (ß2AR) need to be to effectively serve as platforms for docking-based virtual screening campaigns? To answer this research question, here, we targeted through controlled virtual screening experiments 23 homology models of the ß2AR endowed with different levels of structural accuracy. Subsequently, we studied the correlation between virtual screening performance and structural accuracy of the targeted models. Moreover, we studied the correlation between virtual screening performance and template/target receptor sequence identity. Our study demonstrates that docking-based virtual screening campaigns targeting homology models of the ß2AR, in the majority of the cases, yielded results that exceeded random expectations in terms of area under the receiver operating characteristic curve (ROC AUC). Moreover, with the most effective scoring method, over one-third and one-quarter of the models yielded results that exceeded random expectation also in terms of enrichment factors (EF1, EF5, and EF10) and BEDROC (α = 160.9), respectively. Not surprisingly, we found a detectable linear correlation between virtual screening performance and structural accuracy of the ligand-binding cavity. We also found a detectable linear correlation between virtual screening performance and structural accuracy of the second extracellular loop (EL2). Finally, our data indicate that, although there is no detectable linear correlation between virtual screening performance and template/ß2AR sequence identity, models built on the basis of templates that show high sequence identity with the ß2AR, especially within the ligand-biding cavity, performed consistently well. Conversely, models with lower sequence identity displayed performance levels that ranged from very good to random, with no apparent correlation with the sequence identity itself.

[The pathway of vasopressin as a pharmacological target in nephrology: a narrative review].

ADH is a hormone secreted by neurohypophysis that plays different roles based on the target organ. At the renal level, this peptide is capable of causing electrolyte-free water absorption, thus playing a key role in the hydro-electrolytic balance. There are pathologies and disorders that jeopardize this balance and, in this field, ADH receptor inhibitors such as Vaptans could play a key role. By inhibiting the activation pathway of vasopressin, they are potentially useful in euvolemic and hypervolemic hypotonic hyponatremia. However, clinical trials in heart failure have not given favourable results on clinical outcomes. Even in SIADH, despite their wide use, there is no agreement by experts on their use. Since vaptans inhibit the cAMP pathway in tubular cells, their use has been proposed to inhibit cystogenesis. A clinical trial has shown favourable effects on ADPKD progression. Because vaptans have been shown to be effective in models of renal cysts disorders other than ADPKD, their use has been proposed in diseases such as nephronophthisis and recessive autosomal polycystic disease. Other possible uses of vaptans could be in kidney transplantation and cardiorenal syndrome. Due to the activity of ADH in coagulation and haemostasis, ADH's activation pathway by Desmopressin Acetate could be a useful strategy to reduce the risk of bleeding in biopsies in patients with haemorrhagic risk.

Chiral analogues of (+)-cyclazosin as potent α1B-adrenoceptor selective antagonist.

(+)-Cyclazosin [(+)-1] is one of most selective antagonists of the α1B-adrenoceptor subtype (selectivity ratios, α1B/α1A = 13, α1B/α1D = 38-39). To improve the selectivity, we synthesized and pharmacologically studied the blocking activity against α1-adrenoceptors of several homochiral analogues of (+)-cyclazosin featuring different substituents on the carbonyl or amine groups, namely (-)-2, (+)-3, (-)-4-(-)-8, (+)-9. Moreover, we studied the activity of some their opposite enantiomers, namely (-)-1, (-)-3, (+)-6, and (-)-9, to evaluate the influence of stereochemistry on selectivity. The benzyloxycarbonyl and methyl (4aS,8aR) analogues (+)-3 and (-)-6 improved in a significant way the α1B selectivity of the progenitor compound: 4 and 14 time vs. the α1D subtype and 35 and 77 times vs. the α1A subtype, respectively. The study confirmed the importance of the hydrophobic cis-octahydroquinoxaline moiety of these molecules for the establishment of interactions with the α1-adrenoceptors as well that of their (4aS,8aR) stereochemistry to grant selectivity for the α1B subtype. Hypotheses on the mode of interaction of these compounds were advanced on the basis of molecular modeling studies performed on compound (+)-3.

Contrast-enhanced ultrasonography in chronic glomerulonephritides: correlation with histological parameters of disease activity.

