Affinity-Driven Aryl Diazonium Labeling of Peptide Receptors on Living Cells.

Peptide-receptor interactions play critical roles in a wide variety of physiological processes. Methods to link bioactive peptides covalently to unmodified receptors on the surfaces of living cells are valuable for studying receptor signaling, dynamics, and trafficking and for identifying novel peptide-receptor interactions. Here, we utilize peptide analogues bearing deactivated aryl diazonium groups for the affinity-driven labeling of unmodified receptors. We demonstrate that aryl diazonium-bearing peptide analogues can covalently label receptors on the surface of living cells using both the neurotensin and the glucagon-like peptide 1 receptor systems. Receptor labeling occurs in the complex environment of the cell surface in a sequence-specific manner. We further demonstrate the utility of this covalent labeling approach for the visualization of peptide receptors by confocal fluorescence microscopy and for the enrichment and identification of labeled receptors by mass spectrometry-based proteomics. Aryl diazonium-based affinity-driven receptor labeling is attractive due to the high abundance of tyrosine and histidine residues susceptible to azo coupling in the peptide binding sites of receptors, the ease of incorporation of aryl diazonium groups into peptides, and the relatively small size of the aryl diazonium group. This approach should prove to be a powerful and relatively general method to study peptide-receptor interactions in cellular contexts.

PRRT with Lu-177 DOTATATE in Treatment-Refractory Progressive Meningioma: Initial Experience from a Tertiary-Care Neuro-Oncology Center.

PURPOSE: Refractory and/or recurrent meningiomas have poor outcomes, and the treatment options are limited. Peptide receptor radionuclide therapy (PRRT) has been used in this setting with promising results. We have documented our experience of using intravenous (IV) and intra-arterial (IA) approaches of Lu-177 DOTATATE PRRT. METHODS: Eight patients with relapsed/refractory high-grade meningioma received PRRT with Lu-177 DOTATATE by IV and an IA route. At least 2 cycles were administered. Time to progression was calculated from the first PRRT session to progression. The response was assessed on MRI using RANO criteria, and visual analysis of uptake was done on Ga-68 DOTANOC PET/CT. Post-therapy dosimetry calculations for estimating the absorbed dose were performed. RESULTS: Median time to progression was 8.9 months. One patient showed disease progression, whereas seven patients showed stable disease at 4 weeks following 2 cycles of PRRT. Dosimetric analysis showed higher dose and retention time by IA approach. No significant peri-procedural or PRRT associated toxicity was seen. CONCLUSION: PRRT is a safe and effective therapeutic option for relapsed/refractory meningioma. The IA approach yields better dose delivery and should be routinely practised.

Cartography of teneurin and latrophilin expression reveals spatiotemporal axis heterogeneity in the mouse hippocampus during development.

Synaptic adhesion molecules (SAMs) are evolutionarily conserved proteins that play an important role in the form and function of neuronal synapses. Teneurins (Tenms) and latrophilins (Lphns) are well-known cell adhesion molecules that form a transsynaptic complex. Recent studies suggest that Tenm3 and Lphn2 (gene symbol Adgrl2) are involved in hippocampal circuit assembly via their topographical expression. However, it is not known whether other teneurins and latrophilins display similar topographically restricted expression patterns during embryonic and postnatal development. Here, we reveal the cartography of all teneurin (Tenm1-4) and latrophilin (Lphn1-3 [Adgrl1-3]) paralog expression in the mouse hippocampus across prenatal and postnatal development as monitored by large-scale single-molecule RNA in situ hybridization mapping. Our results identify a striking heterogeneity in teneurin and latrophilin expression along the spatiotemporal axis of the hippocampus. Tenm2 and Tenm4 expression levels peak at the neonatal stage when compared to Tenm1 and Tenm3, while Tenm1 expression is restricted to the postnatal pyramidal cell layer. Tenm4 expression in the dentate gyrus (DG) exhibits an opposing topographical expression pattern in the embryonic and neonatal hippocampus. Our findings were validated by analyses of multiple RNA-seq datasets at bulk, single-cell, and spatial levels. Thus, our study presents a comprehensive spatiotemporal map of Tenm and Lphn expression in the hippocampus, showcasing their diverse expression patterns across developmental stages in distinct spatial axes.

