Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 10 de 10
Filter
1.
Bioorg Med Chem Lett ; 21(13): 4059-65, 2011 Jul 01.
Article in English | MEDLINE | ID: mdl-21640588

ABSTRACT

A series of N-aryl pyridinone inhibitors of p38 mitogen activated protein (MAP) kinase were designed and prepared based on the screening hit SC-25028 (1) and structural comparisons to VX-745 (5). The focus of the investigation targeted the dependence of potency and metabolic stability on the benzyloxy connectivity, the role of the C-6 position and the substitution pattern on the N-phenyl ring. Further optimization produced the highly selective and potent pyridinones 2 and 3. These inhibitors exhibited activity in both acute and chronic models of inflammation.


Subject(s)
Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Pyridones/chemical synthesis , Pyridones/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Animals , Disease Models, Animal , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Humans , Inhibitory Concentration 50 , Male , Microsomes, Liver/enzymology , Molecular Structure , Pyridazines/chemistry , Pyridazines/pharmacology , Pyridones/chemistry , Pyrimidines/chemistry , Pyrimidines/pharmacology , Rats , Rats, Sprague-Dawley
2.
J Pharmacol Exp Ther ; 333(3): 797-807, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20237073

ABSTRACT

Activation of the p38 kinase pathway in immune cells leads to the transcriptional and translational regulation of proinflammatory cytokines. Mitogen-activated protein kinase-activated protein kinase 2 (MK2), a direct downstream substrate of p38 kinase, regulates lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6) production through modulating the stability and translation of these mRNAs. Developing small-molecule inhibitors of MK2 may yield anti-inflammatory efficacy with a different safety profile relative to p38 kinase inhibitors. This article describes the pharmacologic properties of a benzothiophene MK2 inhibitor, PF-3644022 [(10R)-10-methyl-3-(6-methylpyridin-3-yl)-9,10,11,12-tetrahydro-8H-[1,4]diazepino[5',6':4,5]thieno[3,2-f]quinolin-8-one]. PF-3644022 is a potent freely reversible ATP-competitive compound that inhibits MK2 activity (K(i) = 3 nM) with good selectivity when profiled against 200 human kinases. In the human U937 monocytic cell line or peripheral blood mononuclear cells, PF-3644022 potently inhibits TNFalpha production with similar activity (IC(50) = 160 nM). PF-3644022 blocks TNFalpha and IL-6 production in LPS-stimulated human whole blood with IC(50) values of 1.6 and 10.3 microM, respectively. Inhibition of TNFalpha in U937 cells and blood correlates closely with inhibition of phospho-heat shock protein 27, a target biomarker of MK2 activity. PF-3644022 displays good pharmacokinetic parameters in rats and is orally efficacious in both the rat acute LPS-induced TNFalpha model and the chronic streptococcal cell wall-induced arthritis model. Dose-dependent inhibition of TNFalpha production in the acute model and inhibition of paw swelling in the chronic model is observed with ED(50) values of 6.9 and 20 mg/kg, respectively. PF-3644022 efficacy in the chronic inflammation model is strongly correlated with maintaining a C(min) higher than the EC(50) measured in the rat LPS-induced TNFalpha model.


Subject(s)
Anti-Inflammatory Agents , Heterocyclic Compounds, 4 or More Rings/pharmacology , Inflammation/drug therapy , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/biosynthesis , Acute Disease , Adenosine Triphosphate/metabolism , Animals , Arthritis, Experimental/drug therapy , Arthritis, Experimental/pathology , Binding, Competitive/drug effects , Cell Wall/chemistry , Chronic Disease , Dose-Response Relationship, Drug , Female , Heterocyclic Compounds, 4 or More Rings/pharmacokinetics , Humans , Inflammation/chemically induced , Lipopolysaccharides/antagonists & inhibitors , Lipopolysaccharides/pharmacology , Male , Protein Kinase Inhibitors/pharmacokinetics , Rats , Rats, Inbred Lew , Rats, Sprague-Dawley , Streptococcus , U937 Cells , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors
3.
J Biol Chem ; 285(18): 13498-506, 2010 Apr 30.
Article in English | MEDLINE | ID: mdl-20167598