PURPOSE: To compare contrast-enhanced ultrasonography (CEUS)-derived time-intensity (TI) curves with histological findings in kidneys of patients affected by chronic glomerulonephritides (GN) in the early stage of disease. METHODS: Research ethics committee approval and patient written informed consent were obtained. Thirty-one patients who showed clinical and laboratory signs of GN, with preserved renal function, were consecutively enrolled. They underwent kidney CEUS, from which TI curves were obtained, and kidney biopsy. TI curves were compared with clinical data, ultrasound (US) Doppler, and histological parameters. RESULTS: The persistence of contrast agent signal during the wash-out phase was found to be correlated with the degree of disease activity (p = 0.016) and in particular with the presence of mesangial hyperplasia (p = 0.008). No correlation was observed between TI curves and clinical or Doppler US-derived parameters. CONCLUSIONS: The persistence of contrast agent signal in the wash-out phase of CEUS appears to reflect a disturbance of perfusion in glomerular capillaries in the early stages of GN. We found that the histological element directly correlated with the prolonged wash-out was mesangial hyperplasia.

Nerve Agents: What They Are, How They Work, How to Counter Them.

Nerve agents are organophosphorus chemical warfare agents that exert their action through the irreversible inhibition of acetylcholinesterase, with a consequent overstimulation of cholinergic transmission followed by its shutdown. Beyond warfare, they have notoriously been employed in acts of terrorism as well as high profile assassinations. After a brief historical introduction on the development and deployment of nerve agents, this review provides a survey of their chemistry, the way they affect cholinergic transmission, the available treatment options, and the current directions for their improvement. As the review illustrates, despite their merits, the currently available treatment options present several shortcomings. Current research directions involve the search for improved antidotes, antagonists of the nicotinic receptors, small-molecule pretreatment options, as well as bioscavengers as macromolecular pretreatment options. These efforts are making good progress in many different directions and, hopefully, will lead to a lower target susceptibility, thus reducing the appeal of nerve agents as chemical weapons.

Defective activation of the MAPK/ERK pathway, leading to PARP1 and DNMT1 dysregulation, is a common defect in IgA nephropathy and Henoch-Schönlein purpura.

Studies on IgA nephropathy (IgAN) have identified, through GWAS, linkage analysis, and pathway scanning, molecular defects in familial and sporadic IgAN patients. In our previous study, we identified a novel variant in the SPRY2 gene that segregates with the disease in one large family. The functional characterization of this variant led us to discover that the MAPK/ERK pathway was defective not only in this family, but also in two sporadic IgAN patients wild type for SPRY2. In the present study, we have deepened the molecular analysis of the MAPK/ERK pathway and extended our evaluation to a larger cohort of sporadic patients and to one additional family. We found that the ERK pathway is defective in IgAN patients and in patients affected by another IgA-mediated disorder, Henoch-Schönlein purpura (HSP). Furthermore, we found that two other proteins, PARP1 and DNMT1, respectively involved in DNA repair and in antibody class switching and methylation maintenance duties, were critically downregulated in IgAN and HSP patients. This study opens up the possibility that defective ERK activation, in some patients, leads to PARP1 and DNMT1 downregulation suggesting that IgAN could be the consequence of a dysregulated epigenetic maintenance leading to the upregulation of several genes. In particular, PARP1 could be used as a potential biomarker for the disease.

Allosteric "beta-blocker" isolated from a DNA-encoded small molecule library.

The ß2-adrenergic receptor (ß2AR) has been a model system for understanding regulatory mechanisms of G-protein-coupled receptor (GPCR) actions and plays a significant role in cardiovascular and pulmonary diseases. Because all known ß-adrenergic receptor drugs target the orthosteric binding site of the receptor, we set out to isolate allosteric ligands for this receptor by panning DNA-encoded small-molecule libraries comprising 190 million distinct compounds against purified human ß2AR. Here, we report the discovery of a small-molecule negative allosteric modulator (antagonist), compound 15 [([4-((2S)-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide], exhibiting a unique chemotype and low micromolar affinity for the ß2AR. Binding of 15 to the receptor cooperatively enhances orthosteric inverse agonist binding while negatively modulating binding of orthosteric agonists. Studies with a specific antibody that binds to an intracellular region of the ß2AR suggest that 15 binds in proximity to the G-protein binding site on the cytosolic surface of the ß2AR. In cell-signaling studies, 15 inhibits cAMP production through the ß2AR, but not that mediated by other Gs-coupled receptors. Compound 15 also similarly inhibits ß-arrestin recruitment to the activated ß2AR. This study presents an allosteric small-molecule ligand for the ß2AR and introduces a broadly applicable method for screening DNA-encoded small-molecule libraries against purified GPCR targets. Importantly, such an approach could facilitate the discovery of GPCR drugs with tailored allosteric effects.

