26 results on '"Sravan Mandadi"'
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2. Real-Time Translocation and Function of PKCβII Isoform in Response to Nociceptive Signaling via the TRPV1 Pain Receptor
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Basil D. Roufogalis, Patricia J. Armati, and Sravan Mandadi
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pain ,protein kinase C ,transient receptor potential vanilloid-1 ,real-time translocation ,dorsal root ganglion neurons ,nociceptive signaling ,Medicine ,Pharmacy and materia medica ,RS1-441 - Abstract
Serine/threonine protein kinase C βII isoform (PKCβII) or the pain receptor transient receptor potential vanilloid 1 (TRPV1) have been separately implicated in mediating heat hyperalgesia during inflammation or diabetic neuropathy. However, detailed information on the role of PKC βII in nociceptive signaling mediated by TRPV1 is lacking. This study presents evidence for activation and translocation of the PKC βII isoform as a signaling event in nociception mediated by activation of TRPV1 by capsaicin. We show that capsaicin induces translocation of cytosolic PKCβII isoform fused with enhanced green fluorescence protein (PKCβII-EGFP) in dorsal root ganglion (DRG) neurons. We also show capsaicin-induced translocation in Chinese Hamster Ovarian (CHO) cells co-transfected with TRPV1 and PKCβII-EGFP, but not in CHO cells expressing PKCβII-EGFP alone. By contrast, the PKC activator phorbol-12-myristate-13-acetate (PMA) induced translocation of PKCβII-EGFP which was sustained and independent of calcium or TRPV1. In addition PMA-induced sensitization of TRPV1 to capsaicin response in DRG neurons was attenuated by PKCβII blocker CGP 53353. Capsaicin response via TRPV1 in the DRG neurons was confirmed by TRPV1 antagonist AMG 9810. These results suggested a novel and potential signaling link between PKCβII and TRPV1. These cell culture models provide a platform for investigating mechanisms of painful neuropathies mediated by nociceptors expressing the pain sensing gene TRPV1, and its regulation by the PKC isoform PKCβII.
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- 2011
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3. Control of Neonatal Spinal Networks by Nociceptors: A Potential Role for TRP Channel Based Therapies
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Sravan Mandadi, Peter Hong, Arjun Sunny Dhoopar, and Patrick Whelan
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Therapeutics. Pharmacology ,RM1-950 ,Pharmacy and materia medica ,RS1-441 - Abstract
Pediatric spinal cord injury (SCI) often leads to increased nociceptive input resulting in aberrant motor output like tremor and spasticity. Acute plasticity within spinal pain and motor networks following pediatric SCI may result in long-term sensorimotor disabilities. Despite this, pediatric SCI remains poorly understood. Part of the problem lies in the paucity of detailed studies aimed at defining sensorimotor control by nociceptors during development. This review provides an overview of work that highlights afferent control of sensorimotor networks by defined nociceptors in the developing spinal cord. Here, we focus on the well established and widely used neonatal sensorimotor model called sacrocaudal afferent (SCA) pathway. Until recently, the identity of specific subclasses of nociceptive afferents in the SCA pathway controlling developing sensorimotor networks was unknown. We highlight here the use of members of the Transient Receptor Potential (TRP) ion channels and mouse genetics to identify specific subsets of nociceptive afferents in the SCA pathway. In addition, we highlight the use of mouse genetics to map sensorimotor networks during development and potential future applications. A neonatal spinal cord model of central neuropathic pain via a defined set of nociceptors is presented as a probe into potential therapeutic avenues in neonatal SCI. Finally, knowledge translation from neonatal basic research to the pediatric population in the clinic is described. In conclusion, studies in neonatal models may lead to therapeutic strategies and pharmaceuticals for chronic pain and motor dysfunction after SCI during development. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.
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- 2013
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4. Abstract 462: A novel and potent Cblb inhibitor demonstrates robust immunological profile and anti-tumor efficacy
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Murugan Chinnapattu, Sandeep Shelke, Prashant Ingale, Nayan Waghmare, Nanasaheb Kadlag, Manoj Pawar, Akshay Kangane, Sachin S. Chaudhari, Jagmohan Saini, Vidya Kattige, Arti Joshi, Colina Dutta, Debjyoti Boral, Sheetal Kadam, Varada Potdar, Jiju Mani, Pooja Sawant, Megha Marathe, Madhavi Mulay, Akshata Virdikar, Sravan Mandadi, Atul Akarte, Anuj Singh, Chandrasekhar Misra, Pandurang Lambade, Chaitanya Tirumalasetty, Raju Patole, Vikas Karande, Dayanidhi Behera, Pankaj Jain, Vishwanath Kurawattimath, Nagaraj Gowda, and Pravin S. Iyer
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Cancer Research ,Oncology - Abstract
Background: Casitas B-lineage lymphoma b (Cbl-b), a RING finger E3 ligase, is a negative regulator of immune cell activation1. Genetic deletion or pharmacological inhibition of Cbl-b resulted in hyper-reactive and co-stimulation independent T cell activation and cytokine production1. In syngeneic tumor models, CD8 T-cell and NK-cell mediated rejection of tumours were observed1. These findings point to Cbl-b as a therapeutic target in cancer immunotherapy. Inhibition of Cbl-b also demonstrated the potential to enhance the efficacy of check-point blockers like anti-PD-1 antibody, an unmet need in the clinic. Methods: Using intuitive medicinal chemistry design supported by computational approaches, we identified a lead Cbl-b inhibitor. SAR was developed using a battery of biochemical assays, functional read-outs and primary human in vitro T-cell activation and exhaustion assays. In vivo efficacy was demonstrated in syngeneic mouse colon tumor model. Results: Our lead Cbl-b inhibitor demonstrated potent binding to Cbl-b, robust anti-tumor cytokine secretion in human and mouse T cells, whole blood and potent reversal of T cell exhaustion. A strong tumor growth inhibition was demonstrated by the lead compound in a mouse colon tumor model. Compared to single agent, a combination of the lead compound with anti-PD-1 antibody induced enhanced complete tumor rejections. Conclusions: We have identified a novel, potent and orally bioavailable Cbl-b inhibitor that demonstrated robust in vitro and in vivo anti-tumor profiles. Acknowledgements: We thank Sanjib Das, Ajit Patil, Gauri Gawas, Savita Pandita, Priya Yadav, Sneha Pusadkar, Mayura Behere, Subhadip Das, Shravankumar Kolli and Radheshyam Yadav for their contributions to the project References: 1. Clinical and Experimental Immunology, 204: 14-31, 2020 Citation Format: Murugan Chinnapattu, Sandeep Shelke, Prashant Ingale, Nayan Waghmare, Nanasaheb Kadlag, Manoj Pawar, Akshay Kangane, Sachin S. Chaudhari, Jagmohan Saini, Vidya Kattige, Arti Joshi, Colina Dutta, Debjyoti Boral, Sheetal Kadam, Varada Potdar, Jiju Mani, Pooja Sawant, Megha Marathe, Madhavi Mulay, Akshata Virdikar, Sravan Mandadi, Atul Akarte, Anuj Singh, Chandrasekhar Misra, Pandurang Lambade, Chaitanya Tirumalasetty, Raju Patole, Vikas Karande, Dayanidhi Behera, Pankaj Jain, Vishwanath Kurawattimath, Nagaraj Gowda, Pravin S. Iyer. A novel and potent Cblb inhibitor demonstrates robust immunological profile and anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 462.
