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Dive into the research topics where Ramakrishna Edupuganti is active.

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Featured researches published by Ramakrishna Edupuganti.


Bioorganic & Medicinal Chemistry | 2014

Synthesis and biological evaluation of pyrido[2,3-d]pyrimidine-2,4-dione derivatives as eEF-2K inhibitors

Ramakrishna Edupuganti; Qiantao Wang; Clint D. J. Tavares; Catrina A. Chitjian; James L. Bachman; Pengyu Ren; Eric V. Anslyn; Kevin N. Dalby

A small molecule library of pyrido[2,3-d]pyrimidine-2,4-dione derivatives 6-16 was synthesized from 6-amino-1,3-disubstituted uracils 18, characterized, and screened for inhibitory activity against eukaryotic elongation factor-2 kinase (eEF-2K). To understand the binding pocket of eEF-2K, structural modifications of the pyrido[2,3-d]pyrimidine were made at three regions (R(1), R(2), and R(3)). A homology model of eEF-2K was created, and compound 6 (A-484954, Abbott laboratories) was docked in the catalytic domain of eEF-2K. Compounds 6 (IC50=420nM) and 9 (IC50=930nM) are found to be better molecules in this preliminary series of pyrido[2,3-d]pyrimidine analogs. eEF-2K activity in MDA-MB-231 breast cancer cells is significantly reduced by compound 6, to a lesser extent by compound 9, and is unaffected by compound 12. Similar inhibitory results are observed when eEF-2K activity is stimulated by 2-deoxy-d-glucose (2-DOG) treatment, suggesting that compounds 6 and 9 are able to inhibit AMPK-mediated activation of eEF-2K to a notable extent. The results of this work will shed light on the further design and optimization of novel pyrido[2,3-d]pyrimidine analogs as eEF-2K inhibitors.


Journal of Biomolecular Screening | 2014

High-Throughput Screens for eEF-2 Kinase

Ashwini K. Devkota; Mangalika Warthaka; Ramakrishna Edupuganti; Clint D. J. Tavares; William H. Johnson; Bulent Ozpolat; Eun Jeong Cho; Kevin N. Dalby

eEF-2 kinase is a potential therapeutic target for breast cancer, gliomas, and depression. No potent inhibitors of eEF-2K have been reported, and thus development of high-throughput assay systems may expedite the process. Two high-throughput assays are described for eEF-2K using recombinant, tag-free enzyme purified from bacteria. The first is a fluorescence-based assay that uses the phosphorylation of a Sox-based peptide substrate by eEF-2K, which results in a 5-fold increase in fluorescence emission, allowing for continuous monitoring of the kinase activity. The second is a luminescence-based assay that produces a luminescence signal, which correlates with the amount of adenosine triphosphate remaining in the kinase reaction. Both assays have been optimized and miniaturized for a 384-well plate format and validated in screens. In conclusion, we demonstrated that a traditional radiolabeled assay can be readily transferred to universal spectroscopic assays that are robust and will facilitate high-throughput screening of larger size libraries for the identification of small-molecule inhibitors and significantly contribute to the development of therapies for targeting eEF2K.


Oncogene | 2017

c-Jun N-terminal kinase promotes stem cell phenotype in triple-negative breast cancer through upregulation of Notch1 via activation of c-Jun

Xiaoming Xie; Tamer S. Kaoud; Ramakrishna Edupuganti; Tinghu Zhang; Takahiro Kogawa; Y. J. Zhao; Gaurav B. Chauhan; D N Giannoukos; Yuan Qi; D Tripathy; Jing Wang; Nathanael S. Gray; Kevin N. Dalby; Chandra Bartholomeusz; Naoto Ueno