Computational Drug Target Screening through Protein Interaction Profiles.

The development of computational methods to discover novel drug-target interactions on a large scale is of great interest. We propose a new method for virtual screening based on protein interaction profile similarity to discover new targets for molecules, including existing drugs. We calculated Target Interaction Profile Fingerprints (TIPFs) based on ChEMBL database to evaluate drug similarity and generated new putative compound-target candidates from the non-intersecting targets in each pair of compounds. A set of drugs was further studied in monoamine oxidase B (MAO-B) and cyclooxygenase-1 (COX-1) enzyme through molecular docking and experimental assays. The drug ethoxzolamide and the natural compound piperlongumine, present in Piper longum L, showed hMAO-B activity with IC50 values of 25 and 65 µM respectively. Five candidates, including lapatinib, SB-202190, RO-316233, GW786460X and indirubin-3'-monoxime were tested against human COX-1. Compounds SB-202190 and RO-316233 showed a IC50 in hCOX-1 of 24 and 25 µM respectively (similar range as potent inhibitors such as diclofenac and indomethacin in the same experimental conditions). Lapatinib and indirubin-3'-monoxime showed moderate hCOX-1 activity (19.5% and 28% of enzyme inhibition at 25 µM respectively). Our modeling constitutes a multi-target predictor for large scale virtual screening with potential in lead discovery, repositioning and drug safety.

Homology modeling of a Class A GPCR in the inactive conformation: A quantitative analysis of the correlation between model/template sequence identity and model accuracy.

With the present work we quantitatively studied the modellability of the inactive state of Class A G protein-coupled receptors (GPCRs). Specifically, we constructed models of one of the Class A GPCRs for which structures solved in the inactive state are available, namely the ß2 AR, using as templates each of the other class members for which structures solved in the inactive state are also available. Our results showed a detectable linear correlation between model accuracy and model/template sequence identity. This suggests that the likely accuracy of the homology models that can be built for a given receptor can be generally forecasted on the basis of the available templates. We also probed whether sequence alignments that allow for the presence of gaps within the transmembrane domains to account for structural irregularities afford better models than the classical alignment procedures that do not allow for the presence of gaps within such domains. As our results indicated, although the overall differences are very subtle, the inclusion of internal gaps within the transmembrane domains has a noticeable a beneficial effect on the local structural accuracy of the domain in question.

Topological Analyses of Protein-Ligand Binding: a Network Approach.

Proteins can be conveniently represented as networks of interacting residues, thus allowing the study of several network parameters that can shed light onto several of their structural and functional aspects. With respect to the binding of ligands, which are central for the function of many proteins, network analysis may constitute a possible route to assist the identification of binding sites. As the bulk of this review illustrates, this has generally been easier for enzymes than for non-enzyme proteins, perhaps due to the different topological nature of the binding sites of the former over those of the latter. The article also illustrates how network representations of binding sites can be used to search PDB structures in order to identify proteins that bind similar molecules and, lastly, how codifying proteins as networks can assist the analysis of the conformational changes consequent to ligand binding.

A SPRY2 mutation leading to MAPK/ERK pathway inhibition is associated with an autosomal dominant form of IgA nephropathy.

IgA nephropathy (IgAN) represents the most common primary glomerulonephritis worldwide with a prevalence of 25-50% among patients with primary glomerulopathies. In ~5-10% of the patients the disease segregates with an autosomal dominant (AD) pattern. Association studies identified loci on chromosomes 1q32, 6p21, 8p23, 17p13, 22q12, whereas classical linkage studies on AD families identified loci on chromosomes 2q36, 4q26-31, 6q22, 17q12-22. We have studied a large Sicilian family where IgAN segregates with an AD transmission. To identify the causal gene, the exomes of two affected and one unaffected individual have been sequenced. From the bioinformatics analysis a p.(Arg119Trp) variant in the SPRY2 gene was identified as the probable disease-causing mutation. Moreover, functional characterization of this variant showed that it is responsible for the inhibition of the MAPK/ERK1/2 pathway. The same effect was observed in two sporadic IgAN patients carriers of wild-type SPRY2, suggesting that downregulation of the MAPK/ERK1/2 pathway represents a common mechanism leading to IgAN.