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- 2023
5. Abstract 463: GRC 54276, a novel small molecule inhibitor of HPK1 has entered phase 1/2 clinical trial insolid malignancies and Hodgkin’s/non Hodgkin’s lymphoma
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Sravan Mandadi, Sanjib Das, Malini Bajpai, Jagmohan Saini, Murugan Chinnapattu, Sanjay Patale, Sandip Patil, Nanasaheb Kadlag, Nayan Waghmare, Balasaheb Gavhane, Ameya Deshpande, Dnyaneshwar Dahale, Vidya Kattige, Priyanka Pangre, Namrata Singh, Ekta Kashyap, Megha Marathe, Jiju Mani, Atul Akarte, Chandrasekhar Misra, Subhadip Das, Anuj Singh, Pandurang Lambade, Avratanu Das, Chaitanya Tirumalasetty, Raju Patole, Nilanjana Biswas, Vikas Karande, Heta Shah, Dayanidhi Behera, Pankaj Jain, Pavankumar Sancheti, Pramod Pawar, Vinod KR, Venkatesha Udupa, Sachin S. Chaudhari, Nagaraj Gowda, and Pravin S. Iyer
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Cancer Research ,Oncology - Abstract
Background: Pre-clinical profile of GRC 54276, a clinical candidate with Phase 1/2 clinical trial ongoing, is presented here. GRC 54276 is a novel small molecule inhibitor of Hematopoietic progenitor kinase 1 (HPK1), a serine/threonine kinase1,2 that negatively regulates T and B cell receptor signaling3. Inhibition of HPK1 is an attractive therapeutic strategy for immuno-oncology based treatment of solid tumors3. Methods: GRC 54276 was designed and developed using SAR based medicinal chemistry design supported by computational approaches. In vitro profiling was done using a battery of biochemical assays, functional read-outs and primary human in vitro T-cell activation assays. In vivo efficacy was demonstrated in mouse colon tumor models of CT26 and MC38-hPD-L1. In vivo inhibition of biomarker pSLP76 Ser(376) by GRC 54276 was determined using CT26 tumor model. Detailed ADME-PK studies have been performed along with safety tolerability studies conducted in mice and monkeys. Results: GRC 54276, demonstrated excellent in vitro immune profile of target engagement and anti-tumor immune response activity in both human and mouse systems. As a single agent, GRC 54276 demonstrated strong inhibition of tumor growth and biomarker pSLP76 Ser(376) in the CT26 tumor model. Enhanced efficacy was demonstrated by combining GRC 54276 with check-point blocking antibodies anti-CTLA4 and Atezolizumab in the CT26 and MC38-hPD-L1 models, respectively. GRC 54276 robustly enhanced complete tumor rejections when combined with Atezolizumab in the MC38-hPD-L1 model, correlating with increased immune effector memory T cells. Pharmacokinetic profile of GRC 54276 is characterized by high permeability, rapid absorption and moderate oral bioavailable across species. GRC 54276 is non-gentoxic with no observed adverse effects in mice and no treatment related cardiovascular or respiratory effects in repeat dose toxicity study in monkeys. Conclusions: GRC 54276 is a novel HPK1 inhibtitor with acceptable pre-clincial profile and is currently undergoing a Phase 1/2 clinical trial. Acknowledgements: We thank Pooja S, Shital M, Rahul B, Ajit J, Sanjay G, Somesh K, Pramod S for their contributions to the project References: 1. F.Kiefer et al., The EMBO Journal 1996 2. Hu et al., Genes and Development 1996 3. Sawasdikosol and Burakoff. eLife 2020;9:e55122 Citation Format: Sravan Mandadi, Sanjib Das, Malini Bajpai, Jagmohan Saini, Murugan Chinnapattu, Sanjay Patale, Sandip Patil, Nanasaheb Kadlag, Nayan Waghmare, Balasaheb Gavhane, Ameya Deshpande, Dnyaneshwar Dahale, Vidya Kattige, Priyanka Pangre, Namrata Singh, Ekta Kashyap, Megha Marathe, Jiju Mani, Atul Akarte, Chandrasekhar Misra, Subhadip Das, Anuj Singh, Pandurang Lambade, Avratanu Das, Chaitanya Tirumalasetty, Raju Patole, Nilanjana Biswas, Vikas Karande, Heta Shah, Dayanidhi Behera, Pankaj Jain, Pavankumar Sancheti, Pramod Pawar, Vinod KR, Venkatesha Udupa, Sachin S. Chaudhari, Nagaraj Gowda, Pravin S. Iyer. GRC 54276, a novel small molecule inhibitor of HPK1 has entered phase 1/2 clinical trial insolid malignancies and Hodgkin’s/non Hodgkin’s lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 463.
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- 2023
6. 848 Novel small molecule HPK1 inhibitor PCC-1 induces strong anti-tumor activity
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Pooja Sawant, Ameya Deshpande, Pravin Iyer, Nagaraj Gowda, Avratanu Das, Megha Marathe, Dayanidhi Behera, Subhadip Das, Sravan Mandadi, Nilanjana Biswas, Priyanka Pangre, Heta Shah, Raju Patole, Murugan Chinnapattu, Vikas Karande, Sanjib Das, Pandurang Lambade, Sachin Sundarlal Chaudhari, Chandrasekhar Misra, Anuj Singh, Chaitanya Tirumalasetty, Jagmohan S. Saini, Varada Potdar, Malini Bajpai, and Atul Akarte
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Pharmacology ,Antitumor activity ,Cancer Research ,Oncology ,Chemistry ,Immunology ,Cancer research ,Molecular Medicine ,Immunology and Allergy ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Small molecule ,RC254-282 - Abstract
BackgroundHematopoietic progenitor kinase 1 (HPK1, MAP4K1), is a negative regulator of T and B cell receptor signaling.1 2 3 A strong anti-tumor immunogenic response and tumor rejection was observed in mice with HPK1 gene knocked out.3 Treatment of HPK1 kinase dead mice with immune check-point blockers (ICBs) demonstrated enhanced tumor growth inhibition.3 Hence, HPK1 is an attractive therapeutic strategy for immuno-oncology based treatment in cancers. In comparison to our previous HPK1 small molecule inhibitor, PCC,4 we present here a differentiated novel HPK1 inhibitor, PCC-1 with good anti-T cell kinases selectivity and stronger anti-tumor efficacy in CT26 tumor model. In addition, using the syngeneic model of MC38 expressing human PD-L1, we present for the first time, the combination efficacy of a HPK1 inhibitor with the clinical ICB, Atezolizumab.MethodsIntuitive medicinal chemistry complemented by structure-based drug design was used to identify & develop potent inhibitors of HPK1 with optimal kinase selectivity, PK and in vivo efficacy profile. The SAR efforts were guided by biochemical assays, functional read-outs and primary human in vitro T-cell activation assays. In vivo target engagement and pharmacodynamic data was generated using CT26 and MC38-hPD-L1 tumor models.ResultsPCC-1 has sub-nanomolar HPK1 inhibition potency and strong target engagement resulting in pSLP76 inhibition, enhanced anti-tumor cytokine production of IL-2 and/or IFNgamma in Jurkat cells, human PBMCs and human whole blood. PCC-1 also demonstrated nanomolar potency in inducing a complete reversal of PGE2 or adenosine mediated immunosuppression. Oral dosing of PCC-1 as a single agent, induced strong tumor growth inhibition (TGI) in the syngeneic model of CT26 and MC38-hPD-L1 tumor models. Combination of PCC-1 with anti-CTLA4 in CT26 tumor model induced significantly greater TGI than anti-CTLA4 alone. Moreover, as a first, the combination of PCC-1 with clinical ICB, Atezolizumab in MC38-hPD-L1 induced enhanced rejection of tumors. These results strongly suggest PCC-1 as a promising candidate for HPK1 inhibition and as a combination partner with ICBs in clinic.ConclusionsPCC-1 is a novel, orally active HPK1 inhibitor that demonstrates excellent stand-alone efficacy and enhances current immunotherapy efficacy. Further evaluation of PCC-1 is ongoing to advance towards clinic.AcknowledgementsWe thank Dnyaneshwar Dahale, Sanjay Patale, Sandip Patil, Vidya Kattige, Jiju Mani, Namrata Singh, Ekta Kashyap, Sandeep Thorat, Pankaj Jain and Pramod Sagar for their contributions to the projectTrial RegistrationN/AReferencesKiefer F, et al. The EMBO Journal 1996.Hu, et al. Genes and Development 1996.Sawasdikosol, Burakoff. eLife 2020;9:e55122.Sachin S Chaudhari, et al. Poster#1709, AACR Annual Meeting April-May 2021.Ethics ApprovalThe studies involving animals have obtained ethics approval from Institutional Animal Ethics Committee (IAEC), The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India, GRC/IAEC/472/2020-1. Participants of the studies have given informed consent before taking part.
