Biomedical subjects

Maria Kornienko

Publications and source records attributed to Maria Kornienko.

4 recordsLinked to original sources

PubMed2005

Biochemical and structural characterization of a novel class of inhibitors of the type 1 insulin-like growth factor and insulin receptor kinases.

The type 1 insulin-like growth factor receptor (IGF-1R) is often overexpressed on tumor cells and is believed to play an important role in anchorage-independent proliferation. Additionally, cell culture studies have indicated that IGF-1R confers increased resistance to apoptosis caused by radiation or chemotherapeutic agents. Thus, inhibitors of the intracellular kinase domain of this receptor may have utility for the clinical treatment of cancer. As part of an effort to develop clinically useful inhibitors of IGF-1R kinase, a novel class of pyrrole-5-carboxaldehyde compounds was investigated. The compounds exhibited selectivity against the closely related insulin receptor kinase intrinsically and in cell-based assays. The inhibitors formed a reversible, covalent adduct at the kinase active site, and treatment of such adducts with sodium borohydride irreversibly inactivated the enzyme. Analysis of a tryptic digest of a covalently modified IGF-1R kinase fragment revealed that the active site Lys1003 had been reductively alkylated with the aldehyde inhibitor. Reductive alkylation of the insulin receptor kinase with one of these inhibitors led to a similarly inactivated enzyme which was examined by X-ray crystallography. The crystal structure confirmed the modification of the active site lysine side chain and revealed details of the key interactions between the inhibitor and enzyme.

Aldehydes↗

PubMed2005

Protein expression plasmids produced rapidly: streamlining cloning protocols and robotic handling.

As many processes in the preclinical drug discovery process become highly parallel, the need to also produce a large number of different proteins in parallel has become acute, such as for protein crystallization and activity screening. In turn, the requisite DNA constructions to produce these proteins must now be done at a rate that requires automated cloning procedures, each with an intrinsic low failure probability per sample. The high-throughput cloning solutions presented here achieve production of 192 different expression plasmids at a success rate of greater than 95% of the targeted open reading frames. Time for completion of the set by one person is reduced to approximately 11 working days, starting with polymerase chain reactions for a number of source clones and ending with purified expression plasmids. Achievement of this throughput utilizes the following: (1) the Beckman Coulter (Fullerton, CA) Biomek FX liquid handler for most manipulations, (2) Gateway cloning technology (Invitrogen Corp., Carlsbad, CA), and (3) computer programs designed for parallel processing of all sample information, including primer design and the resulting DNA and protein sequence assembly. Exemplary data are presented for discovery of a form of the Rho-kinase that crystallizes (ROCK2).

Automation↗

PubMed2003

Structure of apo, unactivated insulin-like growth factor-1 receptor kinase at 1.5 A resolution.

The crystal structure of the wild-type unactivated kinase domain (IGFRK-0P) of insulin-like growth factor-1 receptor has been reported previously at 2.7 A resolution [Munshi et al. (2002), J. Biol. Chem. 277, 38797-38802]. In order to obtain a high-resolution structure, a number of variants of IGFRK-0P were prepared and screened for crystallization. A double mutant with E1067A and E1069A substitutions within the kinase-insert region resulted in crystals that diffracted to 1.5 A resolution. Overall, the structure of the mutant IGFRK-0P is similar to that of the wild-type IGFRK-0P structure, with the exception of the previously disordered kinase-insert region in the wild type having become fixed. In addition, amino-acid residues 947-952 at the N-terminus are well defined in the mutant structure. The monomeric protein structure is folded into two lobes connected by a hinge region, with the catalytic center situated at the interface of the two lobes. Two molecules of IGFRK-0P in the asymmetric unit are associated as a dimer and two different types of dimers with their ATP-binding clefts either facing towards or away from each other are observed. The current refined model consists of a dimer and 635 water molecules.

Amino Acid Substitution↗

PubMed2002

Crystal structure of the Apo, unactivated insulin-like growth factor-1 receptor kinase. Implication for inhibitor specificity.

The x-ray structure of the unactivated kinase domain of insulin-like growth factor-1 receptor (IGFRK-0P) is reported here at 2.7 A resolution. IGFRK-0P is composed of two lobes connected by a hinge region. The N-terminal lobe of the kinase is a twisted beta-sheet flanked by a single helix, and the C-terminal lobe comprises eight alpha-helices and four short beta-strands. The ATP binding pocket and the catalytic center reside at the interface of the two lobes. Despite the overall similarity to other receptor tyrosine kinases, three notable conformational modifications are observed: 1) this kinase adopts a more closed structure, with its two lobes rotated further toward each other; 2) the conformation of the proximal end of the activation loop (residues 1121-1129) is different; 3) the orientation of the nucleotide-binding loop is altered. Collectively, these alterations lead to a different ATP-binding pocket that might impact on inhibitor designs for IGFRK-0P. Two molecules of IGFRK-0P are seen in the asymmetric unit; they are associated as a dimer with their ATP binding clefts facing each other. The ordered N terminus of one monomer approaches the active site of the other, suggesting that the juxtamembrane region of one molecule could come into close proximity to the active site of the other.

Adenosine Triphosphate↗