SandboxPKA: Difference between revisions

The N-terminal half of the protein includes an N-terminal “cap” of 80 residues that is important for autoinhibition, followed by an SH3 domain, an SH2 domain, and a tyrosine kinase domain. The C-terminal half of c-Abl includes binding elements for SH3 domains, nuclear localization and export signals, a DNA binding functionality, and an actin binding domain. Human cells express two splice variants of c-Abl, Abl 1a and Abl 1b, which differ only in the very N-terminal region. Abl 1b is myristoylated, whereas Abl1a is not.  

The fusion of the gene encoding c-Abl with the breakpoint cluster region (BCR) gene, results in the formation of a fusion protein, BCR-Abl, in which all of c-Abl is preserved without mutation, except for the “cap” region upstream of the SH3 domain.

<StructureSection load='1OPK' size='350' side='right' caption='c-Abl tyrosine kinase: SH3 doamain is shown in red while SH2 domain is shown in purple. Catalytic domain is represented by blue. Also it is possible to observe myristoiled group in the N-terminal domain of c-Abl tyrosin kinase' scene='SandboxPKA/Abl1/4'>

Protein kinases are characterized by an architecture that enables distal parts of the enzyme to be linked by conserved hydrophobic elements. Kynase domain is composed by N-lobe and a C-lobe, and the adenine ring of ATP is buried at the base of the cleft between the two lobes.  

The smaller N-lobe is dominated by a five-stranded b-sheet, which is coupled to a helical subdomain that typically consists of the C-helix.  There are several highly conserved sequence motifs are embedded within  this lobe:

• '''<scene name='Dasatinib/Mpl/4'>P-Loop Movement</scene>'''<  often referred to as the Walker-A motif (GxxxxGKT/S) <Ref>PMID: 12468712</Ref>. In this loop there is a higly conserved residue (usually Lys) which is able to form a salt bridge with C-helix.  

• '''C-helix''':  is a unique helix present in the N-lobe. It is very dynamic and plays a key role as a regulatory element in the protein kinase molecule. C-helix occupies a strategically important position between the two lobes. The C-helix connects many different parts of the molecule and serves as a ‘‘signal integration motif’’ <Ref>PMID: 11749372</Ref> The C-helix contains another conserved residue, Glu or Asp, that bridges to Lys located in P-loop. This bridge is really important for catalysis process.  

- The '''F-helix''' serves as a central scaffold for assembly of the entire molecule. Two non consecutive hydrophobic structures termed ‘‘spines’’ anchor all the elements important for catalysis to the F-helix. They are regulatory and catalytic spine.  

- The '''catalytic loop''' is composed by β6 and β7, whereas β8 and β9 strands flank DGF motif, where aspartic/glutamic residue is critical for recognizing one of the ATP-bound Mg++ ions

- The '''<scene name='Dasatinib/Mact/1'>Activation Loop Movement</scene>''' contains Tyr412 responsible of activation of kinase activity.  

- The unactivated, autoinhibited conformation [in which the Asp-810-Phe-811-Gly-812 (DFG) triad at the beginning of the A-loop is in the “DFG-out”, can <scene name='Dasatinib/Mdfg/3'>move</scene> when ATP Tyr412 is phosphorlated, rising an active conformation. <Ref>PMID: 19164557</Ref>

- The '''myristoyl group''' complement activation loop in turning on and off c-abl protein. It has been shown to be the key regulator of this kinase.   

It is responsible of both, ATP binding as well as protein binding. The crystal structure of the catalytic domain of Abl was reported by Schindler et. al in 2000. The binding of STI-571 promotes the adoption by the kinase of an inactive conformation in which a centrally located <scene name='SandboxPKA/Ac/1'>activation loop</scene> is not phosphorylated.

Catalitic subunit of c-Abl protein is composed by two different regions: <br>

• '''Protein-binding pocket''': lamina-B domain

The unactivated conformation ("DGF out") is due to Phe (in the DGF triad) is oriented near the ATP-binding pocket. When Tyr 412 is phosphorylated, “DFG-in” conformation buries the Phe away from the ATP-binding pocket and the A-loop extends over the C terminus of the catalytic domain). The protein can be considered to be in equilibrium among these conformations, with a shift to the activated form upon phosphorylation.<scene name='Dasatinib/Mtot/2'>(Morphs of the movement)</scene>

== '''Reaction''' ==

Protein kinases are a group of enzymes that possess a catalytic subunit that transfers the gamma (terminal) phosphate from nucleotide triphosphates (often ATP) to one or more amino acid residues in a protein substrate side chain, resulting in a conformational change affecting side protein function.