ABSTRACT

The classical nuclear factor kappaB (NF-kappaB) signaling pathway is under the control of the IkappaB kinase (IKK) complex, which consists of IKK-1, IKK-2, and NF-kappaB essential modulator (NEMO). This complex is responsible for the regulation of cell proliferation, survival, and differentiation. Dysregulation of this pathway is associated with several human diseases, and as such, its inhibition offers an exciting opportunity for therapeutic intervention. NEMO binding domain (NBD) peptides inhibit the binding of recombinant NEMO to IKK-2 in vitro. However, direct evidence of disruption of this binding by NBD peptides in biological systems has not been provided. Using a cell system, we expanded on previous observations to show that NBD peptides inhibit inflammation-induced but not basal cytokine production. We report that these peptides cause the release of IKK-2 from an IKK complex and disrupt NEMO-IKK-2 interactions in cells. We demonstrate that by interfering with NEMO-IKK-2 interactions, NBD peptides inhibit IKK-2 phosphorylation, without affecting signaling intermediates upstream of the IKK complex of the NF-kappaB pathway. Furthermore, in a cell-free system of IKK complex activation by TRAF6 (TNF receptor-associated factor 6), we show that these peptides inhibit the ability of this complex to phosphorylate downstream substrates, such as p65 and inhibitor of kappaB alpha (IkappaB alpha). Thus, consistent with the notion that NEMO regulates IKK-2 catalytic activity by serving as a scaffold, appropriately positioning IKK-2 for activation by upstream kinase(s), our findings provide novel insights into the molecular mechanisms by which NBD peptides exert their anti-inflammatory effects in cells.


Subject(s)
Anti-Inflammatory Agents/pharmacology , I-kappa B Kinase/metabolism , I-kappa B Kinase/pharmacology , Multiprotein Complexes/metabolism , Peptides/pharmacology , Transcription Factor RelA/metabolism , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/metabolism , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Cytokines/metabolism , Humans , I-kappa B Kinase/antagonists & inhibitors , I-kappa B Kinase/chemistry , Multiprotein Complexes/antagonists & inhibitors , Peptides/chemistry , Peptides/metabolism , Phosphorylation , Protein Binding/drug effects , Protein Structure, Tertiary , TNF Receptor-Associated Factor 6/metabolism , Transcription Factor RelA/antagonists & inhibitors
4.
Protein Expr Purif ; 69(1): 54-63, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19781647

ABSTRACT

Janus-associated kinases (JAKs) play critical roles in cytokine signaling, and have emerged as viable therapeutic targets in inflammation and oncology related diseases. To date, targeting JAK proteins with highly selective inhibitor compounds have remained elusive. We have expressed the active kinase domains for both JAK2 and JAK3 and devised purification protocols to resolve the non-, mono- (Y1007) and diphosphorylated (Y1007 and Y1008) states of JAK2 and non- and monophosphorylated states of JAK3 (Y980). An optimal purified protein yield of 20, 29 and 69mg per 20L cell culture was obtained for the three JAK2 forms, respectively, and 12.2 and 2.3mg per 10L fermentation for the two JAK3 forms allowing detailed biochemical and biophysical studies. To monitor the purification process we developed a novel HPLC activity assay where a sequential order of phosphorylation was observed whereby the first tyrosine residue was completely phosphorylated prior to phosphorylation of the tandem tyrosine residue. A Caliper-based microfluidics assay was used to determine the kinetic parameters (K(m) and k(cat)) for each phosphorylated state, showing that monophosphorylated (Y1007) JAK2 enzyme activity increased 9-fold over that of the nonphosphorylated species, and increased an additional 6-fold for the diphosphorylated (Y1007/Y1008) species, while phosphorylation of JAK3 resulted in a negligible increase in activity. Moreover, crystal structures have been generated for each isolated state of JAK2 and JAK3 with resolutions better than 2.4A. The generation of these reagents has enabled kinetic and structural characterization to inform the design of potent and selective inhibitors of the JAK family.


Subject(s)
Janus Kinase 2/chemistry , Janus Kinase 2/isolation & purification , Janus Kinase 3/chemistry , Janus Kinase 3/isolation & purification , Amino Acid Sequence , Biocatalysis , Chromatography, High Pressure Liquid , Crystallization , Electrophoresis, Polyacrylamide Gel , Fermentation , Humans , Kinetics , Molecular Sequence Data , Phosphorylation , Protein Structure, Tertiary
5.
J Pharmacol Exp Ther ; 331(3): 882-95, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19720877

