Targeting the JAK/STAT Pathway: A Combined Ligand- And Target-Based Approach

Janus kinases (JAKs) are a family of proinflammatory enzymes able to mediate the immune responses and the inflammatory cascade by modulating multiple cytokine expressions as well as various growth factors. In the present study, the inhibition of the JAK-signal transducer and activator of transcription (STAT) signaling pathway is explored as a potential strategy for treating autoimmune and inflammatory disorders. A computationally driven approach aimed at identifying novel JAK inhibitors based on molecular topology, docking, and molecular dynamics simulations was carried out. For the best candidates selected, the inhibitory activity against JAK2 was evaluated in vitro. Two hit compounds with a novel chemical scaffold, 4 (IC50 = 0.81 μM) and 7 (IC50 = 0.64 μM), showed promising results when compared with the reference drug Tofacitinib (IC50 = 0.031 μM).This study was funded by the University of Valencia and Generalitat Valenciana (GVA) through postdoctoral grants no. UVINV_POSTDOC18-785681 and APOSTD/2019/055 (M.G-L.) and by the University of Bologna through research grant no. RFO2019 (P.R., S.C., and M.R.)

Inside perspective of the synthetic and computational toolbox of JAK inhibitors: Recent updates

The mechanisms of inflammation and cancer are intertwined by complex networks of signaling pathways. Dysregulations in the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway underlie several pathogenic conditions related to chronic inflammatory states, autoimmune diseases and cancer. Historically, the potential application of JAK inhibition has been thoroughly explored, thus triggering an escalation of favorable results in this field. So far, five JAK inhibitors have been approved by the Food and Drug Administration (FDA) for the treatment of different diseases. Considering the complexity of JAK-depending processes and their involvement in multiple disorders, JAK inhibitors are the perfect candidates for drug repurposing and for the assessment of multitarget strategies. Herein we reviewed the recent progress concerning JAK inhibition, including the innovations provided by the release of JAKs crystal structures and the improvement of synthetic strategies aimed to simplify of the industrial scale-up

표적암치료를 위한 저분자 STAT3 저해제인 옥사디아졸계 ODZ10117의 개발

학위논문 (박사) -- 서울대학교 대학원 : 의과대학 의과학과, 2020. 8. 예상규.STAT3 is a transcription regulator involved in many intracellular functions, including cell proliferation, differentiation, survival, angiogenesis, and immune response. Persistently activated STAT3 is a promising target for a new class of anticancer drug development and cancer therapy, as it is associated with tumor initiation, progression, malignancy, drug resistance, cancer stem cell properties, and recurrence. Here, I discovered 3-(2,4-dichloro-phenoxymethyl)-5-trichloromethyl-[1,2,4]oxadiazole (ODZ10117) as a small molecule inhibitor of STAT3 and suggested that it may have an effective therapeutic utility for the STAT3-targeted cancer therapy. ODZ10117 targeted the SH2 domain of STAT3 regardless of other STAT family proteins and upstream regulators of STAT3, leading to inhibition of the tyrosine phosphorylation and transcriptional activity of STAT3. The inhibitory effect of ODZ10117 on STAT3 was stronger than the known STAT3 inhibitors such as S3I-201, STA-21, and nifuroxazide. Furthermore, I demonstrated the therapeutic efficacy of ODZ10117 by targeting STAT3. ODZ10117 suppressed the cancer cell migration and invasion, induced apoptotic cell death, and reduced tumor growth in both in vitro and in vivo models of breast cancer and glioblastoma. In addition, ODZ10117 suppressed stem cell properties in glioma stem cells (GSCs). To confirm these results, I demonstrated two different types of xenograft model. First, I have shown that extended the survival rate and reduced lung metastasis in models of breast cancer. Next, the administration of ODZ10117 showed significant therapeutic efficacy in mouse xenograft models of GSCs. In conclusion, I believe this study provides insight in to a promising therapeutic candidate for cancers by targeting STAT3.신호변환 및 전사활성인자 3 (STA3)는 많은 종양에서 과발현하고 있으며, 종양 미세 환경에서 STAT3는 다양한 경로를 통해 지속적으로 활성화되며, 일반적으로 지속적으로 활성화된 STAT3는 종양의 형성, 진행, 악성도, 재발 및 약물 저항성, 암 줄기세포 특성과 연관되어 있다. 따라서, STAT3는 암 치료에서 성공 가능성이 매우 높은 단백질로 새로운 종류의 항암제 개발과 암치료에 유망한 표적이다. 이 연구에서 STAT3의 소분자 억제제로서 3-(2,4-디클로로-페녹시메틸)-5-트리클로로메틸-[1,2,4]옥사디아졸 (ODZ10117)을 발견하였고 이는 STAT3 표적 암치료에 효과적인 치료 효용이 있을 수 있음을 시사하였다. 먼저, ODZ10117은 다양한 종류의 암, 특히 유방암과 신경교모세포종에서 STAT3 활성화를 효과적으로 억제하는 것을 확인하였으며, 흥미롭게도 다른 STAT 계열 단백질 및 STAT3 상위 신호전달계에 관계없이 STAT3의 SH2 도메인을 표적으로 하여 STAT3의 타이로신 인산화, 핵 내로의 이동 및 전사 활성을 억제하는 것을 확인하였다. 또한, STAT3에 대한 ODZ10117의 억제 효과는 STAT3 억제제인, S3I-201, STA-21 및 니프록사지드와 같은 잘 알려진 STAT3 억제제 보다 STAT3의 활성화 저해 능력이 뛰어났다. ODZ10117은 암세포의 이동과 침윤을 억제하고, 세포사멸을 유도하였으며, 종양의 성장을 감소시켰다. 이러한 결과를 확인하기 위해, 본 연구에서는 두가지 암 종류에 해당하는 이종 이식 모델을 연구하였다. 첫번째로, 유방암 모델에서 ODZ10117의 억제 효과는 종양형성을 억제하였으며, 마우스의 생존율을 높이고 폐 전이를 감소시키는 것을 확인하였다. 다음으로, ODZ10117의 투여는 신경 교종 줄기세포의 마우스 이종 이식 모델에서도 종양형성을 저해하고 생존율을 높이는 등 치료 효과를 보였다. 결론적으로, 새롭게 발굴한STAT3의 소분자 억제 화합물인 ODZ10117은 종양에서 STAT3의 활성화 억제를 통해 항암치료에 대한 새로운 치료 전략이 될 수 있음을 시사한다. 나아가, 종양 미세 환경에서 STAT3의 역할과 종양 미세 환경에서의 ODZ10117의 새로운 역할을 규명함으로써, 암세포 뿐만 아니라, 종양 미세 환경에서도 STAT3 저해제의 항암작용 역할을 기대할 수 있다.Introduction 1 Materials and methods 8 Results 20 Figures 34 Discussion 74 References 79 Abstract in Korean 86Docto