c-Jun N-terminal kinase (JNK) plays a vital role in malignant transformation of different cancers, and JNK is highly activated in basal-like triple-negative breast cancer (TNBC). However, the roles of JNK in regulating cancer stem-like cell (CSC) phenotype and tumorigenesis in TNBC are not well defined. JNK is known to mediate many cellular events via activating c-Jun. Here, we found that JNK regulated c-Jun activation in TNBC cells and that JNK activation correlated with c-Jun activation in TNBC tumors. Furthermore, the expression level of c-Jun was significantly higher in TNBC tumors than in non-TNBC tumors, and high c-Jun mRNA level was associated with shorter disease-free survival of patients with TNBC. Thus, we hypothesized that the JNK/c-Jun signaling pathway contributes to TNBC tumorigenesis. We found that knockdown of JNK1 or JNK2 or treatment with JNK-IN-8, an adenosine triphosphate-competitive irreversible pan-JNK inhibitor, significantly reduced cell proliferation, the ALDH1+ and CD44+/CD24− CSC subpopulations, and mammosphere formation, indicating that JNK promotes CSC self-renewal and maintenance in TNBC. We further demonstrated that both JNK1 and JNK2 regulated Notch1 transcription via activation of c-Jun and that the JNK/c-Jun signaling pathway promoted CSC phenotype through Notch1 signaling in TNBC. In a TNBC xenograft mouse model, JNK-IN-8 significantly suppressed tumor growth in a dose-dependent manner by inhibiting acquisition of the CSC phenotype. Taken together, our data demonstrate that JNK regulates TNBC tumorigenesis by promoting CSC phenotype through Notch1 signaling via activation of c-Jun and indicate that JNK/c-Jun/Notch1 signaling is a potential therapeutic target for TNBC.


Cancer Research | 2016

Definition of a Novel Feed-Forward Mechanism for Glycolysis-HIF1α Signaling in Hypoxic Tumors Highlights Aldolase A as a Therapeutic Target.

Geoffrey Grandjean; Petrus R. de Jong; Brian James; Mei Yee Koh; Robert Lemos; John Kingston; Alexander E. Aleshin; Laurie A. Bankston; Claudia P. Miller; Eun Jeong Cho; Ramakrishna Edupuganti; Ashwini K. Devkota; Gabriel Stancu; Robert C. Liddington; Kevin N. Dalby; Garth Powis

The hypoxia-inducible transcription factor HIF1α drives expression of many glycolytic enzymes. Here, we show that hypoxic glycolysis, in turn, increases HIF1α transcriptional activity and stimulates tumor growth, revealing a novel feed-forward mechanism of glycolysis-HIF1α signaling. Negative regulation of HIF1α by AMPK1 is bypassed in hypoxic cells, due to ATP elevation by increased glycolysis, thereby preventing phosphorylation and inactivation of the HIF1α transcriptional coactivator p300. Notably, of the HIF1α-activated glycolytic enzymes we evaluated by gene silencing, aldolase A (ALDOA) blockade produced the most robust decrease in glycolysis, HIF-1 activity, and cancer cell proliferation. Furthermore, either RNAi-mediated silencing of ALDOA or systemic treatment with a specific small-molecule inhibitor of aldolase A was sufficient to increase overall survival in a xenograft model of metastatic breast cancer. In establishing a novel glycolysis-HIF-1α feed-forward mechanism in hypoxic tumor cells, our results also provide a preclinical rationale to develop aldolase A inhibitors as a generalized strategy to treat intractable hypoxic cancer cells found widely in most solid tumors. Cancer Res; 76(14); 4259-69. ©2016 AACR.


Frontiers in Molecular Biosciences | 2015

Using docking and alchemical free energy approach to determine the binding mechanism of eEF2K inhibitors and prioritizing the compound synthesis.

Qiantao Wang; Ramakrishna Edupuganti; Clint D. J. Tavares; Kevin N. Dalby; Pengyu Ren