ABSTRACT

Signal transduction through the p38 mitogen-activated protein (MAP) kinase pathway is central to the transcriptional and translational control of cytokine and inflammatory mediator production. p38 MAP kinase inhibition hence constitutes a promising therapeutic strategy for treatment of chronic inflammatory diseases, based upon its potential to inhibit key pathways driving the inflammatory and destructive processes in these debilitating diseases. The present study describes the pharmacological properties of the N-phenyl pyridinone p38 MAP kinase inhibitor benzamide [3- [3-bromo-4-[(2,4-difluorophenyl)methoxy]-6-methyl-2- oxo-1(2H)-pyridinyl]-N,4-dimethyl-, (-)-(9CI); PH-797804]. PH-797804 is an ATP-competitive, readily reversible inhibitor of the alpha isoform of human p38 MAP kinase, exhibiting a K(i) = 5.8 nM. In human monocyte and synovial fibroblast cell systems, PH-797804 blocks inflammation-induced production of cytokines and proinflammatory mediators, such as prostaglandin E(2), at concentrations that parallel inhibition of cell-associated p38 MAP kinase. After oral dosing, PH-797804 effectively inhibits acute inflammatory responses induced by systemically administered endotoxin in both rat and cynomolgus monkeys. Furthermore, PH-797804 demonstrates robust anti-inflammatory activity in chronic disease models, significantly reducing both joint inflammation and associated bone loss in streptococcal cell wall-induced arthritis in rats and mouse collagen-induced arthritis. Finally, PH-797804 reduced tumor necrosis factor-alpha and interleukin-6 production in clinical studies after endotoxin administration in a dose-dependent manner, paralleling inhibition of the target enzyme. Low-nanomolar biochemical enzyme inhibition potency correlated with p38 MAP kinase inhibition in human cells and in vivo studies. In addition, a direct correspondence between p38 MAP kinase inhibition and anti-inflammatory activity was observed with PH-797804, thus providing confidence in dose projections for further human studies in chronic inflammatory disease.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Benzamides/therapeutic use , Pyrones/therapeutic use , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Adolescent , Adult , Animals , Anti-Inflammatory Agents, Non-Steroidal/blood , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Arthritis, Experimental/drug therapy , Arthritis, Experimental/enzymology , Arthritis, Experimental/immunology , Arthritis, Experimental/metabolism , Arthritis, Rheumatoid/drug therapy , Arthritis, Rheumatoid/enzymology , Arthritis, Rheumatoid/immunology , Benzamides/blood , Benzamides/chemistry , Benzamides/pharmacology , Bone Density/drug effects , Bone Marrow Cells/drug effects , Bone Marrow Cells/enzymology , Bone Marrow Cells/immunology , Cell Line , Cytokines/biosynthesis , Cytokines/blood , Dinoprostone/biosynthesis , Drug Evaluation, Preclinical , Female , Humans , Lipopolysaccharides/pharmacology , Macaca fascicularis , Male , Mice , Mice, Inbred DBA , Middle Aged , Monocytes/drug effects , Monocytes/enzymology , Monocytes/immunology , Osteoclasts/drug effects , Osteoclasts/enzymology , Osteoclasts/immunology , Pyridones , Pyrones/blood , Pyrones/chemistry , Pyrones/pharmacology , Rats , Rats, Inbred Lew , Systemic Inflammatory Response Syndrome/drug therapy , Systemic Inflammatory Response Syndrome/enzymology , Systemic Inflammatory Response Syndrome/immunology , Young Adult
6.
Biochemistry ; 48(27): 6402-11, 2009 Jul 14.
Article in English | MEDLINE | ID: mdl-19496616

ABSTRACT

PH-797804 is a diarylpyridinone inhibitor of p38alpha mitogen-activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. On the basis of structural comparison with a different biaryl pyrazole template and supported by dozens of high-resolution crystal structures of p38alpha inhibitor complexes, PH-797804 is predicted to possess a high level of specificity across the broad human kinase genome. We used a structural bioinformatics approach to identify two selectivity elements encoded by the TXXXG sequence motif on the p38alpha kinase hinge: (i) Thr106 that serves as the gatekeeper to the buried hydrophobic pocket occupied by 2,4-difluorophenyl of PH-797804 and (ii) the bidentate hydrogen bonds formed by the pyridinone moiety with the kinase hinge requiring an induced 180 degrees rotation of the Met109-Gly110 peptide bond. The peptide flip occurs in p38alpha kinase due to the critical glycine residue marked by its conformational flexibility. Kinome-wide sequence mining revealed rare presentation of the selectivity motif. Corroboratively, PH-797804 exhibited exceptionally high specificity against MAP kinases and the related kinases. No cross-reactivity was observed in large panels of kinase screens (selectivity ratio of >500-fold). In cellular assays, PH-797804 demonstrated superior potency and selectivity consistent with the biochemical measurements. PH-797804 has met safety criteria in human phase I studies and is under clinical development for several inflammatory conditions. Understanding the rationale for selectivity at the molecular level helps elucidate the biological function and design of specific p38alpha kinase inhibitors.