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- 2021
7. Abstract 1804: IND-ready clinical candidate for HPK1 developed with excellent efficacy and safety profile
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Sravan Mandadi, Sanjib Das, Jagmohan Saini, Sachin S. Chaudhari, Murugan Chinnapattu, Ameya Deshpande, Dnyaneshwar Dahale, Malini Bajpai, Priyanka Pangre, Namrata Singh, Ekta Kashyap, Megha Marathe, Jiju Mani, Atul Akarte, Chandrasekhar Misra, Subhadip Das, Anuj Singh, Avratanu Das, Pandurang Lambade, Chaitanya Tirumalasetty, Raju Patole, Nilanjana Biswas, Vikas Karande, Heta Shah, Dayanidhi Behera, Pankaj Jain, Pavankumar Sancheti, Somesh Kakade, Pramod K. Pawar, Vinod KR, Venkatesha Udupa, Nagaraj Gowda, and Pravin S. Iyer
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Cancer Research ,Oncology - Abstract
Background Hematopoietic progenitor kinase 1 (HPK1) is a member of the mitogen-activated protein kinase (MAP4K) family of protein serine/threonine kinases1,2 and is a negative regulator of T and B cell receptor signaling3. Inhibition of HPK1 is an attractive therapeutic strategy for immuno-oncology based treatment of solid tumors3. We present in vitro, in vivo, pharmacokinetic (PK) and early safety profiles of a novel and differentiated HPK1 inhibitor GRC 54276. Methods GRC 54276 is our clinical candidate, designed and developed using intuitive medicinal chemistry design and supported by computational approaches. SAR studies included a battery of biochemical assays, functional read-outs and primary human in vitro T-cell activation assays. In vivo efficacy was demonstrated in syngeneic mouse tumor models, both as a single agent and combination with immune check-point blockers (ICB), mouse anti-CTLA4 antibody or Atezolizumab (human anti-PD-L1 antibody). ADME-PK properties was evaluated cross-species. GLP and non-GLP safety tolerability studies were conducted in mice and monkeys. Results GRC 54276 demonstrated sub-nanomolar HPK1 potency, strong target engagement of pSLP76 inhibition, anti-tumor cytokines (IL-2 and IFN-γ) induction, reversal of immunosuppression by prostaglandin E2 (PGE2) or adenosine in both human and mouse systems. GRC 54276 demonstrated very strong tumor growth inhibition (TGI) efficacy as single agent and significantly enhanced TGI in combination with ICB antibodies anti-CTLA4 (CT26 model) or Atezolizumab (MC38-hPD-L1 model). The in vivo TGI efficacy mechanistically correlated with increased immune responses of cytokine induction, infiltration of cytotoxic T cells, tumor rejections accompanied by immune memory T cells induction. Pharmacokinetic profile of GRC 54276 included cross-species oral bioavailability (30 to 100%), predominant clearance by CYP3A4 with no significant inhibition of major CYP isoforms, negative activation potency in human PXR assay at several-fold over EDmax exposures. Safety profile demonstrated that GRC 54276 is non-genotoxic in the bone marrow micronucleus assay in mice and has no hERG liability. The no observed adverse effect levels in the 14-day and 17-day exploratory studies in mice and monkeys were 50 and 15 mg/kg/day, respectively. Conclusions GRC 54276, our clinical candidate is potent, selective, orally bioavailable HPK1 inhibitor demonstrating strong single-agent and combination efficacy, low DDI liability accompanied by acceptable early safety profile in mice and monkeys. GRC 54276 is undergoing IND enabling studies to advance to Phase 1 clinical trial. Acknowledgements We thank Vidya Kattige, Pooja Sawant, Shital More, Rahul B. Bhadane, Ajit Jagadale, Sanjay Gaikwad, Pramod Sagar for their contributions to the project References 1. The EMBO Journal 1996 2. Genes and Development 1996 3. eLife 2020;9:e55122 Citation Format: Sravan Mandadi, Sanjib Das, Jagmohan Saini, Sachin S. Chaudhari, Murugan Chinnapattu, Ameya Deshpande, Dnyaneshwar Dahale, Malini Bajpai, Priyanka Pangre, Namrata Singh, Ekta Kashyap, Megha Marathe, Jiju Mani, Atul Akarte, Chandrasekhar Misra, Subhadip Das, Anuj Singh, Avratanu Das, Pandurang Lambade, Chaitanya Tirumalasetty, Raju Patole, Nilanjana Biswas, Vikas Karande, Heta Shah, Dayanidhi Behera, Pankaj Jain, Pavankumar Sancheti, Somesh Kakade, Pramod K. Pawar, Vinod KR, Venkatesha Udupa, Nagaraj Gowda, Pravin S. Iyer. IND-ready clinical candidate for HPK1 developed with excellent efficacy and safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1804.
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- 2022
8. Abstract 1709: Novel small molecule HPK1 inhibitor induces immunogenic anti-tumor effects
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Pavankumar Sancheti, Somesh Kakade, Pooja Sawant, Vidya Ganapati Kattige, Vinod K. R, Pandurang Lambade, Sandeep Thorat, Raju Patole, Chandrasekhar Misra, Dayanidhi Behera, Nagaraj Gowda, Pravin Iyer, Varada Potdar, Malini Bajpai, Nilanjana Biswas, Sravan Mandadi, Subhadip Das, Sandeep Mahankali, Atul Akarte, Anuj Singh, Pankaj Jain, Pramod K. Pawar, Avratanu Das, Venkatesha Udupa, Priyanka Pangre, Sachin Sundarlal Chaudhari, and Heta Shah
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Antitumor activity ,Cancer Research ,Oncology ,Chemistry ,Cancer research ,Small molecule - Abstract
Background Hematopoietic progenitor kinase 1 (HPK1, MAP4K1), a member of the MAP4K family of protein serine/threonine kinases1,2 is a negative regulator of TCR and BCR3. Immunosuppressive mediators PGE2 and adenosine regulate activation of HPK13. In vivo anti-tumor activity by HPK1 gene deletion, kinase dead HPK1 and small molecule HPK1 inhibitors have been demonstrated in multiple immunogenic syngeneic tumor models3,4. Enhanced anti-tumor efficacy in these models was achieved by combining HPK1 inhibitors with immune check-point blockers (ICBs) like anti-PD-1, anti-PD-L1or anti-CTLA4 antibodies3,4. HPK1 inhibition is a target for immuno-oncology treatment in cancers responsive or non-responsive to current ICBs. Methods Creative medicinal chemistry design complemented by structured-based support was used to identify & develop inhibitors of HPK1 with pico/nano-molar potency, optimal kinase selectivity, PK and efficacy profile. Our SAR efforts were guided by biochemical assays, functional read-outs and primary human in vitro T-cell activation assays. In vivo target engagement and pharmacodynamic data was generated using mouse models of the lung (LLC) and colon (MC38 and CT26) cancer. Results We describe novel and potent HPK1 inhibitors that can inhibit pSLP76 and enhance IL-2 production in Jurkat cells, IL-2 and IFN-γ production in human PBMCs, whole blood and primary T cells and TNF-α production by dendritic cells following stimulation. A reversal of PGE2 or adenosine mediated immunosuppression was also achieved by the inhibitors. These compounds on oral dosing showed strong tumor growth inhibition (TGI) in syngeneic models refractory to ICBs (subcutaneous LLC) as well as responsive to ICBs (subcutaneous MC38 and CT26; orthotopic LLC). TGI was accompanied by the immune response of increased cytokine (IL-2/IFNγ) levels and tumor infiltrating lymphocytes (TILs). Combination with ICBs induced enhanced TGI correlating with enhanced cytokine induction and TILs. We confirmed the immune-mediated mechanism of inhibitors by their lack of efficacy in immune-compromised mice. The lead compound demonstrated cardiac safety (hERG assay), lack of genotoxicity and very good safety margins in the mouse exploratory toxicology studies up to 28-days. Conclusion We have discovered a novel, orally active HPK1 inhibitor that demonstrates excellent stand-alone efficacy in multiple tumor models and also offers the potential to enhance current immunotherapy regimens in both responsive and refractory cancers. Further evaluation of our lead molecule towards the clinic is underway. Acknowledgements Sanjib D, Megha M, Jiju M, Sheetal K, Arti J, Swayam M, Srinivas K, Pradeep V, Vikram B, Abhay K, Jagmohan S, Ravi T, Mohammad Y, Rahul B, Ajit J, Sanjay G, Pramod S. References 1. Kiefer et al., EMBO Journal 1996; 2. Hu et al., Genes and Development 1996; 3. Sawasdikosol and Burakoff. eLife 2020;9:e55122; 4. AACR Annual Meeting June 22-24 2020 Citation Format: Sachin S. Chaudhari, Malini Bajpai, Sravan Mandadi, Vidya G. Kattige, Sandeep Thorat, Varada Potdar, Priyanka Pangre, Pooja Sawant, Chandrasekhar Misra, Subhadip Das, Atul Akarte, Anuj Singh, Sandeep Mahankali, Pandurang Lambade, Avratanu Das, Raju Patole, Venkatesha Udupa, Pavankumar Sancheti, Pramod K. Pawar, Somesh Kakade, Vinod K. R, Nilanjana Biswas, Heta Shah, Dayanidhi Behera, Pankaj Jain, Nagaraj Gowda, Pravin S. Iyer. Novel small molecule HPK1 inhibitor induces immunogenic anti-tumor effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1709.