JAK2 Alterations in Acute Lymphoblastic Leukemia: Molecular Insights for Superior Precision Medicine Strategies

published: 12 July 2022Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, arising from immature lymphocytes that show uncontrolled proliferation and arrested differentiation. Genomic alterations affecting Janus kinase 2 (JAK2) correlate with some of the poorest outcomes within the Philadelphia-like subtype of ALL. Given the success of kinase inhibitors in the treatment of chronic myeloid leukemia, the discovery of activating JAK2 point mutations and JAK2 fusion genes in ALL, was a breakthrough for potential targeted therapies. However, the molecular mechanisms by which these alterations activate JAK2 and promote downstream signaling is poorly understood. Furthermore, as clinical data regarding the limitations of approved JAK inhibitors in myeloproliferative disorders matures, there is a growing awareness of the need for alternative precision medicine approaches for specific JAK2 lesions. This review focuses on the molecular mechanisms behind ALL-associated JAK2 mutations and JAK2 fusion genes, known and potential causes of JAK-inhibitor resistance, and how JAK2 alterations could be targeted using alternative and novel rationally designed therapies to guide precision medicine approaches for these high-risk subtypes of ALL.Charlotte EJ. Downes, Barbara J. McClure, Daniel P. McDougal, Susan L. Heatley, John B. Bruning, Daniel Thomas, David T. Yeung and Deborah L. Whit

Development of efficient docking strategies and structure-activity relationship study of the c-Met type II inhibitors

c-Met is a transmembrane receptor tyrosine kinase and an important therapeutic target for anticancer drugs. In the present study, we systematically investigated the influence of a range of parameters on the correlation between experimental and calculated binding free energies of type II c-Met inhibitors. We especially focused on evaluating the impact of different force fields, binding energy calculation methods, docking protocols, conformation sampling strategies, and conformations of the binding site captured in several crystallographic structures. Our results suggest that the force fields, the protein flexibility, and the selected conformation of the binding site substantially influence the correlation coefficient, while the sampling strategies and ensemble docking only mildly affect the prediction accuracy. Structure-activity relationship study suggests that the structural determinants to the high binding affinity of the type II inhibitors originate from its overall linear shape, hydrophobicity, and two conserved hydrogen bonds. Results from this study will form the basis for establishing an efficient computational docking approach for c-Met type II inhibitors design