Safety and efficacy of peptide receptor radionuclide therapy in neuroendocrine tumors: A single center experience.

Peptide receptor radionucleotide therapy (PRRT) with 177Lu-dotatate is widely used for the treatment of patients with neuroendocrine tumors (NETs). We analyzed data from 104 patients with NETs treated with 177Lu -dotatate at a US academic center between December 2017 and October 2020 to better understand patterns of long-term efficacy, safety, and toxicity in the real-world setting. 177Lu-dotatate (200 mCi) was administered every eight weeks for four doses. The most common sites of primary disease were small intestine NETs (n = 49, 47%), pancreatic NETs (n = 32, 31%), and lung NETs (n = 7, 7%). Twenty-seven percent had Ki-67 <3%, 49% had Ki-67 between 3-20%, and 13.5% had Ki-67 >20%. The cohort had been pretreated with a median of two prior lines of treatment. Forty percent had received prior liver-directed treatment. Seventy-four percent of patients completed all four doses of treatment. The objective response rate was 18%. The median time-to-treatment failure/death was significantly longer for small-bowel NETs when compared to pancreatic NETs (37.3 months vs. 13.2 months, p = 0.001). In a multivariate model, Ki-67, primary site, and liver tumor burden ≥50% were found to independently predict time-to-treatment failure/death. Around 40% of patients experienced adverse events of ≥grade 3 severity. Treatment-related adverse events leading to discontinuation of therapy happened in 10% of patients. Preexisting mesenteric/peritoneal disease was present in 33 patients; seven of these patients developed bowel-related toxicities including two grade 5 events. We also report two cases of delayed-onset minimal change nephrotic syndrome, which occurred 14 and 27 months after the last dose of PRRT. Lastly, we describe six patients who developed rapid tumor progression in the liver leading to terminal liver failure within 7.3 months from the start of PRRT, and identify potential risk factors associated with this occurrence, which will need further study.

Hyaline fibromatosis syndrome: a rare, yet recognizable syndrome.

BACKGROUND: Hyaline fibromatosis syndrome is a rare autosomal recessive disorder caused by ANTXR2 pathogenic variants. The disorder is characterized by the deposition of amorphous hyaline material in connective tissues. The hallmarks of the disease are joint contractures, generalized skin stiffness, hyperpigmented papules over extensor surfaces of joints, fleshy perianal masses, severe diarrhea, and gingival hypertrophy. The severity of the disease varies and prognosis is poor. No specific treatment is yet available. Most patients with the severe form of the condition pass away before the second year of age. In this study, we describe the clinical and molecular findings of a cohort of seven hyaline fibromatosis syndrome patients who were diagnosed and followed up at a single tertiary reference center in Turkey. METHODS: Genomic DNA was extracted by standard salting out method from peripheric blood samples of three patients. In one patient DNA extraction was performed on pathology slides since peripheric blood DNA was not available. All coding exons of the ANTXR2 were amplified and sequenced on ABI Prism 3500 Genetic Analyser. RESULTS: Sanger sequencing was performed in 3 patients and homozygous c.945T>G p.(Cys315Trp), c.1073dup p.(Ala359CysfsTer13), and c.1074del p.(Ala359HisfsTer50) variants were identified in ANTXR2. All patients passed away before the age of five years. CONCLUSIONS: HFS is a rare, progressive disorder with a broad phenotypic spectrum. HFS can be recognized easily with distinctive clinical features. Nevertheless, it has poor prognosis with increased mortality due to severe clinical decompensation.

Investigating the Influence of ANTXR2 Gene Mutations on Protective Antigen Binding for Heightened Anthrax Resistance.

