Mythily Srinivasan

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https://hdl.handle.net/1805/17678

Targeting Neuroinflammation Using Novel Glucorticoid-Induced Leucine Zipper Analogs

Unresolved inflammation and altered immune signals contribute to the pathology of many chronic diseases. Dr. Srinivasan's research includes the design and development of immunotherapeutic peptides using motif-mediated protein-protein interactions as drug targets.

Recently, she investigated approaches to suppress neuroinflammation in Alzheimer's disease, the most common cause of dementia that is projected to affect over seven million individuals in the United States by 2025. These studies resulted from earlier observations that the blockade of T cell co-receptor CD28 by CD80 competitive antagonist peptides unregulated glucocorticoid-induced leucine zipper (GILZ)-an anti-inflammatory molecule that directly inhibits the pro-inflammatory transcription factor, nuclear factor-kappa B (NF-kB).

Adopting data from the GILZ and NF-kB interaction complex, Dr. Srinivasan developed modified analogs of the p65 binding motif of GIlZ. Efficacy studies, in collaboration with Dr. Debomoy Lahiri, Professor of Neuroscience, and Dr. Deborah Hickman of the IU School of Medicine, suggested that the novel therapeutics exhibited suppressive potential in cellular and animal models of Alzheimer's disease.

Together with the Indiana University Research and Technology Corporation, Dr. Srinivasan initiated a startup venture, Provaidya LLC, to develop the patented GILZ analogs as investigational new drugs and received phase I funding from the National Institutes of Aging.

Dr. Srinivasan's work to develop peptide therapeutics to suppress neuroinflammation is another example of how IUPUI faculty are TRANSLATING RESEARCH INTO PRACTICE.