Targeting STAT3 and STAT5 in Cancer

Every minute, 34 new patients are diagnosed with cancer globally. Although over the past 50 years treatments have improved and survival rates have increased dramatically for several types of cancers, many remain incurable. Several aggressive types of blood and solid cancers form when mutations occur in a critical cellular signaling pathway, the JAK-STAT pathway; (Janus Kinase-Signal Transducer and Activator of Transcription). Currently, there are no clinically available drugs that target the oncogenic STAT3/5 proteins in particular or their Gain of Function hyperactive mutant products. Here, we summarize targeting approaches on STAT3/5, as the field moves towards clinical applications as well as we illuminate on upstream or downstream JAK-STAT pathway interference with kinase inhibitors, heat shock protein blockers or changing nuclear import/export processes. We cover the design paradigms and medicinal chemistry approaches to illuminate progress and challenges in understanding the pleiotropic role of STAT3 and STAT5 in oncogenesis, the microenvironment, the immune system in particular, all culminating in a complex interplay towards cancer progression

JAK-STAT Signaling and Inhibition in Immunological Diseases

Janus kinaasi (JAK) – signaalinvälittäjä ja transkriptioaktivaattori (STAT) reitti on yksi keskeisimmistä signaalinvälitysreiteistä kehoissamme. JAK-STAT reitti säätelee lukuisia biologisia toimintoja, kuten immuunivastetta, aineenvaihduntaa, hematopoieesia sekä kehitystä. Mutaatiot JAKeissa, STATeissa tai niiden toimintaa säätelevissä proteiineissa voivat aiheuttaa virheellisen JAK-STAT signalointivasteen, joka voi johtaa vakaviin sairauksiin. Myös tulehduksellisissa sairauksissa tyypillisesti esiintyvä sytokiiniympäristö voi aiheuttaa JAK-STAT reitin yliaktivaation. Janus kinaasit nähtiinkin potentiaalisina lääkekehityskohteina jo pian niiden tunnistamisen jälkeen. Tällä hetkellä kliinisessä käytössä on jo useita hyväksyttyjä JAK-estäjiä tulehduksellisten sairauksien ja hematologisten syöpien hoitoon. Tässä väitöskirjassa tutkittiin JAK-STAT reittien aktivaatiota harvinaisessa ja aiemmin määrittelemättömässä, immunologisessa sairaudessa virtaussytometria- menetelmää hyödyntäen. Toisessa osatyössä tutkittiin JAK-estäjä tofasitinibin in vivo vaikutusta eri JAK-STAT reitteihin lääkettä käyttävillä nivelreumapotilailla samankaltaisella metodologialla. Lisäksi analysoimme ja vertailimme keskenään 20 kliinisissä tutkimuksissa arvioidun JAK-estäjän selektiivisyyttä in vitro käyttäen sekä entsyymiaktiivisuutta että sytokiini-inhibitiota mittaavia menetelmiä. Työssä kuvaillaan uusi mutaatio IRF2BP2 geenissä, joka löydettiin kahdelta potilaalta, joilla oli sekä tulehduksellisia oireita että lymfopeniaa. Osoitimme, että IRF2BP2 proteiini on tärkeä erityisesti JAK-STAT välitteisen interferonivasteen säätelyssä. JAK-estäjillä tehdyissä tutkimuksissa osoitettiin, että tofasitinibi estää useiden JAK-STAT reittien aktivaation, mutta estämisen voimakkuus riippuu sekä tutkittavasta sytokiinireitistä että solupopulaatiosta. Toisaalta tofasitinibin sytokiinien estoprofiili myös jossain määrin riippuu tutkimusmenetelmästä, sillä eroavaisuuksia löydettiin in vivo ja in vitro mittausten välillä. JAK-estäjien vertailututkimus osoitti, että kliinisten JAK-estäjien selektiivisyydessä on eroja. Selektiivisyyden määrittäminen vaatii kuitenkin useiden mittausmenetelmien käyttämistä yhdistettynä farmakokineettisiin muuttujiin. Edellä kuvatut tulokset lisäävät ymmärrystä JAK-STAT reitin ja sen estämisen merkityksestä immunologisissa sairauksissa. Immunologisia sairauksia ja JAK-estäjiä karakterisoimalla voidaan tulevaisuudessa saavuttaa entistä kohdennetumpia ja siten tehokkaampia ja turvallisempia hoitoja erilaisista immuunijärjestelmän sairauksista kärsiville potilaille.Janus kinase (JAK) - signal transducer and activator of transcription (STAT) pathway is one of the key signaling pathways in our bodies. JAK-STAT pathway regulates numerous biological functions including the immune response, metabolism, hematopoiesis, and development. Mutations in JAKs, STATs, or their regulatory proteins can cause defective JAK-STAT signaling responses resulting in human diseases. In addition, the cytokine environment typically present in inflammatory diseases can cause overactivation of the JAK-STAT pathway. Consequently, Janus kinases were identified as potential drug targets shortly after their discovery. To date, several JAK inhibitors are in clinical use for the treatment of both inflammatory diseases and hematological malignancies. In this thesis project, a rare, previously uncharacterized, immunological disease was investigated focusing on the activation of JAK-STAT pathways, which was studied using flow cytometry. In the second study, we analyzed the in vivo effects of a JAK inhibitor, tofacitinib, on different JAK-STAT pathways in patients with rheumatoid arthritis using similar methodology. In addition, the selectivity of 20 clinical JAK inhibitors was analyzed and compared by using in vitro methods that measure enzymatic activity and cytokine inhibition. The results presented in this thesis describe a new mutation in the IRF2BP2 gene found in two patients having both inflammatory symptoms and lymphopenia. We showed that IRF2BP2 protein is particularly important in the regulation of the JAK- STAT-mediated interferon response. Studies done with JAK inhibitors showed that tofacitinib inhibits the activation of several JAK-STAT pathways, but the extent of inhibition depends on both the cytokine pathway and the cell population studied. On the other hand, the cytokine inhibition profile of tofacitinib also depends to some extent on the methodology, as differences were found between the in vivo and in vitro measurements. Comparison of 20 JAK inhibitors revealed inhibitors to possess distinct selectivity profiles and demonstrated that selectivity determination requires use of multiple measurement methods combined with pharmacokinetic variables. The results described above add to our understanding of the role of JAK-STAT pathway and its inhibition in immunological diseases. By characterizing immunological diseases and JAK inhibitors, more targeted and thus more effective and safer treatments can be achieved in the future for patients with various diseases of the immune system