Bacillus anthracis is the bacterium responsible for causing the zoonotic disease called anthrax. The disease presents itself in different forms like gastrointestinal, inhalation, and cutaneous. Bacterial spores are tremendously adaptable, can persist for extended periods and occasionally endanger human health. The Anthrax Toxin Receptor-2 (ANTXR2) gene acts as membrane receptor and facilitates the entry of the anthrax toxin into host cells. Additionally, mutations in the ANTXR2 gene have been linked to various autoimmune diseases, including Hyaline Fibromatosis Syndrome (HFS), Ankylosing Spondylitis (AS), Juvenile Hyaline Fibromatosis (JHF), and Infantile Systemic Hyalinosis (ISH). This study delves into the genetic landscape of ANTXR2, aiming to comprehend its associations with diverse disorders, elucidate the impacts of its mutations, and pinpoint minimal non-pathogenic mutations capable of reducing the binding affinity of the ANTXR2 gene with the protective antigen. Recognizing the pivotal role of single-nucleotide polymorphisms (SNPs) in shaping genetic diversity, we conducted computational analyses to discern highly deleterious and tolerated non-synonymous SNPs (nsSNPs) in the ANTXR2 gene. The Mutpred2 server determined that the Arg465Trp alteration in the ANTXR2 gene leads to altered DNA binding (p = 0.22) with a probability of a deleterious mutation of 0.808; notably, among the identified deleterious SNPs, rs368288611 (Arg465Trp) stands out due to its significant impact on altering the DNA-binding ability of ANTXR2. We propose these SNPs as potential candidates for hypertension linked to the ANTXR2 gene, which is implicated in blood pressure regulation. Noteworthy among the tolerated substitutions is rs200536829 (Ala33Ser), recognized as less pathogenic; this highlights its potential as a valuable biomarker, potentially reducing side effects on the host while also reducing binding with the protective antigen protein. Investigating these SNPs holds the potential to correlate with several autoimmune disorders and mitigate the impact of anthrax disease in humans.

Development of a synthetic relaxin-3/INSL5 chimeric peptide ligand for NanoBiT complementation binding assays.

INSL5 and relaxin-3 are relaxin family peptides with important roles in gut and brain function, respectively. They mediate their actions through the class A GPCRs RXFP4 and RXFP3. RXFP4 has been proposed to be a therapeutic target for colon motility disorders whereas RXFP3 targeting could be effective for neurological conditions such as anxiety. Validation of these targets has been limited by the lack of specific ligands and the availability of robust ligand-binding assays for their development. In this study, we have utilized NanoBiT complementation to develop a SmBiT-conjugated tracer for use with LgBiT-fused RXFP3 and RXFP4. The low affinity between LgBiT:SmBiT should result in a low non-specific luminescence signal and enable the quantification of binding without the tedious separation of non-bound ligands. We used solid-phase peptide synthesis to produce a SmBiT-labelled RXFP3/4 agonist, R3/I5, where SmBiT was conjugated to the B-chain N-terminus via a PEG12 linker. Both SmBiT-R3/I5 and R3/I5 were synthesized and purified in high purity and yield. Stable HEK293T cell lines expressing LgBiT-RXFP3 and LgBiT-RXFP4 were produced and demonstrated normal signaling in response to the synthetic R3/I5 peptide. Binding was first characterized in whole-cell binding kinetic assays validating that the SmBiT-R3/I5 bound to both cell lines with nanomolar affinity with minimal non-specific binding without bound and free SmBiT-R3/I5 separation. We then optimized membrane binding assays, demonstrating easy and robust analysis of both saturation and competition binding from frozen membranes. These assays therefore provide an appropriate rigorous binding assay for the high-throughput analysis of RXFP3 and RXFP4 ligands.

Targeting the relaxin-3/RXFP3 system: a patent review for the last two decades.

INTRODUCTION: The neuropeptide relaxin-3/RXFP3 system belongs to the relaxin/insulin superfamily and is involved in many important physiological processes, such as stress responses, appetite control, and motivation for reward. Although relaxin-3 is the endogenous agonist for RXFP3, it can also bind to and activate RXFP1 and RXFP4. Consequently, research has been focused on the development of RXFP3-specific peptides and small-molecule ligands to validate the relaxin-3/RXFP3 system as a novel drug target. AREAS COVERED: This review provides an overview of patents on the relaxin-3/RXFP3 system covering ligand development and pharmacological studies since 2003. Related patents and literature reports were obtained from established sources including SciFinder, Google Patents, and Espacenet for patents and SciFinder, PubMed, and Google Scholar for literature reports. EXPERT OPINION: There has been an increasing amount of patent activities around relaxin-3/RXFP3, highlighting the importance of this novel neuropeptide system for drug discovery. The development of relaxin-3 derived peptides and small-molecule modulators, as well as behavioral studies in rodents, have shown that the relaxin-3/RXFP3 system is a promising drug target for treating various metabolic and neuropsychiatric diseases including obesity, anxiety, and alcohol addiction.