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- 2021
9. Modulatory and plastic effects of kinins on spinal cord networks
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J. Ejdrygiewicz, Patrick J. Whelan, Heather Leduc-Pessah, Tuan Trang, Peter Hong, Simon A. Sharples, and Sravan Mandadi
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0301 basic medicine ,biology ,Physiology ,Chemistry ,TRPV1 ,Spinal cord ,medicine.disease ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,medicine.anatomical_structure ,Nociception ,nervous system ,Anterior Horn Cell ,medicine ,Nociceptor ,Glial cell line-derived neurotrophic factor ,biology.protein ,Posterior Horn Cell ,Neuroscience ,Spinal cord injury ,030217 neurology & neurosurgery - Abstract
Key points Inflammatory kinins are released following spinal cord injury or neurotrauma. The effects of these kinins on ongoing locomotor activity of central pattern generator networks are unknown. In the present study, kinins were shown to have short- and long-term effects on motor networks. The short-term effects included direct depolarization of interneurons and motoneurons in the ventral horn accompanied by modulation of transient receptor potential vanilloid 1-sensitive nociceptors in the dorsal horn. Over the long-term, we observed a bradykinin-mediated effect on promoting plasticity in the spinal cord. In a model of spinal cord injury, we observed an increase in microglia numbers in both the dorsal and ventral horn and, in a microglia cell culture model, we observed bradykinin-induced expression of glial-derived neurotrophic factor. Abstract The expression and function of inflammatory mediators in the developing spinal cord remain poorly characterized. We discovered novel, short and long-term roles for the inflammatory nonapeptide bradykinin (BK) and its receptor bradykinin receptor B2 (B2R) in the neuromodulation of developing sensorimotor networks following a spinal cord injury (SCI), suggesting that BK participates in an excitotoxic cascade. Functional expression of B2R was confirmed by a transient disruptive action of BK on fictive locomotion generated by a combination of NMDA, 5-HT and dopamine. The role of BK in the dorsal horn nociceptive afferents was tested using spinal cord attached to one-hind-limb (HL) preparations. In the HL preparations, BK at a subthreshold concentration induced transient disruption of fictive locomotion only in the presence of: (1) noxious heat applied to the hind paw and (2) the heat sensing ion channel transient receptor potential vanilloid 1 (TRPV1), known to be restricted to nociceptors in the superficial dorsal horn. BK directly depolarized motoneurons and ascending interneurons in the ventrolateral funiculus. We found a key mechanism for BK in promoting long-term plasticity within the spinal cord. Using a model of neonatal SCI and a microglial cell culture model, we examined the role of BK in inducing activation of microglia and expression of glial-derived neurotrophic factor (GDNF). In the neonatal SCI model, we observed an increase in microglia numbers and increased GDNF expression restricted to microglia. In the microglia cell culture model, we observed a BK-induced increased expression of GDNF via B2R, suggesting a novel mechanism for BK spinal-mediated plasticity.
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- 2016
10. Identification of multisegmental nociceptive afferents that modulate locomotor circuits in the neonatal mouse spinal cord
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Patrick J. Whelan, Pina Colarusso, Sravan Mandadi, Allan I. Basbaum, Michelle A. Tran, Joao M. Braz, and Peter Hong
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General Neuroscience ,TRPV1 ,Central pattern generator ,Anatomy ,Biology ,Spinal cord ,Electrophysiology ,Anterograde tracing ,Bursting ,medicine.anatomical_structure ,Calcium imaging ,Nociception ,medicine ,Neuroscience - Abstract
Compared to proprioceptive afferent collateral projections, less is known about the anatomical, neurochemical, and functional basis of nociceptive collateral projections modulating lumbar central pattern generators (CPG). Quick response times are critical to ensure rapid escape from aversive stimuli. Furthermore, sensitization of nociceptive afferent pathways can contribute to a pathological activation of motor circuits. We investigated the extent and role of collaterals of capsaicin-sensitive nociceptive sacrocaudal afferent (nSCA) nerves that directly ascend several spinal segments in Lissauer's tract and the dorsal column and regulate motor activity. Anterograde tracing demonstrated direct multisegmental projections of the sacral dorsal root 4 (S4) afferent collaterals in Lissauer's tract and in the dorsal column. Subsets of the traced S4 afferent collaterals expressed transient receptor potential vanilloid 1 (TRPV1), which transduces a nociceptive response to capsaicin. Electrophysiological data revealed that S4 dorsal root stimulation could evoke regular rhythmic bursting activity, and our data suggested that capsaicin-sensitive collaterals contribute to CPG activation across multiple segments. Capsaicin's effect on S4-evoked locomotor activity was potent until the lumbar 5 (L5) segments, and diminished in rostral segments. Using calcium imaging we found elevated calcium transients within Lissauer's tract and dorsal column at L5 segments when compared to the calcium transients only within the dorsal column at the lumbar 2 (L2) segments, which were desensitized by capsaicin. We conclude that lumbar locomotor networks in the neonatal mouse spinal cord are targets for modulation by direct multisegmental nSCA, subsets of which express TRPV1 in Lissauer's tract and the dorsal column. J. Comp. Neurol. 521:2870-2887, 2013. © 2013 Wiley Periodicals, Inc.
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- 2013
11. Anandamide modulates carotid sinus nerve afferent activity via TRPV1 receptors increasing responses to heat
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Patrick J. Whelan, Ekaterina Rodikova, Erin V. Ferguson, Sravan Mandadi, Marie-Noelle Fiamma, Arijit Roy, and Richard J. A. Wilson
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Male ,medicine.medical_specialty ,Hot Temperature ,Mice, 129 Strain ,Polyunsaturated Alkamides ,Physiology ,medicine.medical_treatment ,TRPV1 ,TRPV Cation Channels ,Peripheral chemoreceptors ,Arachidonic Acids ,In Vitro Techniques ,Rats, Sprague-Dawley ,Mice ,chemistry.chemical_compound ,Physiology (medical) ,Internal medicine ,medicine ,Animals ,Neurons, Afferent ,Respiratory system ,Mice, Knockout ,Cannabinoids ,Carotid sinus ,Anandamide ,Endocannabinoid system ,Rats ,Mice, Inbred C57BL ,Carotid Sinus ,Endocrinology ,medicine.anatomical_structure ,nervous system ,chemistry ,lipids (amino acids, peptides, and proteins) ,Carotid body ,Cannabinoid ,Endocannabinoids - Abstract
Abnormal respiratory chemosensitivity is implicated in recurrent apnea syndromes, with the peripheral chemoreceptors, the carotid bodies, playing a particularly important role. Previous work suggests that supraphysiological concentrations of the endocannabinoid endovanilloid and TASK channel blocker anandamide (ANA) excite carotid bodies, but the mechanism(s) and physiological significance are unknown. Given that carotid body output is temperature-sensitive, we hypothesized that ANA stimulates carotid body chemosensory afferents via temperature-sensitive vanilloid (TRPV1) receptors. To test this hypothesis, we used the dual-perfused in situ rat preparation to confirm that independent perfusion of carotid arteries with supraphysiological concentrations of ANA strongly excites carotid sinus nerve afferents and that this activity is sufficient to increase phrenic activity. Next, using ex vivo carotid body preparations, we demonstrate that these effects are mediated by TRPV1 receptors, not CB1 receptors or TASK channels: in CB1-null mouse preparations, ANA increased afferent activity across all levels of Po2, whereas in TRPV1-null mouse preparations, the stimulatory effect of ANA was absent. In rat ex vivo preparations, ANA's stimulatory effects were mimicked by olvanil, a nonpungent TRPV1 agonist, and suppressed by the TRPV1 antagonist AMG-9810. The specific CB1 agonist oleamide had no effect. Physiological levels of ANA had no effect alone but increased sensitivity to mild hyperthermia. AMG-9810 blocked ANA's effect on the temperature response. Immunolabeling and RT-PCR demonstrated that TRPV1 receptors are not expressed in carotid body glomus cells but reside in petrosal sensory afferents. Together, these results suggest that ANA plays a physiological role in augmenting afferent responses to mild hyperthermia by activating TRPV1 receptors on petrosal afferents.