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Preclinical characterization of drug like glucocorticoid induced leucine zipper peptide analogs
(Office of the Vice Chancellor for Research, 2014-04-11) Srinivasan, Mythily; Blackburn, C.; Lahiri, Debomoy K.
Many intermolecular interactions in a eukaryotic cell are mediated through protein-peptide interactions. For efficient interaction, the peptide scans the protein surface for a large enough pocket into which it anchors through a small number of residues/core motif that contribute maximally to the free energy of binding. Of special significance is the preponderance of proline rich sequences that preferentially adopt the left-handed polyproline type II (PPII) helical conformation in the interface peptides. Availability of both side chain and backbone carbonyls for interaction makes PPII helix an excellent recognition motif. Glucocorticoid induced leucine zipper (GILZ), is a glucocorticoid responsive protein that has been shown to suppress immuno-inflammatory responses by preventing the nuclear translocation of the p65 subunit of the transcription factor nuclear factor-kappa B (NF-κB). Mutational and binding studies localized the sites of interaction to the proline rich region at the carboxy terminus of GILZ and the transactivation domain of p65. Similar to most intermolecular interactions mediated by proline rich motifs the strength of interaction between the GILZ and the p65 proteins is in the micromolar concentration suggesting weak binding kinetics. A widely used strategy in the discovery of peptide drugs involves exploitation of the complementary surfaces of the naturally occurring binding partners. We observed that a synthetic peptide (GILZ-P) derived from the proline rich region of GILZ suppressed immune mediated inflammatory responses in mice. Here we characterize GILZ-P structurally and evaluate its toxicity and efficacy in mature human macrophage like THP-1 cells. We show that the GILZ-P adopts an extended polyproline type II helical conformation. Functionally GILZ-P is non-toxic, suppresses NF B activation on by activated macrophages suggesting a therapeutic potential in pathologies wherein persistent inflammation plays critical role in the disease initiation and/or progression.
Modulating NK-mediated Immunity by Lunakine
(Office of the Vice Chancellor for Research, 2014-04-11) Chang, Hua-Chen; Tung, Chun-Yu; Lewis, David; Han, Ling; Srinivasan, Mythily; Robertson, Michael J.; Yeh, Wu-Kuang
Despite the plethora of immune modulating agents available in cancer treatment, their effectiveness relies on a functional immune system. However, the adverse side effects by chemotherapy impede the therapeutic benefits from immunotherapy. It remains a major challenge to prevent relapse for cancer patients who have already undergone rigorous chemotherapy. Lunasin, a 43-amino acid peptide, was originally isolated from soybeans. Our team has recently discovered a novel function of lunasin as an immune modulating agent that exerts robust synergistic effects imposed by several therapeutic cytokines. Such synergism strongly augments IFNγ and granzyme B expression by Natural Killer (NK) cells, which is associated with increased tumoricidal activity. The combination regimen with lunasin and cytokine is capable of restoring NK activation from lymphoma patients with chemotherapy-induced immune dysfunction. Our results support the potential application of lunasin to improve the therapeutic effects of existing cytokine treatment that has been used to eliminate residual tumors cells from lymphoma patients after chemotherapy. We designate lunakine as new formulation by combing lunasin and selected cytokine (filed for US Patent Cooperation Treat). In working with Indiana University and Technology Corporation (IURTC), we have started a startup company, Immune Peptide Therapeutics (IPT), LLC. Our mission is to develop a more efficacious immunotherapy that prevents relapse and confers progression-free survival for cancer patients. With the support from FORCES, our team has successfully developed a second generation of lunasin called IPT.103 that deviates from its parental type. Activity of IPT.103 has been tested in vitro with EC50 of 0.78 μM as compared to 4.54 μM for lunasin, indicating an improved potency to induce IFNγ production by NK cells. The newly developed peptide IPT.103 is expected to strengthen the intellectual property (IP) position for commercialization. We are currently working on tumor models for preclinical assessment of IPT’s regimens in immunotherapy for lymphoma.
Soluble toll like receptor 2 (TLR-2) is increased in saliva of children with dental caries
(2014-08) Zhao, Alyssa; Blackburn, Corinne; Chin, Judith; Srinivasan, Mythily