Subject(s)
Benzamides/pharmacology , Computational Biology , Protein Kinase Inhibitors/pharmacology , Pyrones/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Benzamides/chemistry , Crystallography, X-Ray , Humans , Hydrogen Bonding , Models, Molecular , Molecular Structure , Phosphorylation , Protein Kinase Inhibitors/chemistry , Pyridones , Pyrones/chemistry , Substrate Specificity , p38 Mitogen-Activated Protein Kinases/metabolism
7.
J Pharmacol Exp Ther ; 330(2): 377-88, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19478133

ABSTRACT

Nuclear factor-kappaB (NF-kappaB) is one of the major families of transcription factors activated during the inflammatory response in asthma and chronic obstructive pulmonary disease. Inhibitory factor-kappaB kinase 2 (IKK-2) has been shown to play a pivotal role in cytokine-induced NF-kappaB activation in airway epithelium and in disease-relevant cells. Nevertheless, the potential toxicity of specific IKK-2 inhibitors may be unacceptable for oral delivery in chronic obstructive pulmonary disease. Therefore, local delivery to the lungs is an attractive alternative that warrants further exploration. Here, we describe potent and selective small-molecule IKK-2 inhibitors [8-(5-chloro-2-(4-methylpiperazin-1-yl)isonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide (PHA-408) and 8-(2-(3,4-bis(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-5-chloroisonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo-[g]indazole-3-carboxamide (PF-184)] that are competitive for ATP have slow off-rates from IKK-2 and display broad in vitro anti-inflammatory activities resulting from NF-kappaB pathway inhibition. Notably, PF-184 has been designed to have high systemic clearance, which limits systemic exposure and maximizes the effects locally in the airways. We used an inhaled lipopolysaccharide-induced rat model of neutrophilia to address whether inhibiting NF-kappaB activation locally within the airways would show anti-inflammatory effects in the absence of systemic exposure. PHA-408, a low-clearance compound previously shown to be efficacious orally in a rodent model of arthritis, dose-dependently attenuated inhaled lipopolysaccharide-induced cell infiltration and cytokine production. Interestingly, PF-184 produced comparable dose-dependent anti-inflammatory activity by intratracheal administration and was as efficacious as intratracheally administered fluticasone propionate (fluticasone). Together, these results support the potential therapeutic utility of IKK-2 inhibition in inflammatory pulmonary diseases and demonstrate anti-inflammatory efficacy of an inhaled IKK-2 inhibitor in a rat airway model of neutrophilia.


Subject(s)
Drug Delivery Systems/methods , I-kappa B Kinase/antagonists & inhibitors , Inflammation Mediators/administration & dosage , Lung Diseases/enzymology , Protein Kinase Inhibitors/administration & dosage , Administration, Oral , Animals , Cells, Cultured , Disease Models, Animal , Humans , I-kappa B Kinase/metabolism , Inflammation/drug therapy , Inflammation/enzymology , Inflammation/immunology , Inflammation Mediators/chemistry , Inflammation Mediators/metabolism , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/enzymology , Leukocytes, Mononuclear/immunology , Lung Diseases/drug therapy , Lung Diseases/immunology , Male , Protein Binding/drug effects , Protein Binding/physiology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Rats
8.
J Pharmacol Exp Ther ; 329(1): 14-25, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19168710

ABSTRACT

Nuclear factor (NF)-kappaB activation has been clearly linked to the pathogenesis of multiple inflammatory diseases including arthritis. The central role that IkappaB kinase-2 (IKK-2) plays in regulating NF-kappaB signaling in response to inflammatory stimuli has made this enzyme an attractive target for therapeutic intervention. Although diverse chemical classes of IKK-2 inhibitors have been identified, the binding kinetics of these inhibitors has limited the scope of their applications. In addition, safety assessments of IKK-2 inhibitors based on a comprehensive understanding of the pharmacokinetic/pharmacodynamic relationships have yet to be reported. Here, we describe a novel, potent, and highly selective IKK-2 inhibitor, PHA-408 [8-(5-chloro-2-(4-methylpiperazin-1-yl)isonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide]. PHA-408 is an ATP-competitive inhibitor, which binds IKK-2 tightly with a relatively slow off rate. In arthritis-relevant cells and animal models, PHA-408 suppresses inflammation-induced cellular events, including IkappaBalpha phosphorylation and degradation, p65 phosphorylation and DNA binding activity, the expression of inflammatory mediators, and joint pathology. PHA-408 was efficacious in a chronic model of arthritis with no adverse effects at maximally efficacious doses. Stemming from its ability to bind tightly to IKK-2, as a novelty, we demonstrated that PHA-408-mediated inhibition of IKK-2 activity correlated very well with its ability to modulate the fate of IKK-2 substrates and downstream transcriptional events. We ultimately directly linked IKK-2 activity ex vivo and in vivo to markers of inflammation with the inhibitor plasma concentrations. Thus, PHA-408 represents a powerful tool to further gain insight into the mechanisms by which IKK-2 regulates NF-kappaB signaling and validates IKK-2 as a therapeutic target.