新規経口Hsp90 阻害剤の創製と単剤及び併用による抗腫瘍効果に関する研究

筑波大学 (University of Tsukuba)201

Molecular Determinants of Orexin Receptor Ligand Interaction : Studies on Ligand Selectivity and Impact of Calcium

Neuropeptides orexin-A and orexin-B, and their receptors OX1 and OX2 were first found as regulators of appetite, and later several other functions have been found as well. Orexin peptides and receptors have been researched, especially in the field of drug discovery; however many relevant biochemical properties remain partly unsolved, including the factors determining orexin ligand binding properties and ligand selectivity. The study of these factors was pursued in this thesis. Ligand selectivity was studied with chimaeric orexin receptors. We mapped some of the molecular determinants of orexin receptors that are needed for the selectivity of orexin agonists and OX1-specific antagonist. The second quarter of the orexin receptors seems to be the most important area both for agonist and antagonist selectivity. However, for antagonist selectivity, the third quarter also seems to have a role. Activated orexin receptors cause a strong elevation of intracellular calcium. However, reduction of extracellular calcium attenuates that increase and other orexin receptor signalling, mediated by certain phospholipases and kinases, for instance. It remains unknown, however, how calcium causes these effects. With [125I]-orexin-A, a clear decrease in binding was observed after reduction of the extracellular calcium concentration. Also, we saw a similar reduction in the activities of phospholipase C (PLC) and adenylyl cyclase (AC). The concentration-relationship of calcium was identical for radioligand binding, PLC activation, and AC stimulation, while AC inhibition was more strongly attenuated. When the driving force for calcium influx was reduced with high-K+ medium, the orexin-A-induced PLC activity was more strongly reduced than orexin-A binding. In addition, inhibition of the orexin receptor-operated calcium channels had a more pronounced effect on the PLC activity than on the binding. It is thus suggested that reduction of extracellular calcium concentration both inhibits orexin binding and attenuates enzymatic activity. Orexin-B has higher binding affinity for OX2 than OX1 receptor and [Ala11, D-Leu15]-orexin-B, an orexin-B variant, has been reported to display even higher OX2-selectivity. We observed that [Ala11, D-Leu15]-orexin-B showed much lower OX2-selectivity than originally reported. In addition, the selectivity of both forms of orexin-B was dependent on the cell line. These findings may be caused by biased agonism of the orexin receptor, meaning that the orexin receptor can be found in multiple conformations, each of which can interact differently with an agonist. This result extends our knowledge of orexin ligand binding properties, and the phenomenon should be considered, for instance, when novel agonists for orexin receptors are screened.Neuropeptidien oreksiini-A ja oreksiini-B, ja niiden reseptoreiden OX1 ja OX2, löydettiin aluksi säätelevän ruokahalua, mutta myöhemmin niille on löydetty myös monia muita tehtäviä. Oreksiinipeptidejä ja -reseptoreita tutkitaan etenkin lääkekehityksessä, mutta silti monet tärkeät biokemialliset ominaisuudet, kuten ligandin sitoutumiseen ja selektiivisyyteen