- Published
- 2012
12. Locomotor networks are targets of modulation by sensory transient receptor potential vanilloid 1 and transient receptor potential melastatin 8 channels
- Author
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S. T. Nakanishi, A. Dhaka, David D. McKemy, Yoshio Takashima, Patrick J. Whelan, Ardem Patapoutian, and Sravan Mandadi
- Subjects
Periodicity ,Patch-Clamp Techniques ,TRPV1 ,TRPM Cation Channels ,TRPV Cation Channels ,In Vitro Techniques ,Motor Activity ,Inhibitory postsynaptic potential ,Sensory receptor ,Article ,Mice ,Transient receptor potential channel ,Locomotor rhythm ,TRPM8 ,Animals ,Mice, Knockout ,Motor Neurons ,Afferent Pathways ,Chemistry ,General Neuroscience ,Central pattern generator ,Hindlimb ,Cold Temperature ,Menthol ,Animals, Newborn ,Spinal Cord ,Sensory System Agents ,Excitatory postsynaptic potential ,Capsaicin ,Neuroscience - Abstract
It is well recognized that proprioceptive afferent inputs can control the timing and pattern of locomotion. C and Adelta afferents can also affect locomotion but an unresolved issue is the identity of the subsets of these afferents that encode defined modalities. Over the last decade, the transient receptor potential (TRP) ion channels have emerged as a family of non-selective cation conductances that can label specific subsets of afferents. We focus on a class of TRPs known as ThermoTRPs which are well known to be sensor receptors that transduce changes in heat and cold. ThermoTRPs are known to help encode somatosensation and painful stimuli, and receptors have been found on C and Adelta afferents with central projections onto dorsal horn laminae. Here we show, using in vitro neonatal mouse spinal cord preparations, that activation of both spinal and peripheral transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential melastatin 8 (TRPM8) afferent terminals modulates central pattern generators (CPGs). Capsaicin or menthol and cooling modulated both sacrocaudal afferent (SCA) evoked and monoaminergic drug-induced rhythmic locomotor-like activity in spinal cords from wild type but not TRPV1-null (trpv1(-/-)) or TRPM8-null (trpm8(-/-)) mice, respectively. Capsaicin induced an initial increase in excitability of the lumbar motor networks, while menthol or cooling caused a decrease in excitability. Capsaicin and menthol actions on CPGs involved excitatory and inhibitory glutamatergic mechanisms, respectively. These results for the first time show that dedicated pathways of somatosensation and pain identified by TRPV1 or TRPM8 can target spinal locomotor CPGs.
- Published
- 2009
13. TRPV3 in keratinocytes transmits temperature information to sensory neurons via ATP
- Author
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Tomoko Fukumi-Tominaga, Aziz Moqrich, Takaaki Sokabe, Ardem Patapoutian, Sravan Mandadi, Koji Shibasaki, Atsuko Mizuno, Makoto Tominaga, Kimiaki Katanosaka, and Kazue Mizumura
- Subjects
Keratinocytes ,Male ,TRPV4 ,Serotonin ,TRPV3 ,Hot Temperature ,Patch-Clamp Techniques ,Sensory Receptor Cells ,Physiology ,Clinical Biochemistry ,Thermosensation ,Glutamic Acid ,TRPV Cation Channels ,Biology ,Mice ,Transient receptor potential channel ,chemistry.chemical_compound ,Adenosine Triphosphate ,Dorsal root ganglion ,Ganglia, Spinal ,Physiology (medical) ,TRP channel ,medicine ,Animals ,Humans ,PPADS ,Patch clamp ,Cells, Cultured ,Skin ,Coculture Techniques ,Cell biology ,ATP ,Mice, Inbred C57BL ,medicine.anatomical_structure ,nervous system ,Biochemistry ,chemistry ,Calcium ,Signal transduction ,Adenosine triphosphate ,Ion Channels, Receptors and Transporters ,Signal Transduction - Abstract
Transient receptor potential V3 (TRPV3) and TRPV4 are heat-activated cation channels expressed in keratinocytes. It has been proposed that heat-activation of TRPV3 and/or TRPV4 in the skin may release diffusible molecules which would then activate termini of neighboring dorsal root ganglion (DRG) neurons. Here we show that adenosine triphosphate (ATP) is such a candidate molecule released from keratinocytes upon heating in the co-culture systems. Using TRPV1-deficient DRG neurons, we found that increase in cytosolic Ca2+-concentration in DRG neurons upon heating was observed only when neurons were co-cultured with keratinocytes, and this increase was blocked by P2 purinoreceptor antagonists, PPADS and suramin. In a co-culture of keratinocytes with HEK293 cells (transfected with P2X2 cDNA to serve as a bio-sensor), we observed that heat-activated keratinocytes secretes ATP, and that ATP release is compromised in keratinocytes from TRPV3-deficient mice. This study provides evidence that ATP is a messenger molecule for mainly TRPV3-mediated thermotransduction in skin. Electronic supplementary material The online version of this article (doi:10.1007/s00424-009-0703-x) contains supplementary material, which is available to authorized users.
- Published
- 2009
14. ThermoTRP Channels in Nociceptors: Taking a Lead from Capsaicin Receptor TRPV1
- Author
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Sravan Mandadi and Basil D. Roufogalis
- Subjects
TRPV4 ,TRPV3 ,TRPA ,TRPV1 ,TRPV ,Article ,Transient receptor potential channel ,ThermoTRP ,nociceptor ,TRPM8 ,Medicine ,Pharmacology (medical) ,pain ,Pharmacology ,business.industry ,phosphorylation ,Chronic pain ,General Medicine ,medicine.disease ,analgesia ,Psychiatry and Mental health ,Neurology ,nervous system ,Nociceptor ,Neurology (clinical) ,Transient receptor potential (TRP) ,business ,Neuroscience ,TRPM - Abstract
Nociceptors with peripheral and central projections express temperature sensitive transient receptor potential (TRP) ion channels, also called thermoTRP's. Chemosensitivity of thermoTRP's to certain natural compounds eliciting pain or exhibiting thermal properties has proven to be a good tool in characterizing these receptors. Capsaicin, a pungent chemical in hot peppers, has assisted in the cloning of the first thermoTRP, TRPV1. This discovery initiated the search for other receptors encoding the response to a wide range of temperatures encountered by the body. Of these, TRPV1 and TRPV2 encode unique modalities of thermal pain when exposed to noxious heat. The ability of TRPA1 to encode noxious cold is presently being debated. The role of TRPV1 in peripheral inflammatory pain and central sensitization during chronic pain is well known. In addition to endogenous agonists, a wide variety of chemical agonists and antagonists have been discovered to activate and inhibit TRPV1. Efforts are underway to determine conditions under which agonist-mediated desensitization of TRPV1 or inhibition by antagonists can produce analgesia. Also, identification of specific second messenger molecules that regulate phosphorylation of TRPV1 has been the focus of intense research, to exploit a broader approach to pain treatment. The search for a role of TRPV2 in pain remains dormant due to the lack of suitable experimental models. However, progress into TRPA1's role in pain has received much attention recently. Another thermoTRP, TRPM8, encoding for the cool sensation and also expressed in nociceptors, has recently been shown to reduce pain via a central mechanism, thus opening a novel strategy for achieving analgesia. The role of other thermoTRP's (TRPV3 and TRPV4) encoding for detection of warm temperatures and expressed in nociceptors cannot be excluded. This review will discuss current knowledge on the role of nociceptor thermoTRPs in pain and therapy and describes the activator and inhibitor molecules known to interact with them and modulate their activity.