Subject(s)
Arthritis/pathology , Enzyme Inhibitors/pharmacology , I-kappa B Kinase/antagonists & inhibitors , NF-kappa B/drug effects , Signal Transduction/drug effects , Animals , Arthritis, Experimental/chemically induced , Arthritis, Experimental/pathology , Blotting, Western , Cells, Cultured , Chromatography, High Pressure Liquid , Electrophoretic Mobility Shift Assay , Enzyme Inhibitors/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Fibroblasts/drug effects , Fibroblasts/metabolism , I-kappa B Kinase/metabolism , Inflammation/chemically induced , Inflammation/pathology , Lipopolysaccharides/pharmacology , Rats , Rats, Inbred Lew , Recombinant Proteins/metabolism , Streptococcus/immunology , Synovial Fluid/cytology , Synovial Fluid/drug effects , Tandem Mass Spectrometry , Tomography, X-Ray Computed , Transcription Factor RelA/genetics , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors
9.
J Med Chem ; 50(11): 2647-54, 2007 May 31.
Article in English | MEDLINE | ID: mdl-17480064

ABSTRACT

A new class of potent kinase inhibitors selective for mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2 or MK-2) for the treatment of rheumatoid arthritis has been prepared and evaluated. These inhibitors have IC50 values as low as 10 nM against the target and have good selectivity profiles against a number of kinases including CDK2, ERK, JNK, and p38. These MK-2 inhibitors have been shown to suppress TNFalpha production in U397 cells and to be efficacious in an acute inflammation model. The structure-activity relationships of this series, the selectivity for MK-2 and their activity in both in vitro and in vivo models are discussed. The observed selectivity is discussed with the aid of an MK-2/inhibitor crystal structure.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyridines/chemical synthesis , Pyrroles/chemical synthesis , Acute Disease , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Crystallography, X-Ray , Humans , Inflammation/drug therapy , Intracellular Signaling Peptides and Proteins , Models, Molecular , Protein Serine-Threonine Kinases/chemistry , Pyridines/chemistry , Pyridines/pharmacology , Pyrroles/chemistry , Pyrroles/pharmacology , Rats , Structure-Activity Relationship , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/biosynthesis , U937 Cells
10.
Biochim Biophys Acta ; 1598(1-2): 88-97, 2002 Jul 29.
Article in English | MEDLINE | ID: mdl-12147348

ABSTRACT

The kinetic mechanism of mitogen-activated protein kinase activated protein kinase-2 (MAPKAPK2) was investigated using a peptide (LKRSLSEM) based on the phosphorylation site found in serum response factor (SRF). Initial velocity studies yielded a family of double-reciprocal lines that appear parallel and indicative of a ping-pong mechanism. The use of dead-end inhibition studies did not provide a definitive assignment of a reaction mechanism. However, product inhibition studies suggested that MAPKAPK2 follows an ordered bi-bi kinetic mechanism, where ATP must bind to the enzyme prior to the SRF-peptide and the phosphorylated product is released first, followed by ADP. In agreement with these latter results, surface plasmon resonance measurements demonstrate that the binding of the inhibitor peptide to MAPKAPK2 requires the presence of ATP. Furthermore, competitive inhibitors of ATP, adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP) and a staurosporine analog (K252a), can inhibit this ATP-dependent binding providing further evidence that the peptide substrate binds preferably to the E:ATP complex.


Subject(s)
Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Binding Sites , Cloning, Molecular , Enzyme Activation , Humans , Intracellular Signaling Peptides and Proteins , Kinetics , Peptide Fragments/chemistry , Peptide Mapping , Phosphorylation , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/isolation & purification , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Substrate Specificity
SELECTION OF CITATIONS
SEARCH DETAIL
...