This prospective, multicenter, randomized study aimed to compare the 1-year clinical outcomes after primary stenting with self-expanding bare metal nitinol stent (SENS) and plain old balloon angioplasty (POBA) in patients with critical limb ischemia (CLI) and below-the-knee (BTK) lesions. Overall, 119 patients with CLI and BTK lesions were randomized to POBA alone  or primary stenting with SENS  after achieving acceptable POBA results in target BTK lesions. Clinical outcomes including amputation and revascularization rates were prospectively compared for 1 year. After 1 year, similar incidence rates of individual clinical endpoints, including cardiac death , myocardial infarction , repeat revascularization , target lesion revascularization , and amputation , were observed. POBA appeared to have acceptable treatment outcomes compared with primary stenting with SENS after 1 year in CLI patients with BTK lesions undergoing percutaneous transluminal angioplasty (PTA). With the advent of newer techniques and growing evidence, endovascular intervention is a frontline treatment strategy for treating below-the-knee (BTK) lesions with critical limb ischemia (CLI) –3. HowevThis randomized clinical trial was conducted in 18 well-experienced tertiary endovascular intervention centers in South Korea to compare the efficacy of SENS implantation to balloon angioplasty alone for BTK lesions following successful balloon angioplasty . The study was conducted in accordance with the ethical guidelines of the 2004 Declaration of Helsinki. Institutional Review Board (IRB) of a Korea University Guro Hospital (KUGH) approved all of the consenting procedures. All patients or their legal guardians were given a thorough written and verbal explanation of the study procedure before obtaining written consent for the participation in this study. The authors of this manuscript have certified that the information contained herein is true and correct as reflected in the records of the IRB (#KUGH MD120008). Angiographic images and data management were analyzed by the Cardiovascular Intervention Research Institution, a core laboratory independent of the sponsor .This trial compared the clinical outcomes of primary stenting and POBA for the treatment of patients with CLI and BTK lesions. Between December 2012 and September 2016, patients with CLI and BTK lesions were enrolled in this prospective, multicenter, randomized trial. Patients were eligible if they had peripheral arterial disease with ischemic rest pain, wounds, such as ulcers or gangrene, that manifested as BTK lesions. More than half of included patients had wounds with a Rutherford classification (RC) of 4–6. If the wound site was related to a vascular origin according to the angiosome concept, patients were included regardless of the RC of their wound. Patients with acute limb ischemia, those with a life expectancy < 1 year, systemic coagulopathy, or intolerance to antiplatelet agents were excluded. Angiographic eligibility criteria included the presence of de novo stenotic lesions involving 50% of the lumen or occlusion of BTK arteries, and patients meeting these criteria were considered acceptable for endovascular therapy .Eligibility was assessed, and informed consent was obtained by the physicians. Randomization was performed in a 1:1 ratio using a computer-generated randomization sequence so that all patients enrolled in this study were randomized to undergo either SENS implantation (stenting group) or POBA alone (POBA group). For a fair comparison, patients who achieved satisfactory results with balloon angioplasty were randomly assigned to either the POBA group or the stenting group. The POBA group ended the procedure after randomization and the stenting group received SENS implantation. To compare groups in which the lesions had a homogeneous character, only a single target lesion < 80 mm in length in each leg of each patient was selected and treated with an individual stent or POBA alone. Between December 2012 and September 2016, 119 patients were included in this study. In total, 61 patients were randomly assigned to undergo POBA alone, and 58 patients were randomly assigned to undergo SENS implantation . Also, 3All patients were administered loading doses of clopidogrel (300–600 mg) and aspirin (200–300 mg) before the procedure. After sheath insertion at the arterial access site, a bolus dose of unfractionated heparin (70–100 units/kg) was administered. All patients received aspirin (100 mg daily) and clopidogrel (75 mg daily) as a dual antiplatelet (DAPT) maintenance regimen for at least 1 month. Cilostazol (100 mg bid or 200 mg dp daily) was prescribed based on the physician’s discretion as a triple antiplatelet on top of clopidogrel-based DAPT. Some physicians used cilostazol-based DAPT 3–6 months after the index procedure to reduce restenosis.PTA was performed using the standard technique. After successfully crossing the target lesion with a 0.014-inch guidewire, either intraluminally or subintimally, prolonged balloon dilatation with an adequately sized balloon  was performed for 2–3 min. Some patients were recanalized via the retrograde or transcollateral approach following unsuccessful reentry into the distal true lumen using an antegrade approach. To minimize crossover from PTA to stent implantation, prolonged balloon dilatation was performed in patients with unsatisfactory results for balloon dilatation. Once the POBA results were acceptable and the procedure was completed, randomization was performed to leave it alone or with additional stenting. In the stenting group, Xpert  SENSs were used for routine primary stenting.The main study end-point was the target extremity amputation rate of the patient groups at 1 year. Imaging follow-up was intended to be an additional study endpoint. However, patient characteristics and limitations of insurance coverage in South Korea with CLI made routine follow-up with imaging studies difficult. In addition, limb salvage is considered the most important target of revascularization in patients with CLI. Hence, to assess more clinically relevant information, target extremity amputation was set as the primary endpoint, revascularization including TLR at 1 year, was set as the secondary endpoint.Death, including cardiac death, myocardial infarction (MI), stroke, and wound recurrence were also assessed during follow-up. Follow-up visits were planned at 1, 6, and 12 months after the index procedure; clinical examinations, laboratory investigations, and imaging studies were performed according to the physician’s advice. One year after the index procedure, follow-up data were collected via face-to-face interviews at the outpatient clinic, review of medical records, and/or telephone conversations with the patients.Data for all endpoints were evaluated using intention-to-treat analysis. All data are expressed as mean ± standard deviation. The unpaired Student’s t-test and Mann–Whitney rank test were used to compare continuous variables. Categorical variables were compared using the chi-squared and Fisher’s exact tests. The Kaplan-Meier method with a log-rank test was used to compare the cumulative incidence of primary and secondary endpoints. A p value < 0.05 was considered statistically significant. All statistical analyses were performed using the SPSS software .The baseline clinical and laboratory characteristics are presented in A clinical investigation was performed on all patients at the 1-year follow-up visit. The 1-year clinical outcomes are presented in The main findings of this study were as follows. In patients with CLI with a BTK lesion undergoing an intervention, (1) clinical outcomes, including amputation and cardiovascular event rates, were similar between the primary stenting and POBA groups; (2) amputation and revascularization rates were relatively low in both groups; and (3) reasonable results could be derived only from short-term clinical follow-up, and imaging follow-up was not compulsory.With the rapid improvement in device technology along with increasing operator experience, interventional treatment is a well-accepted procedure in patients with a BTK lesion and CLI –11. MultThe most critical limitations of previous studies on patients with CLI were the format of the study and the heterogeneous strategies, including primary and bail-out stenting in patients undergoing stenting with BTK lesions –15. In tCLI with a BTK lesion is usually an extensive and multilevel arterial disease that results in adverse clinical outcomes . SENS haIn this study, the primary stenting (0.0%) and POBA (1.6%) groups showed a low major amputation rate. Even with the use of advanced techniques, the development of an interventional method alone is insufficient to explain the significantly low limb recovery rates demonstrated in both groups. The development of wounds and CLI are related to diabetes . AdditioThis study also elucidated the importance of setting an appropriate endpoint for the treatment of BTK lesions with CLI. In the coronary era, clinical practices have shifted toward minimizing the use of routine imaging tests and focusing on clinical symptoms, and this has resulted in good patient outcomes . In patiThis study had some limitations. First, it addressed only BMS and POBA alone. However, there are several newer devices, such as drug-coated balloons, DES, and polymer-free stents, and a study comparing these devices should be conducted. Second, the relatively small sample size and short duration of the study period did not provide strong evidence of the actual clinical efficacy and safety of the procedures. Third, although this was a multicenter study and the general goal of CLI treatment was early-phase ulcer healing, large-scale and long-term clinical investigations are required. Fourth, this study was designed to focus on a “patient-centric” concept; hence, follow-up imaging modalities, such as computed tomography angiography and conventional angiography, were not routinely performed. These investigations were only performed in the event of wound recurrence or at the physician’s discretion, regardless of ischemic symptoms. Even if restenosis occurred, follow-up imaging tests may not have been performed if the wound did not recur or patients did not complain of definite claudication during the follow-up period after the index procedure. Thus, the actual restenosis rate may not have been reflected. Finally, due to the characteristics of the lesions in the patients with CLI, there were patients with iliac artery or femoral artery lesions . The stuIn this prospective randomized multicenter study, during the 1-year follow-up, primary stenting with SENS and POBA alone demonstrated a significantly low rate of clinical adverse events in treating patients with CLI and BTK lesions. These results indicate that POBA may be an acceptable option in treating CLI with BTK lesions avoiding stent implantation. Also, based on the non-inferior results of the stenting group, SENS implantation could remain the preferred treatment option in special circumstances such as persistent recoil and flow-limiting dissection despite prolonged balloon dilatation in the treatment of CLI with BTK lesions. Long-term follow-up studies with larger study populations and routine imaging follow-up tests are necessary to elucidate the final conclusions.S1 Checklist(DOCX)Click here for additional data file.S1 Protocol(DOC)Click here for additional data file.