Dysfunction of the adhesion G protein-coupled receptor latrophilin 1 (ADGRL1/LPHN1) increases the risk of obesity.

Obesity is one of the diseases with severe health consequences and rapidly increasing worldwide prevalence. Understanding the complex network of food intake and energy balance regulation is an essential prerequisite for pharmacological intervention with obesity. G protein-coupled receptors (GPCRs) are among the main modulators of metabolism and energy balance. They, for instance, regulate appetite and satiety in certain hypothalamic neurons, as well as glucose and lipid metabolism and hormone secretion from adipocytes. Mutations in some GPCRs, such as the melanocortin receptor type 4 (MC4R), have been associated with early-onset obesity. Here, we identified the adhesion GPCR latrophilin 1 (ADGRL1/LPHN1) as a member of the regulating network governing food intake and the maintenance of energy balance. Deficiency of the highly conserved receptor in mice results in increased food consumption and severe obesity, accompanied by dysregulation of glucose homeostasis. Consistently, we identified a partially inactivating mutation in human ADGRL1/LPHN1 in a patient suffering from obesity. Therefore, we propose that LPHN1 dysfunction is a risk factor for obesity development.

Advances in Radionuclide Therapies for Patients with Neuro-endocrine Tumors.

PURPOSE OF REVIEW: To provide insights into the role of peptide receptor radionuclide therapy (PRRT) in patients with advanced neuroendocrine tumors (NET) and an overview of possible strategies to combine PRRT with locoregional and systemic anticancer treatments. RECENT FINDINGS: Research on combining PRRT with other treatments encompasses a wide variety or treatments, both local (transarterial radioembolization) and systemic therapies, chemotherapy (i.e., capecitabine and temozolomide), targeted therapies (i.e., olaparib, everolimus, and sunitinib), and immunotherapies (e.g., nivolumab and pembrolizumab). Furthermore, PRRT shows promising first results as a treatment prior to surgery. There is great demand to enhance the efficacy of PRRT through combination with other anticancer treatments. While research in this area is currently limited, the field is rapidly evolving with numerous ongoing clinical trials aiming to address this need and explore novel therapeutic combinations.

Alpha-emitter Peptide Receptor Radionuclide Therapy in Neuroendocrine Tumors.

Peptide receptor radionuclide therapy (PRRT) has become mainstream therapy of metastatic neuroendocrine tumors not controlled by somatostatin analog therapy. Currently, beta particle-emitting radiopharmaceuticals are the mainstay of PRRT. Alpha particle-emitting radiopharmaceuticals have a theoretic advantage over beta emitters in terms of improved therapeutic efficacy due to higher cancer cell death and lower nontarget tissue radiation-induced adverse events due to shorter path length of alpha particles. We discuss the available evidence for and the role of alpha particle PRRT.

Mapping the interaction site for ß-arrestin-2 in the prokineticin 2 receptor.

G protein-coupled receptors (GPCRs) are a family of cell membrane receptors that couple and activate heterotrimeric G proteins and their associated intracellular signalling processes after ligand binding. Although the carboxyl terminal of the receptors is essential for this action, it can also serve as a docking site for regulatory proteins such as the ß-arrestins. Prokineticin receptors (PKR1 and PKR2) are a new class of GPCRs that are able to activate different classes of G proteins and form complexes with ß-arrestins after activation by the endogenous agonists PK2. The aim of this work was to define the molecular determinants within PKR2 that are required for ß-arrestin-2 binding and to investigate the role of ß-arrestin-2 in the signalling pathways induced by PKR2 activation. Our data show that PKR2 binds constitutively to ß-arrestin-2 and that this process occurs through the core region of the receptor without being affected by the carboxy-terminal region. Indeed, a PKR2 mutant lacking the carboxy-terminal amino acids retains the ability to bind constitutively to ß-arrestin-2, whereas a mutant lacking the third intracellular loop does not. Overall, our data suggest that the C-terminus of PKR2 is critical for the stability of the ß-arrestin-2-receptor complex in the presence of PK2 ligand. This leads to the ß-arrestin-2 conformational change required to initiate intracellular signalling that ultimately leads to ERK phosphorylation and activation.