A-484954 is a known eEF2K inhibitor with submicromolar IC50 potency. However, the binding mechanism and the crystal structure of the kinase remains unknown. Here, we employ a homology eEF2K model, docking and alchemical free energy simulations to probe the binding mechanism of eEF2K, and in turn, guide the optimization of potential lead compounds. The inhibitor was docked into the ATP-binding site of a homology model first. Three different binding poses, hypothesis 1, 2, and 3, were obtained and subsequently applied to molecular dynamics (MD) based alchemical free energy simulations. The calculated relative binding free energy of the analogs of A-484954 using the binding pose of hypothesis 1 show a good correlation with the experimental IC50 values, yielding an r2 coefficient of 0.96 after removing an outlier (compound 5). Calculations using another two poses show little correlation with experimental data, (r2 of less than 0.5 with or without removing any outliers). Based on hypothesis 1, the calculated relative free energy suggests that bigger cyclic groups, at R1 e.g., cyclobutyl and cyclopentyl promote more favorable binding than smaller groups, such as cyclopropyl and hydrogen. Moreover, this study also demonstrates the ability of the alchemical free energy approach in combination with docking and homology modeling to prioritize compound synthesis. This can be an effective means of facilitating structure-based drug design when crystal structures are not available.


ACS Medicinal Chemistry Letters | 2015

Quantification of a Pharmacodynamic ERK End Point in Melanoma Cell Lysates: Toward Personalized Precision Medicine.

Mangalika Warthaka; Charles H. Adelmann; Tamer S. Kaoud; Ramakrishna Edupuganti; Chunli Yan; William H. Johnson; Scarlett B. Ferguson; Clint D. J. Tavares; Lindy J. Pence; Eric V. Anslyn; Pengyu Ren; Kenneth Y. Tsai; Kevin N. Dalby

Protein kinases are mutated or otherwise rendered constitutively active in numerous cancers where they are attractive therapeutic targets with well over a dozen kinase inhibitors now being used in therapy. While fluorescent sensors have capacity to measure changes in kinase activity, surprisingly they have not been utilized for biomarker studies. A first-generation peptide sensor for ERK based on the Sox fluorophore is described. This sensor called ERK-sensor-D1 possesses high activity toward ERK and more than 10-fold discrimination over other MAPKs. The sensor can rapidly quantify ERK activity in cell lysates and monitor ERK pathway engagement by BRAF and MEK inhibitors in cultured melanoma cell lines. The dynamic range of the sensor assay allows ERK activities that have potential for profound clinical consequences to be rapidly distinguished.


ChemBioChem | 2014

Reversible Covalent Inhibition of eEF-2K by Carbonitriles

Ashwini K. Devkota; Ramakrishna Edupuganti; Chunli Yan; Yue Shi; Jiney Jose; Qiantao Wang; Tamer S. Kaoud; Eun Jeong Cho; Pengyu Ren; Kevin N. Dalby

eEF‐2K is a potential target for treating cancer. However, potent specific inhibitors for this enzyme are lacking. Previously, we identified 2,6‐diamino‐4‐(2‐fluorophenyl)‐4H‐thiopyran‐3,5‐dicarbonitrile (DFTD) as an inhibitor of eEF‐2K. Here we describe its mechanism of action against eEF‐2K, on the basis of kinetic, mutational, and docking studies, and use chemoinformatic approaches to identify a similar class of carbonitrile‐containing compounds that exhibit the same mechanism of action. We show that DFTD behaves as a reversible covalent inhibitor of eEF‐2K with a two‐step mechanism of inhibition: a fast initial binding step, followed by a slower reversible inactivation step. Molecular docking suggests that a nitrile group of DFTD binds within 4.5 Å of the active site Cys146 to form a reversible thioimidate adduct. Because Cys146 is not conserved amongst other related kinases, targeting this residue holds promise for the development of selective covalent inhibitors of eEF‐2K.


Bioorganic & Medicinal Chemistry | 2017

Discovery of a potent inhibitor of MELK that inhibits expression of the anti-apoptotic protein Mcl-1 and TNBC cell growth

Ramakrishna Edupuganti; Juliana M. Taliaferro; Qiantao Wang; Xuemei Xie; Eun Jeong Cho; Fnu Vidhu; Pengyu Ren; Eric V. Anslyn; Chandra Bartholomeusz; Kevin N. Dalby