Intrauterine adhesions (IUAs) are the formation of scar tissues in the endometrial cavity. The fibrous tissue in the uterus decreases the space inside the uterine cavity. It includes both endometrium and myometrium. It may lead to hypomenorrhea or amenorrhea, pain, difficulty in conceiving, and recurrent abortion. IUA is caused by uterine tissue damage mostly during surgical procedures such as dilatation and curettage. Other causes may include pregnancy-related complications, miscarriage, abnormal bleeding, infections, fibroid removal, and cesarean section (C-section). Patients generally do not have any symptoms and hence are unaware of the condition. The main therapeutic procedure presently used is hysteroscopic transcervical resection of adhesion (TCRA) with hormonal therapy and nondegradable stent as postoperative adjuvant therapy. It has some major limitations such as failure to prevent recurrence and preserve fertility along with difficulty in endometrial tissue repair due to its anatomical site. These limitations have forced the researchers to think about a better treatment modality. In recent times, a better treatment modality has evolved with stem cell therapy. Therefore, this review presents the recent and advanced therapeutic modalities for the treatment of IUAs. Intrauterine adhesions (IUAs) also called Asherman’s syndrome is one of the most severely undiagnosed conditions among women. It is caused as a result of trauma to the endometrial tissue resulting in hypomenorrhea or amenorrhea, recurrent abortion, and infertility . It coulMostly being asymptomatic, the diagnosis accidental. The new endoscopic procedure that has enabled easy access inside the uterine cavity has made diagnosis and management very easy. Hysteroscopy is the gold standard diagnostic modality . It can Stem cells are completely or partially undifferentiated cells that can differentiate into a particular type of cell. They can be cultured in long-term in vitro. They have cell surface markers and in vivo reconstruction capacity. Embryonic stem cells and adult stem cells are the two types of stem cells . EmbryonIUAs are one of the most undiagnosed and underdiagnosed conditions and have only limited treatment modalities. These treatment modalities have limitations that are responsible for the poor prognosis of this condition. Newer treatment modalities are under research; therefore, this review provides detail about the new treatment modality that may overcome the drawbacks of the present treatment technique.We undertook a systematic search through PubMed and CENTRAL in November 2022 using keywords such as "Asherman syndrome" and "stem cell therapy" (((Asherman syndrome [Title / Abstract) OR (stem cell therapy (Title / Abstract])) OR (koch*[Title / Abstract])) OR ("Asherman syndrome" [MesH terms|) AND (("stem cell therapy" [Title / Abstract) OR (ScT [Title/ Abstract])) OR ("stem cell Therapy" [MesH ferms]). We additionally searched for key references from bibliographies of the relevant studies. the search was updated in February 2023. One reviewer independently monitored the retrieved studies against the inclusion criteria, in the beginning, based on the title and abstract and then on the full text. Another reviewer also reviewed approximately 20% of these studies to validate the inclusion of studies, as shown in Figure Stem cell therapy in the regeneration of endometrial tissue has shown great results in both animal and human models . DiffereStem cell transplantation has many therapeutic approaches toward tissue repair and hence is an excellent alternative form of treatment. Recently, postoperative stem cell therapy is a great approach to resolving reproduction-related abnormalities in IUA patients. The uterine sources of stem cells are mesenchymal stem cells (MSCs)/stromal cells, epithelial stem cells (ESCs), and endothelial stem cells. All of these cells have the capacity for endometrial repair . NonuterEndogenous stem cellsMesenchymal Stem CellsMSCs act as pericytes and perivascular cells and are seen on the outer surface of the small blood vessels that promote endometrial cell regrowth, maturation, and hemostasis . PropertCD140b also called platelet-derived growth factor receptor-β, CD146, stage-specific embryonic antigen (SSEA-1), and leucine-rich repeat-containing G-protein (LGR5) are some of the specific MSC markers . These mEpithelial Stem CellsESCs are found in the basalis layer of the endometrium and are not shed off during menstruation. These cells are large, single-cell-derived colony forming units (CFUs) and have high proliferating capacity . These cEndothelial Stem CellsThe endometrium cells have two types of populations, main populations and side populations. The endometrial side population has a great clonogenic capacity but lacks a niche. The endometrial main population lacks clonogenic capacity but has a great niche. When both EMP and ESP are co-cultured, they show great success in forming uterine environment .Exogenous stem cellsBone-Marrow-Derived Stem CellsStem cells from the bone marrow can differentiate into multiple nonhematopoietic cell lineages. Studies have suggested endometrial cells and stromal cells can be formed from donor-derived bone marrow cells . The tisUmbilical Cord MSCsUCMSCs have a high cartilage differentiation rate and high proliferative capabilities . They arGenes for Endometrial RegenerationWingless-related integration site (Wnt), receptor tyrosine kinase (c-kit) (CD117), octamer-binding transcription factor-4 (Oct-4), CD34, and Musashi-1 are some of the many genes/proteins that are responsible for the complete regeneration of the endometrium. A signal cascade regenerated by the Wnt gene secretes a protein that binds to the surface marker of the frizzled family and decides the future of the cells. Wnt7a is responsible for the regeneration of endometrium, and progesterone suppresses it in the latter half of the menstruation and causes the epithelium to secretory phenotype transformation . MülleriMesenchymal to Epithelial TransitionStem cells have a unique feature of cellular transdifferentiation where one somatic cell differentiates from another without going into any intermediate pluripotent state. This feature plays a great role in the repair of the endometrium. Three periods in women’s life where uterine regeneration is significant are embryogenesis, menstruation, and postpartum . Within Immunomodulatory and Antimicrobial Effects of Stem Cellstolerogenic Treg cells, which are suppressor T-cells that downregulate effector T-cells, in the adaptive system [A dysregulated inflammatory response to an infection causes a life-threatening condition called sepsis. Delays in therapy lead to increased mortality as well as irreparable damage to numerous organs owing to reduced oxygen levels in the tissues and lack of blood flow. Numerous researches have shown a report on the immunomodulatory, anti-inflammatory, anti-apoptotic, and anti-microbial capabilities of MSCs. Therefore, the application of MSCs has been considered a very promising and productive strategy therapeutically. MSCs prevent the growth of B- and T-cells and their development into pro-inflammatory Th1 and Th17 cells as well as e system . Insteade system . When ime system . Stem ceIn Situ Uterine StentsThe application of in situ intrauterine stents can avoid adhesion formation by separating the injured endometrium as well as works as a vehicle for delivering stem cells in various therapies . ArtificPolymer-based hydrogels: Many polymers have been studied and used as a treatment for IUAs; some of them are Pluronic F-127, ascorbyl palmitate (AP), polyethylene glycol (PEG), poly-lactic-co-glycolic acid (PLGA), and heparin poloxamer (HP). These polymers can encapsulate many components like growth factors, drugs, stem cells, etc. . One sucPolysaccharide-based hydrogels: Hyaluronic acid gel (HA-GEL) is one of the most promising polysaccharide-based hydrogels. It is loaded with umbilical cord-derived MSCs that reconstruct and regenerate the endometrium .Protein-based hydrogels: Collagen is one of the most used protein-based biomaterials due to its abundant bioavailabilities. UCMSCs are encapsulated with collagen scaffold and are used in case of recurrence of IUAs postadhesiolysis surgery .Asherman syndrome is one of the most underrated gynecological problems with very common etiologies. It is mostly caused by postoperative complications. It remains undiagnosed as most of the time it is asymptomatic. There is no appropriate treatment for it without any limitation. TCRA is the most widely used treatment modality but it has its limitation such as recurrence and nonrestoration of the endometrium. With advancing technologies, many newer therapies are under research for it. One such treatment modality is stem cell therapy. It has shown wonderful results in animal and human models. Exogenous and endogenous stem cells along with gene modulation and intrauterine stents are some of the units of stem cell therapy. With these advancing technologies and researches stem cells may fulfill the need of the hour in this condition.


This case report presents an 82-year-old woman with decreased visual acuity. Clinical and multimodal evaluation revealed an elevated macular lesion with choroidal thickening. Fluorescein angiography and optical coherence tomography (OCT) showed characteristic patterns of choroidal metastasis. Thoracic CT scan confirmed a nodular mass in the right hilus, suggesting bronchogenic neoplastic process. A pleural biopsy demonstrated malignant neoplasia with characteristics of adenocarcinoma, and immunohistochemistry was positive for thyroid transcription factor 1 (TTF-1). These findings emphasize the critical role of ophthalmological examinations in early detection and intervention, as choroidal tissue is highly susceptible to metastasis, particularly from breast and lung cancers. Additionally, the poor prognosis associated with metastatic lung cancer underscores the urgency for prompt action. The choroid is the most common place for intraocular metastasis . Lung caWe present the case of an 82-year-old woman with choroidal metastasis as the initial presentation of lung cancer through the assessment of the clinical records and complementary exams , ocular ultrasound, fluorescein angiography, and computed tomography (CT) scan).An 82-year-old woman who was subject to a Yttrium aluminum garnet (YAG) on the right eye presented to the emergency department due to maintenance of decreased visual acuity, with a best corrected visual acuity (BCVA) of 20/63. Fundus examination showed an elevated macular lesion Figure . The lefA complementary study of the right eye with OCT showed a choroidal elevation with a bumpy pattern and subretinal fluid Figure . FluoresAdditionally, she underwent an ocular ultrasound showing an elevated mass in the posterior pole of the eyeball that was consistent with a choroidal thickening with contrast enhancement shown in brain and orbit CT scan Figure . Due to HER2 gene (c.2313_2324dup) but no mutations in EGFR, KRAS, or BRAF genes were found.Pleural biopsy confirmed an adenocarcinoma with positive thyroid transcription factor-1 (TTF-1) staining. Next-generation sequencing identified a mutation in the With a diagnosis of stage IV non-small cell lung cancer with choroidal and pleural metastasis, the patient was referred for palliative care in the Oncology and Internal Medicine departments. No specific ophthalmologic treatment was administered for the choroidal mass. Unfortunately, the patient passed away five months after the initial diagnosis.Intraocular metastasis most commonly affects the uveal tissue , with thOur findings are consistent with previous reports indicating that choroidal metastasis from lung cancer often presents as a unifocal and unilateral pattern of metastasis . This paThe prognosis for patients with choroidal metastasis from lung cancer is generally poor, as the presence of such metastases signifies a hematogenous spread of the cancer . StudiesOur findings corroborate the well-established understanding that uveal tissue, particularly the choroid, is highly susceptible to intraocular metastasis. Decreased visual acuity in a patient with a macular lesion and a choroidal hypofluorescent pattern is frequently associated with choroidal metastasis from a primary occult cancer. Breast cancer and lung cancer represent the most common primary malignancies leading to choroidal metastasis. Given the poor prognosis associated with lung cancer uveal metastasis, early detection and prompt intervention are crucial for optimizing patient outcomes.