The enzymes are classified into two broad groups, characterised with respect to substrate specificity:

 

 

- '''Tyrosine specific kinases''': c-Abl is included in this group

There is a highly conserved spatial motif that was found in every active kinase, but missing in inactive kinases.  This motif comprises four non-consecutive hydrophobic residues, two from the N-lobe and two from the C-lobe. It cannot be identified from sequence alone, and the roles of these four residues had never been considered in previous analyses of protein kinase structure and function. Because the middle part of this motif, the C-helix and the activation loop can be very mobile, the hydrophobic spine can be dynamically assembled or disassembled, thereby regulating the protein kinase activity. In this R-spine we can find a backbone of the His/Tyr, anchored to the F-helix which serves as the base of the R-Spine.

Like the R-spine, it comprises residues from both lobes; however, what distinguishes it from the R-spine is that this spine is completed by the adenine ring of ATP. It was thus termed as the catalytic (C) spine. The two C-spine residues in the N-Lobe, Val in β2 and Ala from the ‘‘AxK’’ motif in β3, are docked directly onto the adenine ring of ATP, whereas in the C-lobe it is Leu that docks directly onto the adenine ring. Leu residue lies in the middle of β7.  Identification of the C-spine shows that this helix contributes to the positioning of ATP with respect to the rigid hydrophobic core of the C-lobe.

1- The "latching" of the myristilated NH2-terminal sequence which is directly linked to a myristilation recognition sequence on the c-lobe of the SH1 kinase domain,

3- The interactions between SH3 domain and the C-lobe of the kinase domain. These interactions clamp the structure and prevent the kinase to switch to an active conformation, a process which requires the phosphorylation of Tyr 412 residue and the "unlatching" of the myristoyl group from the C-Lobe of the kinase domain. The attachment of proline-rich SH2 and SH3 ligands leads to the complete switch of the protein to an open, active conformation of the kinase. The NH2-terminal myristilation (autoregulatory role) is deleted during the t(9;22) translocation. <ref>http://atlasgeneticsoncology.org/Genes/ABL.html</ref>

Imatinib achieves BCR-ABL kinase inhibition by binding to the inactive, unphosphorylated conformation of the kinase (DFG-out) , thereby reducing the availability of the catalytically active phosphorylated conformation (DFG-in), necessary for ATP binding. This blocks signal transduction, ultimately resulting in inhibition of proliferation and loss of viability and proliferation.<ref>PMID:16783341</ref>. Imatinib also inhibits the abl protein of non-cancer cells but cells normally have additional redundant tyrosine kinases which allow them to continue to function even if abl tyrosine kinase is inhibited.

Imatinib (a 2-phenylaminopyrimidine derivative) is used for treating CML, gastrointestinal stromal tumours and other diseases. By 2011, Gleevec has been FDA approved to treat ten different cancers.  

<br>'''Pharmacokinetics:''' Imatinib is rapidly absorbed when given by mouth, and is highly bioavailable: 98% of an oral dose reaches the bloodstream. Metabolism of imatinib occurs in the liver and is mediated by several isozymes of the cytochrome P450 system. The main metabolite, N-demethylated piperazine derivative, is also active.  The half-lives of imatinib and its main metabolite are 18 and 40 hours, respectively. <ref>PMID:16485879</ref>

'''[[Dasatinib]]''' is also a second-generation TKI. Dasatinib binds kinases of the SCR family (0,2 nM<IC50<1,1nM). It binds both the active and inactiv forms of the Abl kinase with a higher affinity (300 times) than Imatinib. Dasatinib is active against most BCR-ABL mutants with the exception of T315I. <ref>PMID: 15256671</ref> Dasatinib is well tollerated but adverse effects can include muscle and joint aches, fatigue, dermatologic complaints such as rash and acne, headaches, and diarrhea <ref>PMID: 20008620</ref>

On September 4, 2012, the U. S. Food and Drug Administration approved bosutinib tablets (Bosulif, Pfizer, Inc.) for the treatment of chronic, accelerated, or blast phase Philadelphia chromosome positive (Ph+) chronic myelogenous leukemia (CML) in adult patients with resistance or intolerance to prior therapy. <ref>http://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm318203.htm</ref>

Ponatinib was identified using structure base drug design and focused synthetic libraries of trisubstituted purine analogs. The substance potently inhibits, on nanomolar scale, Src and Bcr-Abl kinases including many common imatinib resistant Bcr-Abl mutations, like T315I mutation. The key structural feature of the molecule is a carbon-carbon triple bond linkage that makes productive hydrophobic contact with the side chain of I315, allowing inhibition of the T315I mutant. The triple bond also acts as an inflexible connector that enforces correct positioning of the two binding segments of AP24534 into their established binding pockets. AP24534 maintains an extensive hydrogen-bonding network and occupies a region of the kinase that overlaps significantly with the imatinib binding site.  <ref>PMID:19878872</ref>

• '''Stopping Imatinib after cytgenetic remission''': in patients who achieve a complete molecular response (CMR), Bcr-Abl transcripts are no longer detectable by PCR.   

Some morphs and scenes were reproduced from the pages from David Canner. Almudena Chaves Pérez; Julio César Corral Serrano; Cristina Murga Montesinos.