- Published
- 2008
15. Increased sensitivity of desensitized TRPV1 by PMA occurs through PKCε-mediated phosphorylation at S800
- Author
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Namie Murayama, Basil D. Roufogalis, Tomoko Tominaga, Mitsuko Numazaki, Patricia J. Armati, Sravan Mandadi, Makoto Tominaga, and Naoaki Saito
- Subjects
medicine.medical_specialty ,Patch-Clamp Techniques ,Recombinant Fusion Proteins ,Molecular Sequence Data ,TRPV1 ,TRPV Cation Channels ,CHO Cells ,Protein Kinase C-epsilon ,Biology ,Kidney ,Transfection ,Cell Line ,Mice ,Phosphoserine ,Cricetulus ,Dorsal root ganglion ,Antibody Specificity ,Cricetinae ,Ganglia, Spinal ,Internal medicine ,Ca2+/calmodulin-dependent protein kinase ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Neurons, Afferent ,Patch clamp ,Phosphorylation ,Protein kinase C ,Kinase ,musculoskeletal, neural, and ocular physiology ,Peptide Fragments ,Cell biology ,Anesthesiology and Pain Medicine ,medicine.anatomical_structure ,Endocrinology ,Amino Acid Substitution ,nervous system ,Neurology ,Tetradecanoylphorbol Acetate ,Calcium ,lipids (amino acids, peptides, and proteins) ,Rabbits ,Neurology (clinical) ,Neuron ,Capsaicin ,Protein Processing, Post-Translational ,HeLa Cells - Abstract
Important mechanisms that regulate inhibitory and facilitatory effects on TRPV1-mediated nociception are desensitization and phosphorylation, respectively. Using Ca2+-imaging, we have previously shown that desensitization of TRPV1 upon successive capsaicin applications was reversed by protein kinase C activation in dorsal root ganglion neurons and CHO cells. Here, using both Ca2+-imaging and patch-clamp methods, we show that PMA-induced activation of PKCepsilon is essential for increased sensitivity of desensitized TRPV1. TRPV1 has two putative substrates S502 and S800 for PKCepsilon-mediated phosphorylation. Patch-clamp analysis showed that contribution of single mutant S502A or S800A towards increased sensitivity of desensitized TRPV1 is indistinguishable from that observed in a double mutant S502A/S800A. Since S502 is a non-specific substrate for TRPV1 phosphorylation by kinases like PKC, PKA or CAMKII, evidence for a role of PKC specific substrate S800 was investigated. Evidence for in vivo phosphorylation of TRPV1 at S800 was demonstrated for the first time. We also show that the expression level of PKCepsilon paralleled the amount of phosphorylated TRPV1 protein using an antibody specific for phosphorylated TRPV1 at S800. Furthermore, the anti-phosphoTRPV1 antibody detected phosphorylation of TRPV1 in mouse and rat DRG neurons and may be useful for research regarding nociception in native tissues. This study, therefore, identifies PKCepsilon and S800 as important therapeutic targets that may help regulate inhibitory effects on TRPV1 and hence its desensitization.
- Published
- 2006
16. Human and mouse mast cells use the tetraspanin CD9 as an alternate interleukin-16 receptor
- Author
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Jing Wang, Kenneth Lai, Basil D. Roufogalis, Feng Yan, Sravan Mandadi, Richard L. Stevens, Kumiko Tanaka, Steven A. Krilis, Beng H. Chong, Jian C. Qi, and Michele C. Madigan
- Subjects
medicine.medical_treatment ,Immunology ,Bone Marrow Cells ,Biology ,Biochemistry ,Tetraspanin 29 ,Wortmannin ,Mice ,Phosphatidylinositol 3-Kinases ,chemistry.chemical_compound ,Tetraspanin ,Antigens, CD ,medicine ,Animals ,Humans ,Calcium Signaling ,Mast Cells ,Cells, Cultured ,Immunobiology ,Interleukin-16 ,Membrane Glycoproteins ,Chemotaxis ,Cell Biology ,Hematology ,Fetal Blood ,medicine.disease ,Mast cell ,Molecular biology ,Cell biology ,Cytokine ,medicine.anatomical_structure ,chemistry ,Cell culture ,embryonic structures ,Mast cell sarcoma ,Signal transduction ,Signal Transduction - Abstract
Interleukin-16 (IL-16) induces the chemotaxis and activation of mast cells (MCs) and other cell types. While it has been concluded that CD4 is the primary IL-16 receptor on T cells, at least one other IL-16 receptor exists. We now show that the IL-16–responsive human MC line HMC-1 lacks CD4, and that the IL-16–mediated chemotactic and Ca2+ mobilization responses of this cell can be blocked by anti-CD9 monoclonal antibodies (mAbs) but not by mAbs directed against CD4 or other tetraspanins. Anti-CD9 mAbs also inhibited the IL-16–mediated activation of nontransformed human cord blood–derived MCs and mouse bone marrow–derived MCs by 50% to 60%. The chemotactic response of HMC-1 cells to IL-16, as well as the binding of the cytokine to the cell's plasma membrane, was inhibited by CD9-specific antisense oligonucleotides. CD9 is therefore essential for the IL-16–mediated chemotaxis and activation of the HMC-1 cell line. In support of this conclusion, IL-16 bound to CD9-expressing CHO cell transfectants. The ability of wortmannin and xestopongin C to inhibit the IL-16–mediated chemotactic response of these cells suggests that the cytokine activates a phosphatidylinositol 3-kinase (PI3K)/inositol trisphosphate–dependent signaling pathway in MCs. This is the first report of a tetraspanin that plays a prominent role in a cytokine-mediated chemotactic response of human MCs.
- Published
- 2005
17. Activation of protein kinase C reverses capsaicin-induced calcium-dependent desensitization of TRPV1 ion channels
- Author
-
Sravan Mandadi, Basil D. Roufogalis, Patricia J. Armati, Makoto Tominaga, Mitsuko Numazaki, and Manjunatha B. Bhat
- Subjects
medicine.medical_specialty ,Physiology ,Receptors, Drug ,TRPV1 ,TRPV Cation Channels ,chemistry.chemical_element ,Calcium ,Tachyphylaxis ,chemistry.chemical_compound ,Dorsal root ganglion ,Cricetinae ,Ganglia, Spinal ,Internal medicine ,medicine ,Animals ,Calcium Signaling ,Enzyme Inhibitors ,Molecular Biology ,Cells, Cultured ,Protein Kinase C ,Protein kinase C ,Ion channel ,Neurons ,Cell Biology ,Recombinant Proteins ,Rats ,Enzyme Activation ,Endocrinology ,medicine.anatomical_structure ,Amino Acid Substitution ,nervous system ,chemistry ,Capsaicin ,Biophysics ,Tetradecanoylphorbol Acetate ,lipids (amino acids, peptides, and proteins) ,Peptides ,Capsazepine - Abstract
Ca2+ selective ion channels of vanilloid receptor subtype-1 (TRPV1) in capsaicin-sensitive dorsal root ganglion (DRG) neurons and TRPV1 transfected Chinese hamster ovarian (CHO) cells are desensitized following calcium-dependent tachyphylaxis induced by successive applications of 100 nM capsaicin. Tachyphylaxis of TRPV1 to 100 nM capsaicin stimuli was not observed in the absence of extracellular calcium. Capsaicin sensitivity of desensitized TRPV1 ion channels recovered on application of phorbol-12-myristate-13-acetate (PMA). PMA-induced recovery of desensitized TRPV1 was primarily due to influx of extracellular calcium observed during re-application of capsaicin following desensitization. Capsazepine blocked the re-sensitization to capsaicin by PMA. Protein kinase C (PKC) inhibitory peptide PKC fragment 19-36 also inhibited re-sensitization to capsaicin by PMA. Reversal of capsaicin-induced desensitization by PMA was prevented by a mutation of TRPV1 where phosphorylation sites serine502 and serine800 were replaced with alanine. This study provides evidence for a role of PKC in reversing capsaicin-induced calcium-dependent desensitization of TRPV1 ion channels.