Background Dental caries is the most common microbial disease affecting mankind. Caries risk assessment methods, identification of biomarkers and vaccine development strategies are being emphasized to control the incidence of the largely preventable disease. Pattern recognition receptors such as the toll like receptors (TLR) have been implicated as modulators of host-microbial interactions. Soluble TLR-2 and its co-receptor, CD14 identified in saliva can bind the cell wall components of cariogenic bacteria and modulate the disease process. The objective of this study is to determine the potential of salivary sTLR-2 and sCD14 as biomarkers of caries activity and indirect measures of the cariogenic bacterial burden. Methods Unstimulated whole saliva was collected from twenty caries free and twenty caries active children between the ages of 5 and 13 years. The concentration of sCD14 and sTLR-2 together with that of the cytokine IL-8 reported to be increased in dental caries was assessed by the enzyme linked immunosorbent assay. Results While the level of sCD14 and that of IL-8 was equivocal between the two groups, the sTLR-2 concentration in caries active saliva was significantly higher than that in caries free saliva. Conclusions The sTLR-2 in saliva could serve as a potential biomarker for caries activity.
Functional characterization of a competitive peptide antagonist of p65 in human macrophage- like cells suggests therapeutic potential for chronic inflammation
(2014-12) Srinivasan, Mythily; Blackburn, Corinne; Lahiri, Debomoy
Glucocorticoid-induced leucine zipper (GILZ) is a glucocorticoid responsive protein that links the nuclear factor-kappa B (NFκB) and the glucocorticoid signaling pathways. Functional and binding studies suggest that the proline-rich region at the carboxy terminus of GILZ binds the p65 subunit of NFκB and suppresses the immunoinflammatory response. A widely-used strategy in the discovery of peptide drugs involves exploitation of the complementary surfaces of naturally occurring binding partners. Previously, we observed that a synthetic peptide (GILZ-P) derived from the proline-rich region of GILZ bound activated p65 and ameliorated experimental encephalomyelitis. Here we characterize the secondary structure of GILZ-P by circular dichroic analysis. GILZ-P adopts an extended polyproline type II helical conformation consistent with the structural conformation commonly observed in interfaces of transient intermolecular interactions. To determine the potential application of GILZ-P in humans, we evaluated the toxicity and efficacy of the peptide drug in mature human macrophage-like THP-1 cells. Treatment with GILZ-P at a wide range of concentrations commonly used for peptide drugs was nontoxic as determined by cell viability and apoptosis assays. Functionally, GILZ-P suppressed proliferation and glutamate secretion by activated macrophages by inhibiting nuclear translocation of p65. Collectively, our data suggest that the GILZ-P has therapeutic potential in chronic CNS diseases where persistent inflammation leads to neurodegeneration such as multiple sclerosis and Alzheimer’s disease.
Literature-Based Discovery of Salivary Biomarkers for Type 2 Diabetes Mellitus
(Libertas Academia, 2015-02) Srinivasan, Mythily; Blackburn, Corinne; Mohamed, Mohamed; Sivagami, A. V.; Blum, Janice; Department of Oral Pathology, Medicine and Radiology, Indiana University School of Dentistry
The alarming increase in type 2 diabetes mellitus (T2DM) underscores the need for efficient screening and preventive strategies. Select protein biomarker profiles emerge over time during T2DM development. Periodic evaluation of these markers will increase the predictive ability of diabetes risk scores. Noninvasive methods for frequent measurements of biomarkers are increasingly being investigated. Application of salivary diagnostics has gained importance with the establishment of significant similarities between the salivary and serum proteomes. The objective of this study is to identify T2DM-specific salivary biomarkers by literature-based discovery. A serial interrogation of the PubMed database was performed using MeSH terms of specific T2DM pathological processes in primary and secondary iterations to compile cohorts of T2DM-specific serum markers. Subsequent search consisted of mining for the identified serum markers in human saliva. More than 60% of T2DM-associated serum proteins have been measured in saliva. Nearly half of these proteins have been reported in diabetic saliva. Measurements of salivary lipids and oxidative stress markers that can exhibit correlated saliva plasma ratio could constitute reliable factors for T2DM risk assessment. We conclude that a high percentage of T2DM-associated serum proteins can be measured in saliva, which offers an attractive and economical strategy for T2DM screening.
GILZ-mimics as novel therapeutic agents for progressive multiple sclerosis
(Office of the Vice Chancellor for Research, 2013-04-05) Srinivasan, Mythily; Dunker, K. A.; Lahiri, Debomoy K.; Pollok, Karen E.
