Results Through Rehabilitation & Research
Assitant Professor, Neurology and Neuroscience
Weill Cornell Medical College
Identification of new protein and enzymatic targets relevant to prevention and treatment of stroke, spinal cord and traumatic brain injury, and neurodegenerative diseases is of particular interest.
(1) More than 70 genes of putative non-heme iron oxygenases have been identified in the human genome, but only a number have the physiological functions established. Hypoxia inducible factor is a heterodimeric transcription factor capable of inducing the expression of a large number of genes involved in hypoxic adaptation. Regulation of HIF occurs primarily through the hydroxylation of its HIF-1 alpha subunit. The oxidative modification of HIF is catalyzed by non heme-iron amino acid hydroxylases, among which prolyl-hydroxylase (PHD) exists in 3 isoforms. The precise role of N-terminal domains in the two of PHDs is still unknown. The development of specific inhibitors for each of PHDs could allow one to understand the role of each enzyme in cell adaptation to hypoxia, i.e. to identification of specific targets for the subsequent drug design. Our laboratory developed new cell-based reporter assays to screen for PHD inhibitors. The assay is based on the constitutive expression of oxygen degradable domain of HIF (1, 2, or 3) fused to firefly luciferase. The assay is order of magnitude more sensitive than the traditionally used HRE-luciferase reporter assay, and permits real-time monitoring of fusion protein stabilization. The time-course of reporter activation can be used to determine PHD inhibition constants. The accomplished HTS allowed us to identify a group of oxyquinolines with branched motifs responsible for specific inhibition of PHD among other enzymes of the same class. Ongoing efforts are focused on the design of new isoform-specific PHD inhibitors may uncover unknown functions each PHD isoform plays in the cell.
(2) The NF-E2-related factor 2 (Nrf2) is a key transcriptional regulator of antioxidant defense and detoxification. Nrf2 activators have been recently shown to prevent the onset of neurodegenerative diseases. To directly monitor stabilization of Nrf2 we fused its Neh2 domain, responsible for the interaction with its nucleocytoplasmic regulator, Keap1, to firefly luciferase (Neh2-luciferase). We showed that Neh2 domain is sufficient for recognition, ubiquitination and proteasomal degradation of Neh2-luciferase fusion protein. The steady-state concentration of Nrf2 (as represented by Neh2 luciferase) established in cells can be manipulated by the addition of compounds affecting the individual steps controlling the Nrf2 stability. The novel reporter allows monitoring the antioxidant response in real-time, right after drug administration, and is suitable both for high throughput screening and elucidation of the mechanism of drug action. The power of the new reporter is illustrated by its application for screening of Spectrum library followed by real-time monitoring of action of selected hits: in addition to the identification of new Nrf2 activators, we for the first time make an insight into the mechanistic details of their action and offer a strategy to discriminate between the action of direct activators such as alkylating agents and those requiring additional transformation steps such as prior oxidation (catechols and diamines) or manipulation of upstream regulatory pathways (via Hsp90 inhibition). The ongoing HTS aims at identification of new structural motifs specifically recognizing Keap1 portion responsible for Nrf2 binding, and thus, working as reversible competitive inhibitors rather than alkylating agents and pro-oxidants.
(3) Besides individual transcription factors and enzymes, mitochondria work as an intracellular oxygen sensing device. Both age-related and acute neurodegeneration is determined by the mitochondria failure to support energy production and matrix redox balance. Understanding of the mechanisms of mitochondrial failure upon different treatments will allow us to determine the targets for medical intervention, which maybe either inside the matrix or upstream mitochondria. Mitochondria have common substrates/products with oxygen sensing proteins such as HIF hydroxylases and adjust the response of oxygen sensors. PHD3 in mice is targeted to mitochondria. The interplay between mitochondria and cytosolic oxygen sensors is just beginning to be recognized. Establishing the links between mitochondria respiratory chain functioning, pore opening and the activity of HIF prolyl hydroxylases is one of the future tasks.
Irina Gazaryan received her M.S. (with honors) and Ph.D. in Chemistry from Moscow State University. She joined the faculty of the School of Chemistry, Moscow State University, in 1984. In 1996 she was awarded D.Sc. degree in Biochemistry following her training as a visiting fellow at the Biotechnological Center in Braunschweig (GBF), Germany (1992-93), the Ohio State University, USA (1994-95), John Innes Center in Norwich, UK (1996). She was awarded a Swedish Royal Society fellowship in 1997 and 1999 (Lund University, Sweden). In 1997 she received the “850th anniversary of Moscow” medal to honor her 15-year service as a faculty member at Moscow State University. In 1998-2000 she served as Russian co-PI on a number of EU-awarded grants (INTAS and INCO-Copernicus). Since 2000 she has worked at Burke Medical Research Institute as a research scientist. In 2007, she became head of the newly launched Enzymology laboratory, leading drug development efforts at Burke. Since 2005 she has been a faculty member of the Department of Neurology and Neuroscience, Weill Medical College of Cornell University. She is coauthor of more than 90 original scientific papers and 6 patents, and serves as a reviewer for Journal of Neuroscience, the International Journal of Biochemistry and Cell Biology, Biophysical Chemistry, etc.
Gazaryan, IG, Krasnikov B, Ashby G, Thorneley R, Kristal B, Brown A (2002) Zinc is a potent inhibitor of thiol oxidoreductase activity and stimulates reactive oxygen species production by lipoamide dehydrogenase. J Biol Chem 277, 10064-72.