- Published
- 2004
18. Gingerols: a novel class of vanilloid receptor (VR1) agonists
- Author
-
MacDonald J. Christie, Vadim N. Dedov, Van Hoan Tran, Colin C. Duke, Basil D. Roufogalis, Mark Connor, and Sravan Mandadi
- Subjects
Pharmacology ,Agonist ,medicine.drug_class ,Chemistry ,Gingerol ,Antagonist ,Biological activity ,Calcium in biology ,chemistry.chemical_compound ,Mechanism of action ,Biochemistry ,medicine ,medicine.symptom ,Receptor ,Capsazepine - Abstract
1. Gingerols, the pungent constituents of ginger, were synthesized and assessed as agonists of the capsaicin-activated VR1 (vanilloid) receptor. 2. [6]-Gingerol and [8]-gingerol evoked capsaicin-like intracellular Ca(2+) transients and ion currents in cultured DRG neurones. These effects of gingerols were blocked by capsazepine, the VR1 receptor antagonist. 3. The potency of gingerols increased with increasing size of the side chain and with the overall hydrophobicity in the series. 4. We conclude that gingerols represent a novel class of naturally occurring VR1 receptor agonists that may contribute to the medicinal properties of ginger, which have been known for centuries. The gingerol structure may be used as a template for the development of drugs acting as moderately potent activators of the VR1 receptor.
- Published
- 2002
19. Astrocytes express functional TRPV2 ion channels
- Author
-
Sravan Mandadi, Koji Shibasaki, and Yasuki Ishizaki
- Subjects
Hot Temperature ,Central nervous system ,TRPV2 ,Biophysics ,TRPV Cation Channels ,Biology ,Biochemistry ,chemistry.chemical_compound ,Transient receptor potential channel ,Mice ,Cerebellum ,medicine ,Animals ,Humans ,Molecular Biology ,Ion channel ,Lysophosphatidylcholines ,Lipid metabolism ,Cell Biology ,Ligand (biochemistry) ,Cell biology ,Mice, Inbred C57BL ,medicine.anatomical_structure ,Lysophosphatidylcholine ,chemistry ,Astrocytes ,Calcium ,Calcium Channels ,Astrocyte - Abstract
Thermosensitive transient receptor potential (thermo TRP) channels are important for sensory transduction. Among them, TRPV2 has an interesting characteristic of being activated by very high temperature (>52 °C). In addition to the heat sensor function, TRPV2 also acts as a mechanosensor, an osomosensor and a lipid sensor. It has been reported that TRPV2 is expressed in heart, intestine, pancreas and sensory nerves. In the central nervous system, neuronal TRPV2 expression was reported, however, glial expression and the precise roles of TRPV2 have not been determined. To explore the functional expression of TRPV2 in astrocytes, the expression was determined by histological and physiological methods. Interestingly, TRPV2 expression was detected in plasma membrane of astrocytes, and the astrocytic TRPV2 was activated by very high temperature (>50 °C) consistent with the reported characteristic. We revealed that the astrocytic TRPV2 was also activated by lysophosphatidylcholine, a known endogenous lipid ligand for TRPV2, suggesting that astrocytic TRPV2 might regulate neuronal activities in response to lipid metabolism. Thus, for the first time we revealed that TRPV2 is functionally expressed in astrocytes in addition to neurons.
- Published
- 2013
20. Control of Neonatal Spinal Networks by Nociceptors: A Potential Role for TRP Channel Based Therapies
- Author
-
Peter Hong, Sravan Mandadi, Patrick J. Whelan, and Arjun Sunny Dhoopar
- Subjects
medicine.medical_specialty ,Pharmaceutical Science ,lcsh:RS1-441 ,lcsh:Pharmacy and materia medica ,Transient receptor potential channel ,Transient Receptor Potential Channels ,Animals ,Humans ,Medicine ,Spasticity ,Spinal cord injury ,Spinal Cord Injuries ,Pharmacology ,business.industry ,lcsh:RM1-950 ,Chronic pain ,Nociceptors ,medicine.disease ,Spinal cord ,medicine.anatomical_structure ,Nociception ,lcsh:Therapeutics. Pharmacology ,Spinal Cord ,Neuropathic pain ,Nociceptor ,Physical therapy ,Neuralgia ,medicine.symptom ,business ,Neuroscience - Abstract
Pediatric spinal cord injury (SCI) often leads to increased nociceptive input resulting in aberrant motor output like tremor and spasticity. Acute plasticity within spinal pain and motor networks following pediatric SCI may result in long-term sensorimotor disabilities. Despite this, pediatric SCI remains poorly understood. Part of the problem lies in the paucity of detailed studies aimed at defining sensorimotor control by nociceptors during development. This review provides an overview of work that highlights afferent control of sensorimotor networks by defined nociceptors in the developing spinal cord. Here, we focus on the well established and widely used neonatal sensorimotor model called sacrocaudal afferent (SCA) pathway. Until recently, the identity of specific subclasses of nociceptive afferents in the SCA pathway controlling developing sensorimotor networks was unknown. We highlight here the use of members of the Transient Receptor Potential (TRP) ion channels and mouse genetics to identify specific subsets of nociceptive afferents in the SCA pathway. In addition, we highlight the use of mouse genetics to map sensorimotor networks during development and potential future applications. A neonatal spinal cord model of central neuropathic pain via a defined set of nociceptors is presented as a probe into potential therapeutic avenues in neonatal SCI. Finally, knowledge translation from neonatal basic research to the pediatric population in the clinic is described. In conclusion, studies in neonatal models may lead to therapeutic strategies and pharmaceuticals for chronic pain and motor dysfunction after SCI during development. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.
- Published
- 2013
21. Identification of multisegmental nociceptive afferents that modulate locomotor circuits in the neonatal mouse spinal cord
- Author
-
Sravan, Mandadi, Peter, Hong, Michelle A, Tran, Joao M, Bráz, Pina, Colarusso, Allan I, Basbaum, and Patrick J, Whelan
- Subjects
Nociception ,Afferent Pathways ,Periodicity ,Sensory Receptor Cells ,Green Fluorescent Proteins ,TRPV Cation Channels ,Mice, Transgenic ,In Vitro Techniques ,Mice ,Animals, Newborn ,Spinal Cord ,Neurofilament Proteins ,Physical Stimulation ,Sensory System Agents ,Animals ,Calcium ,Capsaicin ,Evoked Potentials ,Locomotion - Abstract
Compared to proprioceptive afferent collateral projections, less is known about the anatomical, neurochemical, and functional basis of nociceptive collateral projections modulating lumbar central pattern generators (CPG). Quick response times are critical to ensure rapid escape from aversive stimuli. Furthermore, sensitization of nociceptive afferent pathways can contribute to a pathological activation of motor circuits. We investigated the extent and role of collaterals of capsaicin-sensitive nociceptive sacrocaudal afferent (nSCA) nerves that directly ascend several spinal segments in Lissauer's tract and the dorsal column and regulate motor activity. Anterograde tracing demonstrated direct multisegmental projections of the sacral dorsal root 4 (S4) afferent collaterals in Lissauer's tract and in the dorsal column. Subsets of the traced S4 afferent collaterals expressed transient receptor potential vanilloid 1 (TRPV1), which transduces a nociceptive response to capsaicin. Electrophysiological data revealed that S4 dorsal root stimulation could evoke regular rhythmic bursting activity, and our data suggested that capsaicin-sensitive collaterals contribute to CPG activation across multiple segments. Capsaicin's effect on S4-evoked locomotor activity was potent until the lumbar 5 (L5) segments, and diminished in rostral segments. Using calcium imaging we found elevated calcium transients within Lissauer's tract and dorsal column at L5 segments when compared to the calcium transients only within the dorsal column at the lumbar 2 (L2) segments, which were desensitized by capsaicin. We conclude that lumbar locomotor networks in the neonatal mouse spinal cord are targets for modulation by direct multisegmental nSCA, subsets of which express TRPV1 in Lissauer's tract and the dorsal column. J. Comp. Neurol. 521:2870-2887, 2013. © 2013 Wiley Periodicals, Inc.