The design of newbiomolecules able to harness immunemechanismsfor the treatment of diseases is a prime challenge for computationaland simulative approaches. For instance, in recent years, antibodieshave emerged as an important class of therapeutics against a spectrumof pathologies. In cancer, immune-inspired approaches are witnessinga surge thanks to a better understanding of tumor-associated antigensand the mechanisms of their engagement or evasion from the human immunesystem. Here, we provide a summary of the main state-of-the-art computationalapproaches that are used to design antibodies and antigens, and inparallel, we review key methodologies for epitope identification forboth B- and T-cell mediated responses. A special focus is devotedto the description of structure- and physics-based models, privilegedover purely sequence-based approaches. We discuss the implicationsof novel methods in engineering biomolecules with tailored immunologicalproperties for possible therapeutic uses. Finally, we highlight theextraordinary challenges and opportunities presented by the possibleintegration of structure- and physics-based methods with emergingArtificial Intelligence technologies for the prediction and designof novel antigens, epitopes, and antibodies. The levels of involvedproteins, their interaction strengths, as well as alterations in interactionpartners build network connectivity at a proteome-wide scale ultimatelydefining cell phenotypes.6Such phenomenaare epitomized by mechanisms by which protein–proteinrecognition, interactions, and (re)organizations shape the functionalnetworks that control immune responses.In this general framework, moleculardesigners have the opportunityto address general and specific issues that will have important returnsin the development of new diagnostics and therapeutics with potentialin personalized medicine.9 The development of new immunotherapies is now realizingits potential in combating drug resistance.10 Moreover, exciting results are reported in the treatment of cancer,where immunooncology is becoming an increasingly important tool forpatients. In this context, the discovery of the mechanisms of negativeimmune regulation and approval of immune checkpoint inhibitors hada primary role in revealing the potentialities of the immune systemcells in counteracting tumor development,12 and the prognostic value of the tumor-associated immune landscapeis now becoming more and more apparent.13 More recently, chimeric antigen receptor-based therapies (CAR-T)were approved for the treatment of some hematologic cancers 15 while showing clinically significant antitumor activities in othermalignancies.17 Interestingly, in these therapies,the unpaired targeting specificity of monoclonal antibodies (mAbs)is combined with the cytotoxicity and long-term persistence providedof T-cells.Vaccination is arguably the most successfulmedical discovery thathas benefited human health globally. Worldwide vaccination campaignshave contained the recent Covid-19 pandemic and eradicated life-threateningdiseases.in silico-led approach to protein-antigen (Ag) selection, termed Reverse Vaccinology(RV), and subsequently, pan-genomic RV,19 which resultedin the first vaccine, Bexsero (approved by the European MedicinesAgency in 2013), against meningococcal serotype B infections.20 The most recent development of RV, termed StructuralVaccinology (SV), exploits atomic-level three-dimensional informationto engineer Ags with improved immunological and/or biochemical properties.21Following the genome era advent, conventional immunologyhas beenovertaken by a more rapid and effective In a time where medical challenges arenumerous, the value of beingable to design protein antigens and antibodies with rational methodsis clearly manifold. Besides providing new approaches to diagnosticsand therapeutics development, this will generate new concepts andmethods of general applicability in the realm of protein-interactionstudies in health and disease, favoring the development of novel precisiontools for chemical and cell biology.23 One particularly interesting field of application entails the manipulationof the activation of cell-surface receptors in response to extracellularsignals. Proteins can, in fact, be designed to engage the extracellulardomains of these receptors, in some cases driving their clusteringon the cell surface, and ultimately generating different downstreamcell cascades different from the ones observable in the absence ofthe binder protein.25In a different context,the possibility to design protein bindersthat may act as therapeutic tools, engaging specific molecular targetsand hijacking pathologic protein pathway, has the potential to providenovel approaches to block the detrimental activities of proteins thatstill remain largely undruggable by classical interventions.Our ability to design moleculesthat target proteins in specificnetworks thus has important implications for basic science and appliedresearch in chemical biology, biochemistry, and molecular medicine.From the fundamental point of view, designed biomolecules can helpadvance our understanding of key interactions for diseases (whichentail both host–pathogen interactions and derailed networksin nontransmissible pathologies) at the atomic level. Indeed, selectiveperturbation of well-defined complexes can aptly translate into animpact on the functional activities of the involved biomolecules,shedding light on their roles in biochemical pathways. From an applicativepoint of view, this knowledge can be translated into new rules forthe efficient discovery of new candidates, such as antigens for vaccinedevelopment, proteins as biological therapeutics, and new molecularprobes for diagnostic development.To make progress along thisavenue, it is first necessary to understandwhat makes a protein substructure a potential (immune) interactionsurface and then translate this information into the design of newsystems that mimic desired interactions.This is an excitingtime for this field of research: fundamentalcontributions have been presented over the past few years in the structure-baseddesign of new antigens, antibodies, and binders in general. The adventof Artificial Intelligence (AI) now enables protein structure predictionand analysis on an unprecedented scale and with accuracy. It is clearthat the combination of AI with biophysics- and biochemistry-basedmethods will shape the next few years, speeding up the discovery ofnovel biologics with a wide range of applications.Here, wewill review some of the main advancements in these fieldswith a specific focus on methods involving the development and applicationof physical chemistry and computational chemistry approaches. A moregeneral overview of methods relying on bioinformatic (and AI-powered)tools is provided, and, where needed, the reader is directed to morespecialized reports. We will then provide some  perspectiveson the evolution and potential future impacts of this field in chemical,cell, and molecular biology.228Antibodiesare not only important investigative tools in (bio)chemistry,molecular biology, and medicine but are now largely employed as therapeuticsin cancer, autoimmune and infectious diseases, and as diagnostic tools.29 Breakthroughs incomputational structure prediction methods, high-throughput sequencing,and data analysis have indeed greatly contributed to in silico antibody development.31Established antibody production processes rely on long andcostlyexperimental procedures, often employing inoculated animals. Thisapproach is sometimes limited by the capacity of animals to elicitantibodies targeting the desired epitope. In this framework, computationalmethods hold the potential of making antibody development more customizable,faster, and cheaper.Computationalmethods for antibody development entail structureprediction, design, and antigen–antibody complex characterizationwith the end goal of designing an optimal Ab sequence complementaryto a desired antigen.32 and usually, five ofthe six CDR regions fall into these representative structures. Thesame does not hold for the CDRH3 loop region, whose extended lengthand conformational variability make structural prediction more elusive.Small changes in heavy/light chain variable domain (VH-VL) orientationshave also been shown to have a significant impact on CDR positionand thus on antibody/antigen affinity. Therefore, efficacious modelingof the VH-VL interface is also a highly relevant matter. Since immunoglobulins share a common Y-shapedfold, most traditionalmethods for modeling antibody structure rely on homology modelingstrategies. Human antibody structure consists of two heavy and twolight chains characterized by a well-conserved constant domain andthe fragment antigen binding (FAB) domain, where conserved frameworkregions alternate with the highly variable loop regions named complementarydetermining regions (CDRs). The sequence/conformational variabilityin CDR regions, resulting from VDJ gene recombination and somatichypermutation, is what makes affinity maturation and diverse antigentargeting possible. Yes, from the molecular designer’s pointof view, this also represents one of the major challenges in antibodystructure prediction. Fortunately, making the issue more manageable,canonical structural clusters of loop conformations/orientations havebeen established34 Among the many computationalframeworks included in Rosetta, there are several protocols specificfor the computational modeling and design of antibodies.36Rosetta is one of the leading computational toolsin biomoleculardesign and protein fold prediction. Initially developed in the groupof David Baker at the University of Washington, it is now managedand developed by RosettaCommons, an international community countingmore than 70 research groups.37 is a template-basedprotocol for structural prediction from sequence. First, selectedtemplates for the framework and five canonical loops are grafted ontoa preliminary model; at this point, the HCDR3 loop is modeled de novo, while the heavy/light chain variable region (VH-VL)orientations are refined. Template selection relies on a BLAST sequencesearch of the PyIgClassify database38 andincludes the sampling of diverse VH-VL orientations. The CDRH3 de novo modeling step employs the next-generation kinematicloop closure algorithm (KlC) in increasing resolution steps togetherwith side-chain packing and minimization. With time, more accurateconformational constraints were introduced to overcome the limitedcapacity of the modeling protocol to sample the so-called kinked conformationsof CDRH3 which were, on the contrary, observed more frequently inantibodies’ native structures.40 Applicabilityto nonhuman or nonmurine antibodies is limited by the availabilityof structures in the PDB database. For such modeling tasks, templatescould be entered manually or selected from a custom-made database.Similarly, the modeling of rare CDR conformations will be more reliablewhen more of those structures will be experimentally solved. Sphinx,a hybrid method combining ab initio and knowledge-basedloop structural prediction, has been shown to outperform Rosetta.41Rosetta Antibody42 which does not rely onhomologous templates. Experimentally determined antibody structuresare fragmented into four backbone regions . These fragments are randomly recombined also consideringdifferent rigid-body VH-VL orientations to have a database of structureswith a target sequence length. Starting from a randomly selected initialconformation, a Monte Carlo simulation is performed for sampling combinationsof backbone fragments, packing of side chains, and minimization ofthe entire structure which outputs a single chain variable fragment(scFv) structure. Given the independence from sequence homology, itallows