Vrzheshch PD, Batanova EA, Mevkh AT, Varfolomeev SD, Gazaryan IG, Thorneley RN. (2003) A novel approach to distinguish between enzyme mechanisms: quasi-steady-state kinetic analysis of the prostaglandin H synthase peroxidase reaction. Biochem J. 372, 713-24.
Shleev SV, Khan IG, Gazaryan IG, Morozova OV, Yaropolov AI. (2003) Novel laccase redox mediators: spectral, electrochemical and kinetic properties. Applied Biochemistry and Biotechnology, 111, 167-8
Shipovskov, Ferapontova EE, Gazaryan IG, Ruzgas T. (2004) Recombinant horseradish peroxidase- and cytochrome c-based two-electrode system for detection of superoxide radicals. Bioelectrochemistry 63, 277– 80.
Klyachko NL, Shchedrina VA, Efimov AV, Kazakov SV, Gazaryan IG, Kristal BS, Brown AM. (2005) pH-Dependent substrate preference of pig heart lipoamide dehydrogenase varies with oligomeric state: Response to mitochondrial matrix acidification. J Biol Chem. 280, 16106-16114.
Kazakov S, Kaholek M, Gazaryan IG, Krasnikov B, Miller K, and Levon K. (2006) Ion concentration of external solution as a characteristic of micro- and nanogel ionic reservoirs. Journal of Physical Chemistry B 110 (31), 15107 – 15116.
Hushpulian DM, Poloznikov AA, Savitski PA, Rozhkova AM, Chubar TA, Fechina VA, Orlova MA, Tishkov VI, Gazaryan IG, Lagrimini LM. (2007) Glutamic acid-141: a heme 'bodyguard' in anionic tobacco peroxidase. Biol Chem. 388(4), 373-380.
Gazaryan IG, Krasinskaya IP, Kristal BS, Brown AM. (2007) Zinc irreversibly damages major enzymes of energy production and antioxidant defense prior to mitochondrial permeability transition. J Biol Chem 282, 24373-24380.
Hushpulian D , Poloznikov AA, Savitski PA, Rozhkova AM, Chubar TA, Fechina VA, Lagrimini LM, Tishkov VI, Gazaryan IG. (2007) Biocatalytic properties of recombinant tobacco peroxidase in chemiluminescent reaction. Biocatalysis and Biotransformation. 25(2-4), 163-170 .
Ratan RR, Siddiq A, Smirnova N, Karpisheva K, Haskew-Layton R, McConoughey S, Langley B, Estevez A, Huerta PT, Volpe B, Roy S, Sen CK, Gazaryan I, Cho S, Fink M, Lamanna J. (2007) Harnessing hypoxic adaptation to prevent, treat, and repair stroke. J. Mol. Med. 85(12), 1331-1338.
Kazakov S, Bonvouloir E, Gazaryan I. (2008) Physicochemical characterization of natural ionic microreservoirs: bacillus subtilis dormant spores. Journal of Physical Chemistry B 112(7), 2233-2244.
Ratan RR, Siddiq A, Aminova L, Langley B, McConoughey S, Karpisheva K, Lee H.-H, Carmichael T, Kornblum H, Coppola G, Geschwind DH, Hoke A, Smirnova N, Rink C, Roy S, Sen C, Beattie M, Hart RP, Grumet M, Sun D, Freeman RS, Semenza GL and Gazaryan I. (2008) Small molecule activation of adaptive gene expression: tilorone or its analogs are novel, potent activators of hypoxia induicible factor-1 that provide prophylaxis against stroke and spinal cord injury. Ann NY Acad Sci 1147, 383-394.
Smirnova NA, Rakhman I, Moroz N, Basso M, Payappilly J, Kazakov S, Hernandez-Guzman F, Gaisina IN, Kozikowski AP, Ratan RR, Gazaryan IG. (2010) Utilization of an in vivo reporter for high throughput identification of branched small molecule regulators of hypoxic adaptation. Chem & Biol. 17(4):380-91.
Faust TW, Chang EH, Kowal C, Berlin R, Gazaryan IG, Bertini E, Zhang J, Sanchez-Guerrero J, Fragoso-Loyo HE, Volpe BT, Diamond B, Huerta PT. (2010) Neurotoxic lupus auto-antibodies alter brain function through two distinct mechanisms. Proc Natl Acad Sci USA 107(43):18569-74.
Haskew-Layton RE, Payappilly JB, Smirnova NA, Ma TC, Chan KK, Murphy TH, Guo H, Langley B, Sultana R, Butterfield DA, Santagata S, Alldred MJ, Gazaryan IG, Bell GW, Ginsberg SD, Ratan RR. (2010) Controlled enzymatic production of astrocytic hydrogen peroxide protects neurons from oxidative stress via an Nrf2-independent pathway. Proc Natl Acad Sci USA 107(40):17385-90.
Prof. R. Ratan (BMRI)
Dr. Bobby Thomas (WMCCU)
Dr. Irina Gaisina (UIC)
Prof. P. Cole (John Hopkins University)
Profs. Natalia Klyachko and Vladimir Tishkov (Moscow State University, Russia)
Thomas Hartman Foundation for Parkinson’s Research, Inc.
$100,000 a year for 2 years, 2010-2012
Novel Activators of Antioxidant Response for Prevention and Treatment of Parkinson’s disease