Multiple sclerosis (MS), a leading cause of neurological disability is an inflammatory demyelinating disease of the central nervous system (CNS). The clinical course of MS is highly variable ranging from isolated neurologic episodes to frequently relapsing or progressive disease. Currently there are no effective treatments for progressive MS. The long-term goal of this project is to evaluate a novel therapeutic strategy for progressive MS. Under physiological conditions signaling via the transcription factor, nuclear factor-kappa B (NF-κB) and glucocorticoid (GC) stimulation pathways regulate the immuno-inflammatory responses of the CNS resident glial cells. While NF-κB induces transcriptional activation, signaling via GC receptor functions to suppress immune responses. Persistent activation of NF-κB in the glial cells precipitates neuronal degeneration and axonal loss characteristic of progressive MS. Interactome analysis between the GC and NF-κB pathways suggested a novel strategy to inhibit NF-κB. Glucocorticoid-induced leucine zipper (GILZ) is a GC inducible protein that binds p65, the functionally critical subunit of NF-κB, and prevent transactivation of pathological mediators. The sites of interaction are localized to the proline rich region of the GILZ protein and the p65 transactivation domain. A 23 residue GILZ peptide prevented nuclear translocation of p65 and suppressed disease in an animal model of MS. Structurally GILZ peptide adopted polyproline type II (PPII) helical conformation, a favorable feature for drug development. The objective of this study is to optimize the lead peptide and develop drug like analogs. Specific features of the GILZ-p65 interactions were adapted in the design of over 25 GILZ analogs such that each exhibit optimum PPII helix, bind p65 transactivation domain and potentially accommodate modified residues that enhance the binding specificity with the p65. The analogs were ranked after passing through the Lipinski filter to determine the drug like properties. The top ranked analogs will be evaluated for functional efficacy.
Enhancement of Cancer Immunotherapy Using Immune Modulating Peptides
(Office of the Vice Chancellor for Research, 2013-04-05) Chang, Hua-Chen; Han, Ling; Lewis, David; Tung, Chun-Yu; Srinivasan, Mythily; Robertson, Michael J.; Yeh, Wu-Kuang
Immune Peptide Therapeutics (IPT) LLC, an Indiana-based small business and its research partner Indiana University previously identified a novel property of lunasin as a distinct class of immune modulating agent that enhances anti-tumor immunity, which may promote disease-free survival by limiting tumor progression, and thus prolong lives of cancer patients. Lunasin, a synthetic 43-amino acid peptide, was originally isolated from soybeans. Our studies have demonstrated that lunasin exerts robust synergistic effects with cytokines on augmenting IFNγ and granzyme B expression by Natural Killer (NK) cells, which is associated with increased tumoricidal activity of NK cells. In addition, this combination regimen is capable of rescuing IFNγ production ex vivo by NK cells from chemotherapy-treated Non-Hodgkin’s Lymphoma (NHL) patients who are immunocompromised with acquired immune deficiency. The long-term goal is to develop an efficacious immunotherapy which will impact the treatment and improve the clinical outcomes for NHL patients. The dose-response study indicates the optimum concentration of lunasin is at the range of μM, which would undermine its use in clinical studies. To enhance the medicinal value lunasin must be optimized for in vitro and in vivo efficacy. The objective is to develop a second generation of lunasin, which will increase its potency to improve the performance. In this study we have implemented several strategies to design and modify the prototype. The newly developed peptide called IPT.103 has 15 amino acids that are in the D-isoform configuration. Activity of IPT.103 has been tested in vitro with EC50 of 0.78 μM as compared to 4.54 μM for lunasin. IPT.103 also has in vivo activity on enhancing the serum levels of IFNγ production using a mouse model. Taken together, we have developed a peptide derivative (IPT.103) that deviates from its parental type lunasin to increase intellectual merit for commercialization as well as support clinical application.
Significance of NF-κΒ as a pivotal therapeutic target in the neurodegenerative pathologies of Alzheimer’s disease and multiple sclerosis
(Taylor and Francis, 2015-04) Srinivasan, Mythily; Lahiri, Debomoy K.; Department of Oral Pathology, Medicine and Radiology, Indiana University School of Dentistry
Introduction: Advances in molecular pathogenesis suggest that the chronic inflammation is a shared mechanism in the initiation and progression of multiple neurodegenerative diseases with diverse clinical manifestations such as Alzheimer’s disease (AD) and Multiple sclerosis (MS). Restricted cell renewal and regenerative capacity make the neural tissues extremely vulnerable to the uncontrolled inflammatory process leading to irreversible tissue damage. Areas covered: A predominant consequence of increased inflammatory signaling is the upregulation of the transcription factor, NF-κB with subsequent neuroprotective or deleterious effects depending on the strength of the signal and the type of NF-κB dimers activated. We discuss the interplay between neuroinflammation and neurodegeneration keeping in focus NF-κB signaling as the point of convergence of multiple pathways associated with the development of the neurodegenerative pathologies, AD and MS. Expert opinion: Considerable interest exists in developing efficient NF-κB inhibitors for neurodegenerative diseases. The review includes an overview of natural compounds and rationally designed agents that inhibit NF-κB and mediate neuroprotection in AD and MS. The key chemical moieties of the natural and the synthetic compounds provide efficient leads for the development of effective small molecule inhibitors that selectively target NF-κB activation; this would result in the desired benefit to risk therapeutic effects.