Radionuclide Theranostics in Neuroendocrine Neoplasms: An Update.

PURPOSE OF REVIEW: This paper aims to address the latest findings in neuroendocrine tumor (NET) theranostics, focusing on new evidence and future directions of combined diagnosis with positron emission tomography (PET) and treatment with peptide receptor radionuclide therapy (PRRT). RECENT FINDINGS: Following NETTER-1 trial, PRRT with [177Lu]Lu-DOTATATE was approved by FDA and EMA and is routinely employed in advanced G1 and G2 SST (somatostatin receptor)-expressing NET. Different approaches have been proposed so far to improve the PRRT therapeutic index, encompassing re-treatment protocols, combinations with other therapies and novel indications. Molecular imaging holds a potential added value in characterizing disease biology and heterogeneity using different radiopharmaceuticals (e.g., SST and FDG) and may provide predictive and prognostic parameters. Response assessment criteria are still an unmet need and new theranostic pairs showed preliminary encouraging results. PRRT for NET has become a paradigm of modern theranostics. PRRT holds a favorable toxicity profile, and it is associated with a prolonged time to progression, reduction of symptoms, and improved patients' quality of life. In light of further optimization, different new strategies have been investigated, along with the development of new radiopharmaceuticals.

Developing insulin-like peptide 5-based antagonists for the G protein-coupled receptor, RXFP4.

Human insulin-like peptide 5 (INSL5) is a gut hormone produced by colonic L-cells, and its biological functions are mediated by Relaxin Family Peptide Receptor 4 (RXFP4). Our preliminary data indicated that RXFP4 agonists are potential drug leads for the treatment of constipation. More recently, we designed and developed a novel RXFP4 antagonist, A13-nR that was shown to block agonist-induced activity in cells and animal models. We showed that A13-nR was able to block agonist-induced increases in colon motility in mice of both genders that express the receptor, RXFP4. Our data also showed that colorectal propulsion induced by intracolonic administration of short-chain fatty acids was antagonized by A13-nR. Therefore, A13-nR is an important research tool and potential drug lead for the treatment of colon motility disorders, such as bacterial diarrhea. However, A13-nR acted as a partial agonist at high concentrations in vitro and demonstrated modest antagonist potency (∼35 nM). Consequently, the primary objective of this study is to pinpoint novel modifications to A13-nR that eliminate partial agonist effects while preserving or augmenting antagonist potency. In this work, we detail the creation of a series of A13-nR-modified analogues, among which analogues 3, 4, and 6 demonstrated significantly improved RXFP4 affinity (∼3 nM) with reduced partial agonist activity, enhanced antagonist potency (∼10 nM) and maximum agonist inhibition (∼80 %) when compared with A13-nR. These compounds have potential as candidates for further preclinical evaluations, marking a significant stride toward innovative therapeutics for colon motility disorders.

The Impact of Posttreatment Imaging in Peptide Receptor Radionuclide Therapy.

Posttreatment imaging of γ-emissions after peptide receptor radionuclide therapy (PRRT) can be used to perform quantitative dosimetry as well as assessment response using qualitative measures. We aimed to assess the impact of qualitative posttreatment imaging on the management of patients undergoing PRRT. Methods: In this retrospective study, we evaluated 100 patients with advanced well-differentiated neuroendocrine tumors undergoing PRRT, who had posttreatment SPECT/CT imaging at 24 h. First, we evaluated the qualitative assessment of response at each cycle. Then using a chart review, we determined the impact on management from the posttreatment imaging. The changes in management were categorized as major or minor, and the cycles at which these changes occurred were noted. Additionally, tumor grade was also evaluated. Results: Of the 100 sequential patients reviewed, most (80% after cycle 2, 79% after cycle 3, and 73% after cycle 4) showed qualitatively stable disease during PRRT. Management changes were observed in 27% (n = 27) of patients; 78% of those (n = 21) were major, and 30% (n = 9) were minor. Most treatment changes occurred after cycle 2 (33% major, 67% minor) and cycle 3 (62% major, 33% minor). Higher tumor grade correlated with increased rate of changes in management (P = 0.006). Conclusion: In this retrospective study, qualitative analysis of posttreatment SPECT/CT imaging informed changes in management in 27% of patients. Patients with higher-grade tumors had a higher rate of change in management, and most of the management changes occurred after cycles 2 and 3. Incorporating posttreatment imaging into standard PRRT workflows could potentially enhance patient management.