one to select templates with low sequence identity but highstructural compatibility. On the other hand, the capacity of representingCDR loops of rare length is limited also due to the requirement thatthe target sequence and template match in length. The more recentimplementation AbPredict2 is available as a Web server.43 Improvements consist of reduced computationalcost, a decrease in the stereochemical strain of the model, and bettersampling of light-heavy chain orientations.A second protocol for antibody modelingimplemented in Rosettais AbPredict,44 Kotai Antibody Builder,45 and PIGS.46 ABodyBuilder is a fully automatedcomputational pipeline comprising steps similar to those already describedfor Rosetta Antibody with template selection relying on sequence similarity,using FREAD47 for all CDR loops modelingand SCWRL448 for side chains.44 It has the advantage of being a fast predictiontool, favoring high-throughput screenings, and implements a data-drivenestimation of model accuracy as a measure of the probability of aregion to be modeled within a defined threshold. Kotai Antibody Builderselects templates for framework and non-H3 CDR region, privilegingstatistically derived rules together along with sequence identityevaluation. For CDRH3 loop modeling, it relies on previously establishedrules for base region type prediction. Subsequent refinement can employthe SPANNER module for fragment-based structural prediction, followedby energy minimization steps and scoring.45 PIGS privileges template construction from a unique structure andrelies on canonical structures for loop modeling.46 The main packages for full antibody modeling together withothers that focus on more specific modeling tasks have been listedin a recent dedicated review.30Other open-sourceautomated similar workflows are ABodyBuilder,49TheAMA II benchmarking of many of these protocols revealed thattheir performance is overall similar, with an average RMSD of about1.1 Å for the entire Fv and with the most critical region beingthe CDRH3 loop region, modeled with an average accuracy of about 2.2Å but up to >5 Å RMSD in some cases.de novo structural prediction.51 Methods optimized for immunoglobulin structural prediction performbetter than AlphaFold2, originally developed for general protein structuralprediction.53 DeepAb, an improved version ofDeepH3, was tested on the Rosetta Antibody benchmark and shown tooutperform grafting-based methods.54 Themethod is based on a deep learning model trained using cross-entropyloss on pairs of heavy/light chain sequences derived from the ObservedAntibody Space55 and can predict relativedistances and orientations between pairs of residues of Fv domainfrom sequence information and previous information on Ab structuralorganization. The final step is a fast Rosetta-based protocol forthe generation of the 3D protein structure. Insights about relevantamino-acid interactions and mutations improving binding affinity couldalso be derived. ABlooper,53 based on anequivariant graph neural network is currently the fastest method forpredicting CDR conformations and was shown to achieve on average lowerRMSD than ABodyBuilder but not DeepAb on models from the Rosetta AntibodyBenchmark. The same group recently released IgFold which also embedsinformation from templates and has lower runtime for structural predictions.56 These methods circumvent the lack of experimentallysolved structures on which grafting methods rely and, on the otherhand, take profit from the large amount of sequence data currentlyavailable. Their short runtime  opens the possibility of high-throughput structural screenings.An extended description of AI-based methods is beyond the scope ofthis review. For further details, the reader is directed to otherreports.57In addition to the above-described grafting-basedtools, deep learning-basedmethods are emerging and are being heavily applied to fast and accurateantibody 360 and of experimentally solving high-resolution structures,developing more and more accurate protein–protein docking methodsis of paramount value. While general-purpose methods could be used,accurate prediction of the antibody–antigen interface is challenginggiven the conformational variability of the Ab loop regions involved.In an attempt to overcome such inherent challenges, tailored dockingprotocols were developed. They can be classified into rigid dockingmethods such as those implemented in ClusPro61 and ZDock,62 only allowing rigid bodymovements of the binding partners, and flexible/semiflexible methodsthat also include backbone/side chain refinement steps such as SnugDock,63 HADDOCK,64 SwarmDock,65 and LightDock.66 These toolsimplement an integrative modeling approach allowing the use of experimentallyderived or sequence-conservation knowledge about the interface . Antibody-specific ML paratopepredictors include PINet,73 PECAN,74 Parapred,75 proABC2,76 and others.While great improvements to apo-antibody structuralpredictionhave been brought about by DL-based methods, reliable structural predictionof the antibody–antigen complex is still highly desirable forthe future. Indeed, atomic-level structural information on antibody–antigencomplexes is instrumental to investigate the interactions involvedin forming the complex and providing a rational framework for thedesign of optimized interactors. Given the difficulty of directlypredicting the multimer structures from sequences77 or also from statistical potentials obtainedfrom known complexes. In the latter case, ML-based methods have beenrecently employed for improving pose ranking.79Another challenging aspect isthat once dockingposes have beengenerated, a good scoring function is needed for discriminating non-nativefrom near-native complexes. In this regard, scoring functions canbe derived more traditionally from interaction energy evaluation431 In general, methods can be classified into de novo or grafting-based design protocols, and different criteria in thedesign process need to be adopted depending on whether the goal isto obtain a single antigen specific antibody or a protein capableof binding a broader spectrum of antigens packing, CDR minimization, and, if needed,docking with epitope- or paratope-based constraints. Minimizationemploys CDR cluster-derived constraints, and a Metropolis Monte Carlocriterion is used for optimization in both the graft design and subsequentsteps. In this context, the total energy or interface energy can beconsidered as the guiding criterion. AbDesign81 appliesto antibody design an approach similar to AbPredict: experimentallydetermined structures are fragmented analogously and then recombinedand grafted onto the target structure. After constrained optimizationof segments, dihedral and coordinate templates where each segmentdeviates <1 Å from the original structure are kept for subsequentsequence optimization according to conformation-dependent position-specificscoring matrices (PSSMs) for each segment. At this point, the predictedapo structure is docked against the target antigen to allow furtherbackbone conformation/sequence optimization. Designs are scored basedon different criteria concerning protein structure quality and shapecomplementarity/binding affinity with the antigen. Benchmarking ofthe method highlighted issues related to the expression of the designedantibodies and the deviation of the experimentally determined structurefrom the predicted one, indicating that rounds of optimization wererequired.Concerning the single-statedesign problem, Rosetta also includesantibody-tailored design protocols. RosettaAntibodyDesign (RAbD)82 A suitable protocol is RECON MSD, which has the goal of optimizingthe sequence so that it can adopt various conformations for bindingmore antigens or more conformational states of a single antigen.83 BROAD is an implementation that reduces computationalcosts.84Protocols for multistate-design tasks aiming at thedevelopmentof antibodies that can bind to multiple antigens, optimizing specificitythrough negative selection, or binding to different antigen conformationsare also implemented. Broadly neutralizing antibodies have the potentialof retaining neutralizing efficacy despite the escape mechanisms engagedby pathogens by targeting conserved residues.85 Startingfrom the target epitope structure, the so-called AbAg database comprisinglinear CDR-like motifs and antigen-like sequences, derived from PDBstructures, is searched for complementary CDR-like fragments. AfterCDR-like fragments are combined, contacts with the epitope are optimized,and CDRs are grafted onto an antibody scaffold. This approach circumventsthe need for approximate energy calculations and sampling of the conformationaland mutational space. The applicability is, of course, dependent onthe availability of paratope-like fragments compatible with the targetepitope.Vendruscolo and collaboratorsrecently reported a novel strategyfor designing antibodies that target epitopes whose structure is known.86 Ig-VAE is a VariationalAutoencoder-based generative model that directly outputs 3D backbonecoordinates that can then be combined with various constraint andoptimization approaches for applications in antibody design. For example,this method was applied to the design of a SARS-Cov-2 ACE2 epitopebinder. The latent space of this model can be exploited to expandthe capacity of generating novel proteins that cannot result fromthe sampling of existing structures. The protein design problem isthus reformulated as a constrained optimization in the latent spaceof a generative model.A different approach to the structure-based antibodydesign computationalproblem was adopted by Huang and colleagues, resorting to the useof emerging AI methods.a priori.87 A furtherimportant improvement to this model would come from applying the antibodydesign problem constraints and conditions derived from the stereoelectronicproperties of the specific target antigen.Another recent report moved in the directionof a generative modelcapable of codesigning the antibody CDR 3D structure and sequence,given a framework region restraint, through iterative refinement stepsthat optimize one in response to the other. This represents an advancesince the optimal 3D antibody structure for a target antigen is seldomknown 88 and Chroma,89 whichhave previously been applied to the design of protein binders holdthe promise to bring advances to the Ab field.Similarly, othergenerative models such as RF Diffusionde novo structural generationof the antibody in the presence of the target epitope/antigen.The paradigmis thus shifting from an unconstrained structuralgeneration complemented with a binding affinity predictor and conformational/sequencesearch to a constrained in silico methods that mimic natural affinity maturationhave been developed. Both physics-based and ML-based methods aim atpredicting the mutations that will enhance antibody stability andaffinity for the antigen.92 Automated tools aiming at increasingantibody developability  can alsobe employed in the design process.51Currently, experimentally or computationally designed antibodiesoften need to undergo other rounds of optimization. In this regard, 95In addition to classical antibodies, single-domain antibodies arealso being developed since they present some advantages with respectto immunoglobulins. For instance, they may be easier to produce andmanage and, given their reduced dimensions, they may encounter feweradverse reactions when administered as biological drugs.55.196 The ideal vaccine should cause a prolonged neutralizingimmune response in the host and is composed of an antigen complementedwith adjuvants.While antibody therapeuticsare responsible for passive immunization,a vaccine is defined as a preparation capable of stimulating an activeimmune response in