vaikuttavat tekijät, ovat osittain selvittämättä. Tässä väitöskirjatyössä tutkittiin näitä tekijöitä. Ligandin selektiivisyyttä tutkittiin kimeerisillä reseptoreilla, joilla kartoitimme oreksiiniagonistien ja OX1-spesifisen antagonistin selektiivisyyteen tarvittavia alueita oreksiinireseptoreissa. Oreksiinireseptoreiden toinen neljännes näyttäisi olevan tärkein alue sekä agonistien että antagonistin selektiivisyydelle. Kuitenkin myös kolmannella neljänneksellä on merkitystä antagonistin selektiivisyydelle. Oreksiinireseptorin aktivoituminen aiheuttaa solunsisäisen kalsiumin lisääntymisen. Tätä, ja mm. tiettyjen fosfolipaasien ja -kinaasien aktiivisuuta, voidaan heikentää vähentämällä solunulkoista kalsiumia. Ei ole kuitenkaan tiedossa, kuinka kalsiumi aiheuttaa nämä vaikutukset. Solunulkoisen kalsiumin vähentäminen vähensi selvästi [125I]-oreksiini-A:n sitoutumista. Vastaavasti, myös fosfolipaasi C:n (PLC) ja adenylaattisyklaasin (AC) aktiivisuudet vähenivät. Kalsiumin vähentäminen heikensi yhtä paljon radioligandin sitoutumista, PLC:n aktivaatiota ja AC:n stimulaatiota, kun taas AC:n inhibitio heikkeni enemmän. Kalsiumin sisäänvirtauksen vähentäminen puskurilla, jossa on paljon K+-ioneja, vähensi enemmän PLC:n aktivoitumista kuin oreksiini-A:n sitoutumista. Lisäksi reseptorin säätelemien kalsium kanavien inhibitiolla oli suurempi vaikutus PLC-aktiivisuuteen kuin sitoutumiseen. Täten vaikuttaisi siltä, että solunulkoisen kalsiumin vähentäminen estää sekä oreksiinin sitoutumista että entsymaattista aktiivisuutta. Oreksiini-B:llä on korkeampi sitoutumisaffiniteetti OX2 kuin OX1 reseptoriin, ja oreksiini-B muunnoksen, [Ala11, D-Leu15]-oreksiini-B:n, on raportoitu olevan vielä enemmän spesifisempi OX2:lle. Huomasimme, että [Ala11, D-Leu15]-oreksiini-B:n OX2-selektiivisyys oli paljon matalampi kuin alun perin on raportoitu. Lisäksi, molempien oreksiini-B muotojen selektiivisyys riippui solulinjasta. Nämä havainnot voivat aiheutua siitä, että oreksiinireseptorilla voi olla useita eri rakenteita, joista kukin vuorovaikuttaa erilailla agonistin kanssa. Tulokset laajentavat tietämystämme oreksiinin sitoutumisominaisuuksista, ja ne pitäisi ottaa huomioon, esim. kun oreksiinireseptorille seulotaan uusia agonisteja

Looking at COVID-19 from a Systems Biology Perspective

The sudden outbreak and worldwide spread of the SARS-CoV-2 pandemic pushed the scientific community to find fast solutions to cope with the health emergency. COVID-19 complexity, in terms of clinical outcomes, severity, and response to therapy suggested the use of multifactorial strategies, characteristic of the network medicine, to approach the study of the pathobiology. Proteomics and interactomics especially allow to generate datasets that, reduced and represented in the forms of networks, can be analyzed with the tools of systems biology to unveil specific pathways central to virus\u2013human host interaction. Moreover, artificial intelligence tools can be implemented for the identification of druggable targets and drug repurposing. In this review article, we provide an overview of the results obtained so far, from a systems biology perspective, in the understanding of COVID-19 pathobiology and virus\u2013host interactions, and in the development of disease classifiers and tools for drug repurposing