Distinct Salivary Biomarker Profile in Chronic Periodontitis
(Office of the Vice Chancellor for Research, 2012-04-13) Prakasam, S.; Srinivasan, Mythily
Background: Saliva has potential to diagnose chronic periodontitis (CP). Changes in tissue-expression of pattern-recognition-receptors (PRRs), which recognize periodontal-pathogens, correlate with CP. It follows that PRRs-expression in nucleated-cells (NCs) shed in saliva and soluble-PRRs may differentiate CP from health. Additionally, cytokines in gingival cervical fluid (GCF) correlate with worsening CP, which may be reflected in saliva. One significant test for biomarkers is changes in response to treatment. Objectives: Comparison of CP salivary-biomarkers profile with health and to study treatment effects of scaling and root planning (SRP). Methods: Unstimulated whole saliva (UWS) collection and recording of routine clinical periodontal parameters was done for two groups (n=16): healthy (H) (minimal clinical loss of attachment (CAL) and clinical inflammation) and CP (≥30% sites with ≥4mm CAL). UWS was collected at 3 different time points: before, 1-week and 6-weeks after SRP from the CP group. NCs and clarified saliva (CS) were separated from UWS. Messenger RNA was extracted from NCs and TLR-2 expression was quantitated through real-time-PCR. CS depleted of immunoglobulin and amylase to prevent large molecule interferences and diluted to 1 μg/ml of salivary-protein in PBS, normalize for variations in liquid volume, was used to quantify biomarkers through ELISA. Statistical significance between H- and CP-groups biomarkers was determined through Mann-Whitney ‘U' test and one tailed paired ‘t' test. Results: Statistically significant differences were noted for clinical profiles of H- and CP-groups and for changes after SRP within CP-group. Salivary sTLR-2, IL-17 and IL-10, were significantly higher, and sCD14, IL-6, IL-4 and TLR-2 mRNA were significantly lower in H compared to CP. In CP, salivary sTLR-2 and IL10 increased significantly at 1- and 6-weeks after SRP, whilst IL-4 decreased significantly at 6-weeks. Conclusions: Salivary biomarkers profiles are distinct between health and CP as well as before and after SRP treatment. sTLR-2, IL-10 and IL-4 may serve as short-term biomarkers for monitoring response to SRP. sCD14, TLR2-mRNA and other cytokines need exploration as long-term response biomarkers. Depletion of amylase and immunoglobulin, and normalization for total salivary protein may be important in biomarkers quantification.
Functional characterization of a competitive peptide antagonist of p65 in human macrophage-like cells suggests therapeutic potential for chronic inflammation
(Dove Medical Press, 2014) Srinivasan, Mythily; Blackburn, Corinne; Lahiri, Debomoy K.; Department of Oral Pathology, Medicine and Radiology, IU School of Dentistry
Glucocorticoid-induced leucine zipper (GILZ) is a glucocorticoid responsive protein that links the nuclear factor-kappa B (NFκB) and the glucocorticoid signaling pathways. Functional and binding studies suggest that the proline-rich region at the carboxy terminus of GILZ binds the p65 subunit of NFκB and suppresses the immunoinflammatory response. A widely-used strategy in the discovery of peptide drugs involves exploitation of the complementary surfaces of naturally occurring binding partners. Previously, we observed that a synthetic peptide (GILZ-P) derived from the proline-rich region of GILZ bound activated p65 and ameliorated experimental encephalomyelitis. Here we characterize the secondary structure of GILZ-P by circular dichroic analysis. GILZ-P adopts an extended polyproline type II helical conformation consistent with the structural conformation commonly observed in interfaces of transient intermolecular interactions. To determine the potential application of GILZ-P in humans, we evaluated the toxicity and efficacy of the peptide drug in mature human macrophage-like THP-1 cells. Treatment with GILZ-P at a wide range of concentrations commonly used for peptide drugs was nontoxic as determined by cell viability and apoptosis assays. Functionally, GILZ-P suppressed proliferation and glutamate secretion by activated macrophages by inhibiting nuclear translocation of p65. Collectively, our data suggest that the GILZ-P has therapeutic potential in chronic CNS diseases where persistent inflammation leads to neurodegeneration such as multiple sclerosis and Alzheimer's disease.

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