the host. Active immunity includes both the humoralresponse (antibody-mediated) and the cellular-mediated immune response.98Traditional vaccine discovery approaches reliedon the direct investigation of pathogen components hypothesized tohave a role in eliciting responses to identify protective antigens.This approach has led to the development of many lifesaving vaccinesbut has shown limitations in targeting infectious diseases or otherpathologies characterized by immune evasion mechanisms. Examples areHIV, influenza, or malaria pathogens whose surface proteins elicitextreme sequence variation or are shielded by glycans . The search for anticancer vaccines, a hot field of study at themoment, may also need different approaches.99 Rappuoli and colleaguesspurreda dramatic change in the field by introducing the paradigm of ReverseVaccinology, which has led to the development of a vaccine againstmeningococcus B. Taking advantage of whole genome sequencing, possibleantigenic species are sorted out with the aid of immuno-bioinformaticstools, subject to different criteria such as surface exposition, conservation,toxicity, and allergenicity. The most promising species are then screenedexperimentally for their immunogenic activity. More recently, theterm Reverse Vaccinology 2.0 has been coined to refer to the advancementsbrought to the field by the analysis of human protective antibodies,sequencing and cloning of B-cell repertoires together with structuralcharacterization of antigens and epitopes.100 The investigation of effective antibody response allows the identificationof promising corresponding antigens/epitopes.In 2000101 NERVE,102 Jenner-predict,103 and VacSol,104 ormachine learning-based methods including VaxiJen,105 Vacceed,106 Vaxign-ML,107 and the Bowman-Heinson109 method. Filtering-based methods give as a result putative vaccinecandidates selecting them only based on predetermined threshold valueson features like the probability of exposure to the immune system,to be an adhesion molecule or more generally involved in virulence,limited homology to host proteins and number of transmembrane domainspredicted using external computational tools or databases .110Various open-sourcetools for complete RV workflows have been designedmainly for bacterial antigen discovery. They can be classified infiltering based methods such as Vaxign,Machine learning-based methods are trainedon a set of bacterialprotective antigens and nonprotective proteins and are employed toperform a ranking of the probability of all searched proteomes beinga protective antigen based on the predicted properties. VaxiJen classifiesproteins based on physicochemical properties only and could be suitablefor predicting also viral or cancer antigens. The Bowman-Heinson methodemploys a support vector machine classifier and was trained on a dataset of bacterial protective antigens (BPAs) and non-bacterial protectiveantigens annotated with 10 features related to antigen properties.111 Results highlighted that none of the methods recognizedall the known BPAs, with the ML-based Heinson-Bowman method beingthe best performing according to the fraction of proteome identifiedas a good candidate and agreement with the testing set. Moreover,there is poor accordance between the outputs of the different tools,suggesting the need to interrogate more than one tool in antigen discoveryresearch. Overall, the urge for improvements in protein annotationtools and incorporation in databases of proteins with negative experimentalresults to be employed as negative test sets for ML-based methodsis highlighted.A comparison of six of these RV tools  in the recognition ofBPAs in 11 pathogens has been reported recently.112 and Vaxign2113 were implemented to address possible limitations.For instance, the Vaxign2 Web server includes a predictive frameworkand postprediction computational tools and can be also employed forthe development of candidate viral vaccines. In the predictive partit incorporates both the filtering-based Vaxign and the ML-based Vaxign-ML107 tools. Postprediction analysis entails theprediction of MHC-I and II epitopes with MEME, epitope mapping ontothe input proteins based on IEDB114 immuneepitope database including B- and T-cell experimentally tested epitopes,calculation of population coverage, and prediction of protein functionand orthologs.Since this benchmark was reported, updated versionsof the testedRV tools such as VaxyJen35.2Computationalepitope prediction is of key importance in vaccine development. Indeed,humoral antibody-mediated immune responses rely on interactions betweenB-cell receptors and antigen epitopes, whereas cellular responsesinclude, among others, T-cell mediated responses that rely on interactionbetween T-cell receptors (TCRs) and MHC-I and MHC-II epitopes presenton antigen presenting cells. Different tools were developed for T-or B-cell epitope predictions.115 but their application is limited by the availability of 3D antibody–antigenstructures. Indeed, B-cell epitopes are more frequently discontinuous, and their prediction poses greatchallenges to current predictive approaches, especially sequence-basedones that are more suitable for linear epitopes. Moreover, doubtshave emerged on whether the question of searching for all the possibleepitopes of an antigen for all possible antibodies is well posed.116 Some evidence suggests that in principle allexposed protein surface patches could represent an epitope, giventhe “right” interacting partner.118 Therefore, antibody-specific prediction methods were proposed.120 Clearly, these implicate that the target antibody is known, whichis not always the case.B-cell epitope predictions canbe classified as sequence-based or structure-based methods. The latterhave been demonstrated to be more reliable,121 CBTope122), some structure-basedmethods , and an antibody-specific B-epitope predictionmethod (EpiPred120) have been benchmarked.116 Results evidenced that while performances supportthe reliability and usability of the methods, they may be suboptimalmost likely because they were trained on limited  data sets.Recently, two sequence-based for the prediction of interface regions in proteins based only onenergetic determinants and available as a Web server.139 is employed to reconstruct a simplified matrix which isthen filtered for retaining only local interactions and to obtainthe so-called MLCE . From this,patches that have the lowest energetic couplings with the surroundingstructures are identified as putative interface regions with the potentialto be recognized by Abs , the matrix (or average matrix) of pairwise nonbondedinteractions is calculated. After eigenvalue decomposition, the moststabilizing eigenvector (or few eigenvectors) Abs see 3. This m141 While othermethods estimated desolvation penalties of protein surfaces by meansof surface-area calculations, here the desolvation free energy penaltyis obtained by probing the protein surface with a neutral low-dielectricsphere, therefore also considering the neighboring environment andlong-range electrostatic effects. It was found that interface regionslargely coincide with those characterized by lower free energy losses.141Another method relyingon energy considerationswas reported byFiorucci and Zacharias.73Among ML-based partner specific methods,PINet, already mentionedas a paratope prediction tool, can predict antibody specific interfaceresidues (both the paratope and epitope side) through a geometricdeep learning approach aimed at dissecting the properties of complementarysurfaces. It has been reported to have state-of-the-art performancesin both antibody–antigen and protein–protein interactioncases.142 Themethod leverages residue specific and environment features to determinethe probability of a certain residue belonging to an epitope basedon the conformation of the protein.Recently, AxIEM has consideredthe issue of predicting “crypticepitopes” of class I viral fusion proteins exposed in metastableconformations important for virulence and that are often the targetof broadly neutralizing antibodies.143 It ranks interaction probability based on a naive Bayesian consensusclassifier that combines outputs from independent modules that evaluatesequence and structural similarity to known protein–proteininteractions, functional association, and a neural network classification.While results should be considered also based on other considerationsnot taken into account, such as subcellular colocalization of thepredicted interactors, this and other similar tools can be usefulfor predicting relevant immunogens on a proteome-wide scale.Interestingly, the SARS-CoV-2–humaninteractome was predictedwith the computational pipeline PEPPI.5.3T-cellepitope immunogenicity encompasses three fundamental steps:antigen processing resulting in the immunogenic peptide, binding ofthe peptide to MHC class I or II molecules on Antigen Presenting Cells(APCs), and binding of peptide-presenting MHC molecules (p-MHC) toTCR.148 Current strategies for the identification of neoantigens rely onadvanced sequencing techniques, including next-generation sequencingor -omics data aimed at individuating peptides distinguished fromself-peptides. These are coupled with computational tools includingthose that we describe below for general purpose prediction of T-cellepitopes immunogenicity.151MHC class I molecules are expressed on the surface of allsomatic cells and present peptides from the intracellular environment,whereas MHC class II molecules are found on immune cells such as B-cells,dendritic cells (DCs), and sample peptides from the extracellularmilieu. CD8 T cells only interact with class I p-MHC and once primedand activated, they generate cytotoxic T cells. On the other hand,class II p-MHC complexes are recognized by CD4 T-cells that can giverise to different types of T helper cells. Among T-cell epitopes,neoepitopes are defined as those distinctive of cancer cells resultingfrom tumor-related genetic aberrations. The identification and targetingof neoepitopes is one of the most actively evolving fields of cancerimmunotherapy aiming at the development of neoantigen based vaccinesand T-cell based therapies.152 andATLAS153 as well as the CEDAR cancer epitopedatabase have been curated.154Importantly, along with the general IEDB epitope database,T-cellepitope databases such as SYFPEITHYInthis section, we will give an overview of computational toolspredicting peptide binding to MHC molecules and effective interactionbetween p-MHC and TCR. 156 and mostly rely on existingexperimental elution or binding affinity data. While the first developedmethods only took into account the presence of the so-called anchoringresidues with suitable spacing, current methods consider fixed-lengthpeptides (mainly 9 residues long) and quantify the contribution ofeach peptide position to the binding considering each residue independentlyin the case of linear methods (such as BIMAS),157 or also take into account nonlinear effects 158 or support vector machines (SVMs).159 Interestingly, it was demonstrated that algorithms takinginto account nonlinear effects do not outperform linear ones in theprediction of binding to MHC class I molecules.160 The hypothesized explanation is that since peptides bindin the groove in an extended conformation, the contribution of eachresidue is dependent on its own interactions in the binding pocketwith other nonlinear effects related to residue–residue interactionshaving a lower magnitude. Also, no significant gain of performancewas obtained with prediction methods taking into account the 3D structureof the complexes with respect to sequence-based methods.161 However, MHC molecules in humans ) are characterized by great allelic variability.Indeed, different HLA molecules from different