- Published
- 2012
22. Rodent Isolated Spinal Cord Preparations to Examine Motor Output
- Author
-
Patrick J. Whelan, Sravan Mandadi, Stan T. Nakanishi, Pengcheng Han, and Jennifer M. Humphreys
- Subjects
medicine.anatomical_structure ,Rodent ,biology ,business.industry ,biology.animal ,Medicine ,Anatomy ,business ,Spinal cord - Published
- 2012
23. A new method to study sensory modulation of locomotor networks by activation of thermosensitive cutaneous afferents using a hindlimb attached spinal cord preparation
- Author
-
Patrick J. Whelan and Sravan Mandadi
- Subjects
Serotonin ,Hot Temperature ,N-Methylaspartate ,Cauda Equina ,Dopamine ,Sensation ,TRPV Cation Channels ,Sensory system ,Hindlimb ,Biology ,Mice ,Transient Receptor Potential Channels ,medicine ,Excitatory Amino Acid Agonists ,Animals ,Neurons, Afferent ,Receptor ,Skin ,Sensory modulation ,General Neuroscience ,Compartment (ship) ,Anatomy ,Thermoreceptors ,Spinal cord ,Stimulation, Chemical ,Cold Temperature ,Electrophysiology ,medicine.anatomical_structure ,Spinal Cord ,Nociceptor ,Capsaicin ,Nerve Net ,Artificial cerebrospinal fluid ,Neuroscience ,Locomotion - Abstract
The use of isolated in vitro spinal cord preparations to examine the underlying networks that control locomotion has become popular. It is also well known that afferent feedback can excite and modulate these networks. However, it is often difficult to selectively activate classes of afferents that subserve specific modalities using in vitro preparations. Here, we describe a technique where afferent receptors that detect temperature were selectively activated. To accomplish this we used an in vitro preparation of the mouse where the spinal cord was isolated (T5-cauda equina) with one hind limb left attached. We designed a special chamber allowing the hind paw to be placed in such a way that it remained attached to the spinal cord but received a separate supply of artificial cerebrospinal fluid (aCSF). This allowed us to alter the temperature of the hind limb compartment without affecting the temperature of the central compartment containing the spinal cord. We also demonstrate using this approach that agonists which activate receptors which detect noxious heat could be intradermally injected into the hind limb without it diffusing into the central compartment.
- Published
- 2009
24. Gingerols: a novel class of vanilloid receptor (VR1) agonists
- Author
-
Vadim N, Dedov, Van H, Tran, Colin C, Duke, Mark, Connor, MacDonald J, Christie, Sravan, Mandadi, and Basil D, Roufogalis
- Subjects
Neurons ,Time Factors ,Dose-Response Relationship, Drug ,Receptors, Drug ,Catechols ,Membrane Potentials ,Rats ,Animals, Newborn ,Ganglia, Spinal ,Papers ,Animals ,Calcium ,Capsaicin ,Fatty Alcohols ,Fura-2 ,Cells, Cultured - Abstract
1. Gingerols, the pungent constituents of ginger, were synthesized and assessed as agonists of the capsaicin-activated VR1 (vanilloid) receptor. 2. [6]-Gingerol and [8]-gingerol evoked capsaicin-like intracellular Ca(2+) transients and ion currents in cultured DRG neurones. These effects of gingerols were blocked by capsazepine, the VR1 receptor antagonist. 3. The potency of gingerols increased with increasing size of the side chain and with the overall hydrophobicity in the series. 4. We conclude that gingerols represent a novel class of naturally occurring VR1 receptor agonists that may contribute to the medicinal properties of ginger, which have been known for centuries. The gingerol structure may be used as a template for the development of drugs acting as moderately potent activators of the VR1 receptor.
- Published
- 2002
25. Capsaicin-induced depolarisation of mitochondria in dorsal root ganglion neurons is enhanced by vanilloid receptors
- Author
-
Vadim N. Dedov, Sravan Mandadi, Alexej Verkhratsky, and Patricia J. Armati
- Subjects
Oligomycin ,Receptors, Drug ,TRPV1 ,Mitochondrion ,Biology ,Antioxidants ,Membrane Potentials ,chemistry.chemical_compound ,Dorsal root ganglion ,Ganglia, Spinal ,medicine ,Humans ,Lymphocytes ,Cells, Cultured ,Membrane potential ,Neurons ,Microscopy, Confocal ,General Neuroscience ,Neurotoxicity ,medicine.disease ,Flow Cytometry ,Cell biology ,Mitochondria ,Mitochondrial respiratory chain ,medicine.anatomical_structure ,nervous system ,chemistry ,Capsaicin ,Anesthesia ,lipids (amino acids, peptides, and proteins) ,Calcium - Abstract
Capsaicin, a pungent ingredient of hot chilli peppers, triggered Ca(2+) influx in dorsal root ganglion (DRG) neurons, which express specific vanilloid receptors of type 1, with ED(50)100 nM. An increase in capsaicin concentration to 10 microM inhibited Ca(2+) clearance from the cytosol, but did not affect the amplitude of intracellular Ca(2+) elevation. In DRG neurons, 10 microM capsaicin also produced a significant drop in mitochondrial membrane potential (Deltapsi), as measured with the mitochondria-specific potentiometric fluorescent dye JC-1. Similar loss of mitochondrial potential upon application of capsaicin was observed in non-neuronal primary (human lymphocytes) and transformed (human myeloid leukaemia cell line, HL-60) cells. The EC(50) values for capsaicin-induced mitochondrial depolarisation were 6.9 microM (DRG neurons), 200 microM (human lymphocytes) and 150 microM (HL-60 cells). Removal of extracellular Ca(2+) or an application of the antioxidant trolox attenuated capsaicin-induced dissipation of Deltapsi in DRG neurons, but not in human lymphocytes and HL-60 cells. Rotenone, an inhibitor of complex I of the mitochondrial respiratory chain, and oligomycin, an inhibitor of F(0)F(1)-ATPase, significantly enhanced the mitochondrial depolarisation produced by capsaicin in DRG neurons. In human lymphocytes and HL-60 cells, only oligomycin potentiated the effect of capsaicin. From our results, we suggest that, in DRG neurons and non-neuronal cells, capsaicin dissipates Deltapsi, possibly due to a direct inhibition of complex I of the mitochondrial respiratory chain. The presence of vanilloid receptor-1 in DRG neurons makes their mitochondria 20-30-fold more sensitive to the depolarising effect of capsaicin compared with non-neuronal cells lacking vanilloid receptor-1. The higher sensitivity of DRG neurons to capsaicin may underlie a selective neurotoxicity of capsaicin towards sensory neurons.
- Published
- 2001
26. Protein kinase C modulation of thermo-sensitive transient receptor potential channels: Implications for pain signaling
- Author
-
Basil D. Roufogalis, Sravan Mandadi, and Patricia J. Armati
- Subjects
medicine.medical_specialty ,business.industry ,TRPV1 ,Chronic pain ,Review Article ,General Medicine ,medicine.disease ,General Biochemistry, Genetics and Molecular Biology ,Transient receptor potential channel ,GPCR ,Nociception ,Endocrinology ,medicine.anatomical_structure ,Dorsal root ganglion ,transient receptor potential channels ,Internal medicine ,TRPM8 ,Medicine ,Analgesia ,Anti-inflammatory ,business ,Neuroscience ,Protein kinase C ,protein kinase C ,G protein-coupled receptor - Abstract
A variety of molecules are reported to be involved in chronic pain. This review outlines the specifics of protein kinase C (PKC), its isoforms and their role in modulating thermo-sensitive transient receptor potential (TRP) channels TRPV1-4, TRPM8, and TRPA1. Anatomically, PKC and thermo-sensitive TRPs are co-expressed in cell bodies of nociceptive dorsal root ganglion (DRG) neurons, which are used as physiological correlates of peripheral and central projections involved in pain transmission. In the past decade, modulation of painful heat-sensitive TRPV1 by PKC has received the most attention. Recently, PKC modulation of other newly discovered thermo-sensitive pain-mediating TRPs has come into focus. Such modulation may occur under conditions of chronic pain resulting from nerve damage or inflammation. Since thermo-TRPs are primary detectors of acute pain stimuli, their modulation by PKC can severely alter their function, resulting in chronic pain. Comprehensive knowledge of pain signaling involving interaction of specific isoforms of PKC with specific thermo-sensitive TRP channels is incomplete. Such information is necessary to dissect out modality specific mechanisms to better manage the complex polymodal nature of chronic pain. This review is an attempt to update the readers on current knowledge of PKC modulation of thermo-sensitive TRPs and highlight implications of such modulation for pain signaling.
- Published
- 2011
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