Gigantism and acromegaly due to Xq26 microduplications and GPR101 mutation.

BACKGROUND: Increased secretion of growth hormone leads to gigantism in children and acromegaly in adults; the genetic causes of gigantism and acromegaly are poorly understood. METHODS: We performed clinical and genetic studies of samples obtained from 43 patients with gigantism and then sequenced an implicated gene in samples from 248 patients with acromegaly. RESULTS: We observed microduplication on chromosome Xq26.3 in samples from 13 patients with gigantism; of these samples, 4 were obtained from members of two unrelated kindreds, and 9 were from patients with sporadic cases. All the patients had disease onset during early childhood. Of the patients with gigantism who did not carry an Xq26.3 microduplication, none presented before the age of 5 years. Genomic characterization of the Xq26.3 region suggests that the microduplications are generated during chromosome replication and that they contain four protein-coding genes. Only one of these genes, GPR101, which encodes a G-protein-coupled receptor, was overexpressed in patients' pituitary lesions. We identified a recurrent GPR101 mutation (p.E308D) in 11 of 248 patients with acromegaly, with the mutation found mostly in tumors. When the mutation was transfected into rat GH3 cells, it led to increased release of growth hormone and proliferation of growth hormone-producing cells. CONCLUSIONS: We describe a pediatric disorder (which we have termed X-linked acrogigantism [X-LAG]) that is caused by an Xq26.3 genomic duplication and is characterized by early-onset gigantism resulting from an excess of growth hormone. Duplication of GPR101 probably causes X-LAG. We also found a recurrent mutation in GPR101 in some adults with acromegaly. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others.).

Computational studies to predict or explain G protein coupled receptor polypharmacology.

Since G protein-coupled receptors (GPCRs) belong to a very large superfamily of evolutionarily related receptors (>800 members in humans), and due to the rapid progress on their structural biology, they are ideal candidates for polypharmacology studies. Broad screening and bioinformatics/chemoinformatics have been applied to understanding off-target effects of GPCR ligands. It is now feasible to approach the question of GPCR polypharmacology using molecular modeling and the available X-ray GPCR structures. As an example, large and sterically constrained adenosine derivatives (potent adenosine receptor ligands with low conformational freedom and multiple extended substituents) were screened for binding at diverse receptors. Unanticipated off-target interactions, including at biogenic amine receptors, were then modeled using a structure-based approach to provide a consistent understanding of recognition. A conserved Asp in TM3 changed its role from counterion for biogenic amines to characteristic H-bonding to adenosine. The same systematic approach could potentially be applied to many GPCRs or other receptors using other sets of congeneric ligands.

Modeling G protein-coupled receptors in complex with biased agonists.

The biological response to the activation of G protein-coupled receptors (GPCRs) typically originates from the simultaneous modulation of various signaling pathways that lead to distinct biological consequences. Hence, 'biased agonists' (i.e., compounds that selectively activate one of the pathways while blocking the others) are highly sought-after molecules to provide fine-tuned pharmacological interventions. This review describes strategies that can be deployed to model the conformation of GPCRs in complex with ligands endowed with specific signaling profiles useful for the generation of hypotheses on the structural requirements for the activation of different signaling pathways or for rational computer-aided ligand discovery campaigns. In particular, it focuses on strategies potentially applicable to model the global or local conformational states of GPCRs stabilized by specific ligands.

The GPCR crystallography boom: providing an invaluable source of structural information and expanding the scope of homology modeling.

G protein-coupled receptors (GPCRs) are integral membrane proteins of high pharmaceutical interest. Until relatively recently, their structures have been particularly elusive, and rhodopsin has been for many years the only member of the superfamily with experimentally elucidated structures. However, a number of recent technical and scientific advancements made the determination of GPCR structures more feasible, thus leading to the solution of the structures of several receptors. Besides providing direct structural information, these experimental GPCR structures also provide templates for the construction of GPCR models. In depth studies have been performed to probe the accuracy of these models, in particular with respect to the interactions with their ligands, and to assess their applicability the rational discovery of GPCR modulators. Given the current state of the art and the pace of the field, the future of GPCR structural studies is likely to be characterized by a landscape populated by an increasingly higher number of experimental and theoretical structures.