individuals will havedifferent peptide preferences, and HLA supertypes with similar bindingpreferences were individuated and clustered. Since experimental dataon which the above-mentioned models were trained are available foronly a few allelic variants, they have a limited capability of predictingbinding for all allelic variants. To overcome this issue, pan-MHCmethods able to predict the binding of a peptide to uncharacterizedHLA molecules have been developed. For example, ML models capableof performing such predictions for MHC I molecules such as NetMHCpan162 and a structure-based threading approach163 were developed by training the algorithm oninformation related to the MHC peptide binding site.164 Similar predictors for MHC class II molecules have beendeveloped and improved versions of the initial models have been implementedmore recently.165 Computational tools for predicting promiscuous sequences with highpopulation coverage are also available.167 Interestingly, a method for predicting allele-specific anchor residuepositions has been proposed for complementing neoantigen prioritizationpipelines.168Binding to MHC I andII is similar but has somerelevant distinctivefeatures: MHC I groove is more closed, it typically hosts peptidesranging from 8 to 14 residues (more often 9), and binding site pocketshave selective physical-chemical features. On the other hand, predictingbinding to MHC II molecules is more challenging, as peptides are typicallylonger, even though only a 9-residue core typically sits in the moreopen binding groove featured with less discriminating interactionproperties. A great number of sequence-based predictors have beendeveloped that can either distinguish a binder from a nonbinder oralso give an estimation of the binding affinity. These are listedin recent reviews163 However, the fact that good presentation on HLAs is necessary butnot sufficient for stimulating T-cell response is particularly truefor responses to neoantigens: owing to their endogenous nature, correspondingT-cells could have been deleted or tolerized to avoid self-immunereactions. Therefore, either mutations with respect to self-peptidesshould increase MHC binding (which is not so common) or (more likely)there should be structural differences in T-cell recognition withrespect to wild type p-MHCs. In general, in the structural predictionof TCR-pMHC binding challenges are posed by the degree of TCR diversityand conformational flexibility of the TCR-pMHC interface. Therefore,at first, efforts were devoted to predicting TCR-pMHC from the proteinsequences. Among sequence-based methods of notice is NetTepi169 which integrates previously developed methodsfor the evaluation of peptide-MHC stability (NetMHCstab) and binding(NetMHCcons), and a model for TCR affinity prediction reported byCalis and colleagues.170 The latter scoresthe peptide sequence based on rules derived from experimental dataevidencing, for example, the relevance of positions 4–6. Othercomputational tools relying only on sequence have been described inmore detail in a recent review.171 Sequencedata driven methods have outlined some amino acidic physicochemicalfeatures related to immunogenicity such as the enrichment of aromatichydrophobic and aromatic residues in the binding interface. However,physics-based and structure-based models can provide a rational backgroundto these findings: for instance, the energetic gain of burying a hydrophobicresidue by complex formation strongly depends on the 3D structuralfeatures. Given these premises, considering structural aspects alongsidesequence holds the promise of giving better prediction of peptideimmunogenicity and TCRs binding specificities.174Predictive tools havemostly taken into peptide presentation onMHC molecules and indeed, binding affinity to MHCs correlated wellwith immunogenicity for viral epitopes.175 It outlines biophysical determinants of neoantigenimmunogenicity such as mutations increasing the exposition of hydrophobicresidues and overall peptide energy. Moreover, by including well-presentedbut not immunogenic as well as non-MHC binding peptides in the trainingdata set it is able to capture both binding to HLA and TCR, thus predictingcases where low affinity to MHC is compensated by strong TCR bindingand vice versa.Baker and co-workers developed a neural network-based methodtrainedon structural and energetic features derived from HLA-A2/peptide complexes.178 Structural modeling was employed for optimizing force field-basedscoring for evaluating the involved interactions. This modeling andscoring approach was employed to predict corresponding pMHC/TCR pairsgiven α and β subunits of MHC and TCR molecules and apool of possible peptide binders.180 Very recently,a customized version of Alpha Fold specifically trained for pMHC-TCRmodeling was employed for the same task with prediction success correlatingwell with 3D model accuracy.181Modeling protocols specializedfor pMHC-TCR structural predictionsimilar to those described for antibodies have been developed.182Theseresults demonstrate recent advances in identifying interactingp-MHC and TCR among possible candidates, but further developmentsare needed for a more general prediction of the target peptides ofa given TCR or if and which TCRs could bind to a novel epitope.6Withthe advent of thenew paradigms of reverse vaccinology 1.0and 2.0, antigen/epitope design strategies have been envisaged toselectively present epitopes that are able to optimally elicit thedesired immune response. The main design approaches are illustratedin 184 This approach may be particularly relevantin the development of vaccines for those diseases where traditionalapproaches are not effective such as HIV, respiratory syncytial virus(RSV), and cancer.In this frame of thought, the idea of presentingonly subdominantepitopes is related to the predominance of responses to immunodominantepitopes in the presence of the whole antigen species and was conceivedto circumvent the capacity of pathogens  to mutateand/or mask immunodominant epitopes to evade the immune system.185 the ideais to present the epitope in the context of a nonantigenic proteinor of an already reactive antigen, thus generating a superantigen.This aim is more straightforward for linear epitopes, while it presentssignificant challenges for conformational discontinuous epitopes.Rosetta includes different protocols for the so-called epitope transplantationtasks.36 The first methods implementedallowed side-chain grafting when only side chains of binder interactingresidues were grafted or backbone grafting when the entire peptidesequence was transplanted onto the scaffold. The choice of the scaffoldprotein was the critical step in that there should be a good superpositionof the grafted motif backbone (side chain grafting) or C- and N-terminalregions (backbone grafting) and the scaffold to allow retaining thedesired epitope conformation and overall protein stability. Our groupdeveloped the protocol SAGE, a computational pipeline that automatesthe prediction of the best grafting positions for linear epitopesonto a selected scaffold protein based on structure and sequence alignment,scoring of the secondary structure compatibility between the transplantedepitope and the scaffold, and scoring of surface exposition of theepitope.186To this end, structure-based computationalmethods in the fieldof protein design have been implemented for the grafting of the desiredepitope into novel scaffolds:187 Importantly,the protocol also implements the possibility of biasing the designprocess in the presence of the binder. The latter outlines how proteinsdesigned in the presence of the binder deviate from the accessibleenergy minimum of the protein fold and highlights the relevance ofintroducing “function constraints” in the process. Thismethod is particularly useful for grafting loop regions for whichproper protein scaffolds are more difficult to find and allows fora global adaptation of the protein backbone to motif incorporation.It has been employed for the design of scaffolds presenting antigenicsite II of the respiratory syncytial virus fusion protein, which isa good proof-of-principle result. The resulting antigen was howevershown to induce only low levels of site-specific neutralizing antibodiesin nonhuman primates, indicating the space for additional optimizationof the design approach.188 Applicabilityof this grafting strategy to more complex conformational epitopesremains difficult.FunFoldDes follows previousapproaches  andinvolves the grafting of the epitope onto a scaffold topology insteadof a specific protein, followed by structure folding (with some topologyconstraints) and sequence design.189 First,a customized topology is assembled to accommodate the motif; thenstructural refinements and sequence design are performed with RosettaFunFoldDes. This computational pipeline was applied for the designof a trivalent vaccine for RSV that combined the above-mentioned scaffoldwith the epitope from site II and two newly designed immunogens carryingstructurally complex epitopes from sites 0 and IV.189 This construct was shown to induce an improved antibodyresponse in immunized mice and nonhuman primates and importantly thepossibility of guiding the induction of epitope-specific antibodies.Interestingly, the recently implemented protocol RosettaSurf190 allows sequence design for protein interfacesby employing information derived from the representation of the proteinas a near-continuous surface featured by shape and electrostatic properties.After epitope grafting onto a novel scaffold, protein surfaces canthen be compared by the implemented surface similarity or complementarityscores, which guide the surface/sequence design process. Importantly,the surface optimization step can be biased by the presence of a binder.Within this frame, TopoBuilder is a protocolthat reverses thecomputational pipeline: given the desired motif a stable protein isbuilt around it as a carrier.192 Alongside more traditionally employed natural protein assemblies,self-assembling nanoparticles have been designed computationally bysymmetrical docking of building blocks, followed by the design ofprotein–protein interacting surfaces.193 These nanoparticles were shown to be versatile and promising platformsfor displaying several antigen molecules on the surface193 including heterotypic antigens195 leading the way to more customizable and easy-to-obtain platforms.The topic has been reviewed recently.198In general, such immunogen design tasks usually require the explorationof a great number of possible models and subsequent rounds of optimizationguided by experimental results. Moreover, the scaffold presentingthe desired epitope could also present some other new epitopes thatcould also cause some immunodominance issues that should be takeninto account. Therefore, the current efforts now focus on improvingor enhancing antibody responses. One possibility that is being exploredis the use of nanoparticles as multivalent antigen carriers. Suchnanosystems exploit inherent immune system features, optimized todetect exogenous particles. Indeed, for their shape and size theydisplay advantageous biodistribution in the lymphatic system and uptakein lymphatic cells and can show increased immune response with respectto soluble antigens thanks to effects such as B-cell receptor cross-linking.199 SARS-Cov-2 spike protein,200 and HIV envelope.201 For achieving theseresults, structural knowledge has guided various rounds of optimizationmostly relying on experimental techniques. Indeed, the applicationof computational structure-based methods for the design of thermostabilizedimmunogens seems still scarce, but as in the design of antibodies,there is great potential in the use of high-throughput protocols forthe prediction