Despite recent advances in molecularly directed therapy, triple negative breast cancer (TNBC) remains one of the most aggressive forms of breast cancer, still without a suitable target for specific inhibitors. Maternal embryonic leucine zipper kinase (MELK) is highly expressed in TNBC, where level of overexpression correlates with poor prognosis and an aggressive disease course. Herein, we describe the discovery through targeted kinase inhibitor library screening, and structure-guided design of a series of ATP-competitive indolinone derivatives with subnanomolar inhibition constants towards MELK. The most potent compound, 17, inhibits the expression of the anti-apoptotic protein Mcl-1 and proliferation of TNBC cells exhibiting selectivity for cells expressing high levels of MELK. These studies suggest that further elaboration of 17 will furnish MELK-selective inhibitors with potential for development in preclinical models of TNBC and other cancers.


Oncotarget | 2017

A c-Jun N-terminal kinase inhibitor, JNK-IN-8, sensitizes triple negative breast cancer cells to lapatinib

Nancy D. Ebelt; Tamer S. Kaoud; Ramakrishna Edupuganti; Sabrina Van Ravenstein; Kevin N. Dalby; Carla L. Van

Triple negative breast cancers (TNBC) have poor prognosis compared to other breast cancer subtypes and represent 15-20% of breast cancers diagnosed. Unique targets and new molecularly-targeted therapies are urgently needed for this subtype. Despite high expression of Epidermal Growth Factor Receptor, inhibitors such as lapatinib have not shown therapeutic efficacy in TNBC patients. Herein, we report that treatment with the covalent JNK inhibitor, JNK-IN-8, synergizes with lapatinib to cause cell death, while these compounds as single agents have little effect. The combination significantly increases survival of mice bearing xenografts of MDA-MB-231 human TNBC cells. Our studies demonstrate that lapatinib treatment increases c-Jun and JNK phosphorylation indicating a mechanism of resistance. Combined, these compounds significantly reduce transcriptional activity of Nuclear Factor kappa B, Activating Protein 1, and Nuclear factor erythroid 2-Related Factor 2. As master regulators of antioxidant response, their decreased activity induces a 10-fold increase in reactive oxygen species that is cytotoxic, and is rescued by addition of exogenous antioxidants. Over expression of p65 or Nrf2 also significantly rescues viability during JNK-IN-8 and lapatinib treatment. Further studies combining JNK-IN-8 and lapatinib may reveal a benefit for patients with TNBC, fulfilling a critical medical need.


Archive | 2015

Chapter 2:Design and Synthesis of Synthetic Receptors for Biomolecule Recognition

Katharine L. Diehl; James L. Bachman; Brette M. Chapin; Ramakrishna Edupuganti; P. Rogelio Escamilla; Alexandra M. Gade; Erik T. Hernandez; Hyun Hwa Jo; Amber M. Johnson; Igor V. Kolesnichenko; Jaebum Lim; Chung-Yon Lin; Margaret K. Meadows; Helen M. Seifert; Diana Zamora-Olivares; Eric V. Anslyn

This chapter describes design concepts for synthetic receptors in supramolecular systems. The foundations of host:guest chemistry are first explored in the context of preorganization and complementarity, followed by discussions of the strength and solvent dependence of binding interactions. Reversible covalent interactions are then described, as are computational methods for predicting host:guest binding. Common synthetic receptor scaffolds such as crown ethers, calixarenes, calixpyrroles, cucurbiturils, and cyclodextrins are reviewed, and their binding properties are described. High-throughput methods, such as the design and use of combinatorial libraries and cross-reactive arrays, are detailed. Finally, thermodynamic concerns such as cooperativity and entropy–enthalpy compensation are discussed.

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Kevin N. Dalby

University of Texas at Austin

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Eric V. Anslyn

University of Texas at Austin

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Tamer S. Kaoud

University of Texas at Austin

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Clint D. J. Tavares

University of Texas at Austin

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Pengyu Ren

University of Texas at Austin

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Qiantao Wang

University of Texas at Austin

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Ashwini K. Devkota

University of Texas at Austin

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Chandra Bartholomeusz

University of Texas MD Anderson Cancer Center

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Eun Jeong Cho

University of Texas at Austin

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Jaebum Lim

University of Texas at Austin

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