Identification of Novel Series of Potent and Selective Relaxin Family Peptide Receptor 1 (RXFP1) Agonists.

Relaxin H2 is a clinically relevant peptide agonist for relaxin family peptide receptor 1 (RXFP1), but a combination of this hormone's short plasma half-life and the need for injectable delivery limits its therapeutic potential. We sought to overcome these limitations through the development of a potent small molecule (SM) RXFP1 agonist. Although two large SM HTS campaigns failed in identifying suitable hit series, we uncovered novel chemical space starting from the only known SM RXFP1 agonist series, represented by ML290. Following a design-make-test-analyze strategy based on improving early dose to man ranking, we discovered compound 42 (AZ7976), a highly selective RXFP1 agonist with sub-nanomolar potency. We used AZ7976, its 10 000-fold less potent enantiomer 43 and recombinant relaxin H2 to evaluate in vivo pharmacology and demonstrate that AZ7976-mediated heart rate increase in rats was a result of RXFP1 agonism. As a result, AZ7976 was selected as lead for continued optimization.

Discovery of Clinical Candidate AZD5462, a Selective Oral Allosteric RXFP1 Agonist for Treatment of Heart Failure.

Optimization of the highly potent and selective, yet metabolically unstable and poorly soluble hRXFP1 agonist AZ7976 led to the identification of the clinical candidate, AZD5462. Assessment of RXFP1-dependent cell signaling demonstrated that AZD5462 activates a highly similar panel of downstream pathways as relaxin H2 but does not modulate relaxin H2-mediated cAMP second messenger responsiveness. The therapeutic potential of AZD5462 was assessed in a translatable cynomolgus monkey heart failure model. Following 8 weeks of treatment with AZD5462, robust improvements in functional cardiac parameters including LVEF were observed at weeks 9, 13, and 17 without changes in heart rate or mean arterial blood pressure. AZD5462 was well tolerated in both rat and cynomolgus monkey and has successfully completed phase I studies in healthy volunteers. In summary, AZD5462 is a small molecule pharmacological mimetic of relaxin H2 signaling at RXFP1 and holds promise as a potential therapeutic approach to treat heart failure patients.

The Origin and Evolution of Sex Peptide and Sex Peptide Receptor Interactions.

Post-mating responses play a vital role in successful reproduction across diverse species. In fruit flies, sex peptide binds to the sex peptide receptor, triggering a series of post-mating responses. However, the origin of sex peptide receptor predates the emergence of sex peptide. The evolutionary origins of the interactions between sex peptide and sex peptide receptor and the mechanisms by which they interact remain enigmatic. In this study, we used ancestral sequence reconstruction, AlphaFold2 predictions, and molecular dynamics simulations to study sex peptide-sex peptide receptor interactions and their origination. Using AlphaFold2 and long-time molecular dynamics simulations, we predicted the structure and dynamics of sex peptide-sex peptide receptor interactions. We show that sex peptide potentially binds to the ancestral states of Diptera sex peptide receptor. Notably, we found that only a few amino acid changes in sex peptide receptor are sufficient for the formation of sex peptide-sex peptide receptor interactions. Ancestral sequence reconstruction and molecular dynamics simulations further reveal that sex peptide receptor interacts with sex peptide through residues that are mostly involved in the interaction interface of an ancestral ligand, myoinhibitory peptides. We propose a potential mechanism whereby sex peptide-sex peptide receptor interactions arise from the preexisting myoinhibitory peptides-sex peptide receptor interface as well as early chance events both inside and outside the preexisting interface that created novel sex peptide-specific sex peptide-sex peptide receptor interactions. Our findings provide new insights into the origin and evolution of sex peptide-sex peptide receptor interactions and their relationship with myoinhibitory peptides-sex peptide receptor interactions.