of stabilizing mutations and in the computational screeningof the properties of stabilized mutants. For example, PROSS is a toolavailable as a Web server that leverages information from proteins’evolutionary sequence variability and is able to predict mutationsthat will increase protein stability based on ΔΔG calculations.202Another immunogendesign strategy includes the thermostabilizationof known poorly stable antigenic proteins. Viral fusion proteins usuallyhave a metastable prefusion conformation that rearranges after fusionof the viral envelope with the host cells and that often does notexpose the epitope targeted by neutralizing antibodies. Therefore,stabilization of this class of proteins aims at retaining the immune-activeprefusion conformations. These approaches have been employed for thedevelopment of prefusion stabilized forms of RSVF,203Moleculardynamics and free energy calculations have been employedto identify mutations increasing the stability of interactions betweenthe pentameric subunits in the icosahedral capsid of the foot andmouth disease virus. This resulted in a safer experimental vaccinewith an improved shelf life.4JHW, and neuraminidase (NA).Indeed, NA head tilting, HA ectodomain tilting, and HA head breathingare characterized at full atomistic resolution. The flexibility ofNA reveals a cryptic epitope targeted by a novel convalescent human-donor-derivedmonoclonal antibody. Importantly, this type of work, combining extensivestructural characterizations of the proteins in a realistic crowdedenvironment and the possibility to analyze multiple copies of thesame molecule in its native conditions, shows the possibility to obtainpreviously unappreciated (and inaccessible) views on the dynamicsof antigenic proteins HA and NA. In this context, MD simulations couldunveil transient intermediates able to induce antibodies against epitopesthat, although conserved, are exposed only transiently in native moleculesand may not be visible in a static 3D representation of the antigen.This, combined with the epitope prediction approaches described above,will help develop antibodies and vaccine components efficiently, forinstance, by guiding the stabilization of specific conformations viamutagenesis.The Amaro grouprecently reported impressive mesoscale, all-atomMD simulations of two evolutionary-linked glycosylated influenza Avirions.Approaches like these will become more and morecommon as we beginentering the era of exascale computing. In this framework, effortsto adapt and port MD simulations to these new architectures will alsoplay a key role.Significant advances will likely stem fromthe integration of AIwith computational biology and biotechnology for the development ofnew therapies and treatments.210One promising area of researchis of course the use of AI to predictstructures as well as design and optimize proteins as antigens, antigen-binders,and/or antibodies.If AI were to maintainits current promise, this may also findtranslation in the development of personalized medicine protocols.By analyzing a patient’s genetic data, medical history, andother factors, AI algorithms could predict which antigens will bemost important to target and, as a consequence, which treatments aremost likely to be effective for that individual.In cancer treatment,AI supported interventions may be translatedinto the identification and molecular profiling of patient-specificcancer cells and then designing antibodies that can selectively bindto and destroy those cells while leaving healthy cells unharmed. Similarly,one could design novel and specific antigens that are able to elicita strong protective immune response.The combination of AI,protein design, and CAR-T cell therapy isan exciting area of research that has the potential to significantlyimprove cancer treatment outcomes. By identifying specific proteinsor antigens that have specific signatures in cancer cells but noton healthy cells, researchers can design new CARs with improved safetyand efficacy.The same type of philosophy can be applied tothe design of newproteins that can be used as CARs.Clearly, limitations stillhave to be considered with a criticaleye. Indeed, AI-based antigen and antibody design will depend on thecorrect understanding and modeling of the biophysical principles thatunderpin complex cell molecular recognition phenomena, a problem thatis still far from being fully solved.In silico developed antibodies, engineered T-cells, andimmunogens could cause unwanted inflammatory reactions, toxicity,resistance phenomena, and other side effects. One advantage of (AI-powered) in silico screenings of increasingly rich databases is thatthey could help reduce or predict these risks. Given the complex interplayof the factors involved and the inherent variation of responses ofdifferent individuals, however, risks cannot be fully eliminated butcan be mitigated by an improved knowledge of adverse response predictors.Furthermore, the computational design of immunologically active moleculesremains difficult and usually involves several rounds of optimizationwith experimental testing to obtain molecules of good fitness andefficacy.In this framework, itis important to note that similar to experimentallyderived immunologically active molecules, unsolved issues remain. To advance along these fascinating avenues, an everincreasingintegration of structural biology, computational biochemistry, computerand data science, and algorithm design will be necessary. New researchersand professionals that already embody this necessity are startingto appear. Importantly, novel (and at present mostly unpredictable)opportunities for the development of new research lines will beginto emerge.We envision that the above-described developmentswill come ofage in the next few years, opening new avenues for fundamental andapplied research and facilitating the creation of new biomoleculeswith specific modes of action to be used both as therapeutics in thetreatment of diverse diseases and as molecular tools to disentanglethe intricacies of immune molecular mechanisms.


Total hip arthroplasty is a widely performed surgical procedure, which enables patients to regain mobility, alleviates pain, and improves overall quality of life. Periarticular multimodal drug infiltration (PAI) is increasingly being used as an effective postoperative pain management, decreasing the systemic consumption of opioids. Extensive postoperative skin necrosis without a deep joint infection as a complication of total hip arthroplasty with PAI has not yet been described.A 71-year-old patient who underwent total hip arthroplasty of the right hip for primary osteoarthritis through the Direct Anterior Approach presented postoperatively a large area of necrotic skin at the incision. Joint infection was excluded. An extensive debridement was performed and the tissue defect was reconstructed by a pedicled anterolateral thigh flap. The skin maintained a satisfactory appearance at 1 year postoperatively, and the hip was pain-free with restored ranges of motion. The patient was able to walk with no support and without limitation.We address the possible risk factors, discuss the use of epinephrine in PAI and explore possible treatment options for such a complication.The online version contains supplementary material available at 10.1186/s12891-023-06643-z. Total hip arthroplasty (THA) is a common surgical treatment which provides reliable functional outcomes in patients suffering from hip osteoarthritis. Effective postoperative pain control is most important to enable prompt rehabilitation, reduce postoperative complications, and reduce the length of hospital stay. Pain control is achieved by several methods, including systemic treatments with Non-Steroidal Anti-inflammatory Drugs, opioids, and regional treatments such as epidural anaesthesia, peripheral nerve blocks, and periarticular multimodal drug infiltration (PAI). Up until recently, the efficacy of PAI in pain relief following total hip arthroplasty during rest and activity, and its impact on the length of hospital stay and total opioid consumption was not fully established. A recent meta-analysis concluded that PAI showed better pain relief and less opioid consumption and is safe and effective following total hip arthroplasty . HoweverThis case is presented in accordance with the 2013 CARE checklist.A 71-year-old male, non-smoker, with a previous history of cardiac ischemia was admitted for a THA. The patient was positioned in the supine position on a traction table. Surgery was performed under general anaesthesia through the Direct Anterior Approach. The patient received a fully hydroxyapatite-coated uncemented stem , an uncemented cup , and a ceramic-highly-crosslinked polyethylene bearing surface Fig. . The patConsidering the presence of a large skin necrosis and the high risk of infection of the underlying hip joint, we decided to perform a single-staged surgical debridement and reconstruction of the skin and soft tissue defect using a pedicled fasciocutaneous anterolateral thigh flap (ALT) of about 15 × 6 cm Fig. ; video. Wound complications after joint replacement can result in significant morbidity to the patient with increased risk on infections, further surgeries, increased recovery period and hospital stay . Wound cInterestingly, in our patient, the skin necrosis was first noted on day 8 postoperatively. The dressings had been changed at day 3 and day 6 and a clean wound with no oozing or redness was described. In other reported cases of skin necrosis following local anaesthetics infiltration with epinephrine, skin changes only appeared around day 5. This difference could be due to the site of injection, which was the penoscrotal junction in one case , and theBased on expertise from our department of infectious diseases, we excluded the possibility of acute periprosthetic joint infection as the bacterial culture of synovial fluid was negative with a WBC count of 4650 cells/µl (< 10 000 cells/µl) in the joint fluid . A broadMalnutrition compromises wound healing as decreased protein reserves affects collagen synthesis and fibroblast proliferation and decreased albumin levels promotes tissue oedema formation . It has PAI is a multimodal drug solution comprised of three active agents, long-acting local anaesthetics such as ropivacaine and bupivacaine, nonsteroidal anti-inflammatory drugs (NSAIDs) and epinephrine. Local anaesthetics block voltage-gated sodium channels in nociceptors, thereby decreasing pain transmission. Injection of NSAIDs such as ketorolac reduces pain by inhibiting the production of proinflammatory mediators such as prostaglandins. Epinephrine is a nonspecific alpha- and beta-adrenergic agonist, widely used in local anaesthesia with the rationale that through local vasoconstriction it decreases absorption thereby increasing the duration of analgesia. While some studies have reported that the inclusion of epinephrine in PAI prolongs the duration of local anaesthetics, other studies have reported no significant increase in the duration when epinephrine is added to ropivacaine due to the intrinsic vasoconstrictive effect of ropivacaine . A recenOther possible causes for wound complications include the use of retractors and their pressure effects on skin and soft tissues. However, we have routinely performed total hip arthroplasty through the Direct Anterior Approach (DAA) for more than 12 years (approx. 400 hips/year), and we have not observed this issue previously . Also, gThe DAA has dramatically gained in popularity over the last 15 years because it is a muscle sparing.approach, with a potential reduction of postoperative pain and length of stay. As the skin is thinner in the anterior aspect of the hip, the risks for wound complications might be increased. However, there have been several studies, reviews and meta-analyses which compared early outcomes of total hip replacement through the main different surgical approaches, and reported similar rates of wound complications  16]. Wi. Wi16]. In this case, we performed surgical debridement and reconstruction of the skin and soft tissue defect using a pedicled fasciocutaneous anterolateral thigh flap. This method is a reliable and versatile option for loco-regional reconstruction due to the large skin and soft tissue availability, reliable blood supply, long vascular pedicle and wide arc of rotation . FurtherTo the best of our knowledge, this case represents the first evidence of a non-infectious and non-warfarin related extensive skin necrosis as a complication following total hip arthroplasty. In such cases, further testing should be performed to rule out possible causes such as infection and malnutrition. We achieved a good outcome with extensive debridement and wound reconstruction using a pedicled fasciocutaenous anterolateral thigh flap. As the exact cause of skin necrosis cannot be determined from a single case report, larger studies are required. Additionally, further clinical trials are needed to establish the risk and benefits of using epinephrine in PAI. Meanwhile, we decided to change our practice and not to use epinephrine anymore.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


In today’s era of individualized precision medicine drug repurposing represents a promising approach to offer patients fast access to novel treatments. Apart from drug repurposing in cancer treatments, cardiovascular pharmacology is another attractive field for this approach. Patients with angina pectoris without obstructive coronary artery disease (ANOCA) report refractory angina despite standard medications in up to 40% of cases. Drug repurposing also appears to be an auspicious option for this indication. From a pathophysiological point of view ANOCA patients frequently suffer from vasomotor disorders such as coronary spasm and/or impaired microvascular vasodilatation. Consequently, we carefully screened the literature and identified two potential therapeutic targets: the blockade of the endothelin-1 (ET-1) receptor and the stimulation of soluble guanylate cyclase (sGC). Genetically increased endothelin expression results in elevated levels of ET-1, justifying ET-1 receptor blockers as drug candidates to treat coronary spasm. sGC stimulators may be beneficial as they stimulate the NO-sGC-cGMP pathway leading to GMP-mediated vasodilatation. The concept of drug repurposing  refers to the identification of a new indication for an already existing and authorised drug. It is a way of making new treatment options available to patients. The overall costs for a new drug to appear on the market can exceed 1 billion US$ and many drugs fail at clinical trials or the approval stage (+ /K+)-ATPase pump system, in order to secure its own survival. In accordance with this hypothesis, numerous cardiovascular system-regulating drugs show anticancer activity, which makes them potential candidates in oncological adjuvant therapy  inhibit L-type Ca2.Nitrates activate mitochondrial aldehyde dehydrogenase and increase intracellular NO availability.3.+ channels and cause hyperpolarization with subsequent inhibition of L-type Ca2+ channels. In addition they have a nitrate-like influence and release NO via cytochrome P450 of the smooth endoplasmic reticulum (SER).Potassium channel openers (PCO) such as nicorandil bind to and open K4.2+ overload.Sodium channel blockers (SCB) such as ranolazine inhibit the late phase of the inward sarcolemmal sodium channel and thereby prevent intracellular CaIn patients with symptoms suggestive of obstructive coronary artery disease who undergo diagnostic coronary angiography about 40% have entirely normal arteries or non-obstructive coronary artery disease –14. ThisOngoing clinical trials in this research area are ILIAS ANOCA [Netherlands Trial Register ], EXAMINE-CAD (EudraCT-No: 2020-004717-12), iCorMicA (NCT04674449), and WARRIOR (NCT03417388). All these studies use common antianginal medications. However, from our experience, 30%–40% of patients with angina and non-obstructive coronary arteries show refractory angina despite the use of the common antianginal drugs . In thosFor this review we screened the literature for possible candidates of drug repurposing in ANOCA treatment and identified two promising targets: Soluble Guanylate Cyclase (sGC) Stimulators and Endothelin-1-receptor (ET-1) blockers, both originally approved for the treatment of pulmonary hypertension (PH). Based on the molecular mechanisms of these drugs as shown in Cyclic guanosine-3’-5’-monophosphate (cGMP) modulators have emerged as one of the most promising compounds in recent cardiovascular drug discovery . Genome-In the mid-1990s, the pharmaceutical company Bayer looked for substances that could increase NO synthesis and thus stimulate sGC in endothelial cells. Surprisingly, they discovered NO-independent sGC stimulators. After optimization of potency, pharmacokinetic properties and specificity of this new class of drugs, riociguat was developed. It was tested in hypertensive rats and showed a dose-dependent blood pressure lowering effect. This effect lasted over numerous weeks, even when the rats were nitrate-tolerant. Riociguat was then investigated in different animal models of PH –34 whereThe Left Ventricular Systolic Dysfunction Associated With Pulmonary Hypertension Riociguat Trial (LEPHT) showed that sGC stimulators are well tolerated in patients with advanced systolic left ventricular dysfunction and secondary PH due to heart failure. In addition, they improve cardiac index, quality of life, and pulmonary as well as systemic vascular resistance at 16 weeks of treatment. These hemodynamic improvements occurred in the absence of changes in heart rate, blood pressure, and pulmonary artery pressure. It suggested likely benefits in patients with heart failure afar those with PH , 36.Vericiguat is structurally and pharmacologically distinct from riociguat and has been optimized for use in chronic heart failure patients. The VICTORIA trial investigated the effect of vericiguat in patients with HFrEF in a randomized, double-blind, placebo-controlled study design over a median of 10.8 months . The incThe vasodilatory effect of sGC stimulator and activators makes them promising drugs for the treatment of angina pectoris. Our research group reported about a clinical case of a 77 year-old woman with refractory angina despite conventional anti-anginal treatment . During With the help of genome-wide association studies (GWAS) various loci have been recognised to be linked to coronary artery disease and myocardial infarction. To find drugs appropriate for repurposing Tragante et al.  formed 2The Coronary Vasomotor Response After Riociguat Exposure (CORONARIES) trial was organized to investigate the effects of riociguat on coronary artery disease. For unknown reasons the study was withdrawn before recruitment. A new attempt to study the potential treatment effects of sGC stimulators and activators on angina symptoms in vasomotor disorders would be highly desirable.Endothelin-1 (ET-1) is a potent endogenous vasoconstrictor and smooth-muscle mitogen. It can add to a rise in vascular tone and vascular smooth muscle hypertrophy. Its pathogenic role in PH has been recognized  as patieEx vivo they were able to evaluate the ET-1 related vascular mechanisms by means of wire myography with endothelin A receptor (ETA) antagonists including zibotentan. Patients with the SNP had unaltered small vessel reactivity to ET-1 with high affinity of ETA antagonists indicating that the ETA receptor might not be down-regulated in those patients. Here lies the potential treatment efficiency of a selective ETA receptor antagonist.However, ET-1 has been associated with the pathogenesis of coronary microvascular dysfunction (CMD) as well , 45. ExpA receptor antagonists, which makes it especially suitable for usage in coronary spasm therapy. Individually tailored usage of zibotentan based on genotyping is being investigated in the PRIZE trial (NCT04097314) which is expected to be completed by the end of 2023. Unfortunately, the incidence of the above mentioned SNP occurs in only about 30% of the population with microvascular angina, resulting in a high screening-failure rate. Those screening failures partake in a sub-study to perhaps identify additional expected ‘responders’ to zibotentan. There is baseline genotyping for other genetic variants in the ET-1 pathway, and phenotyping patients by quantification of microvascular disease from analysis of cMRI data. Results from this sub-study will provide a genotype database that might discover novel variants for the pathogenesis of coronary microvascular spasm which might point to other ETA receptor antagonist super-responders, and enabling more patients with microvascular spasms to profit from this repurposed drug  since it has a good chance for clinical translation needing further phase 2 studies. It is the most selective of all orally active ETsed drug .Apart from the awaited results of the PRIZE trial, there are already a few published case reports demonstrating a good therapeutic response to bosentan in patients with coronary spasm –53.Since the endothelium plays an important role in the regulation of vascular tone by synthesizing vasodilator prostaglandins like prostacyclin, synthetic prostacylins might be useful in ANOCA treatment as well and represent additional candidates for drug repurposing. Agents like iloprost, which is also approved for the treatment of PH would be of interest.How can a new indication for an already existing drug be identified? In order to discover a new target for an existing drug, different approaches are available. The general methods to identify a candidate molecule include the analysis of side effects, analysis of preclinical models as well as of the drug’s efficacy in clinical trials . However1.The analyses of molecular docking, where the prediction of a binding-site complementarity between ligand (drug) and a target (receptor) –64 is in2.Genome wide association studies (GWAS), which identify genetic variants as novel targets, which could be shared between diseases treated by drugs and disease phenotypes –67.3.Pathway or network mapping, a method favourable to use when GWAS hits are not druggable. It is a pathway-based strategy that can provide either upstream or downstream information on the GWAS-associated target. The network mapping refers to constructing a drug or a disease network based on gene expression patterns, disease pathology, protein interactions or GWAS data in order to find candidates –70.4.Retrospective clinical analysis, which requires accessible databases . However, this approach often faces problems, since ethical and legal obstacles limit access to these data –73.Novel approaches include the use of immortalized human cancer cell lines (cell viability assays) to pair genomic and pharmacological data , drug repurposing in cardiology appears particularly promising. Treatment of patients with coronary vasomotion disorders like in ANOCA remains challenging due to a lack of evidence-based therapy guidelines and a high proportion of patients showing refractory angina despite the use of common antianginal drugs like CCBs, beta-blockers and others. Considering the mechanisms of action two drug candidates already approved for the treatment of PH may be promising for drug repurposing in ANOCA treatment: sGC stimulators/activators and ET-1 receptor blockers. Clinical trials assessing the safety and efficacy of those candidates in the context of refractory angina due to ANOCA are needed.