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This Sandbox is Reserved from January 19, 2016, through August 31, 2016 for use for Proteopedia Team Projects by the class Chemistry 423 Biochemistry for Chemists taught by Lynmarie K Thompson at University of Massachusetts Amherst, USA. This reservation includes Sandbox Reserved 425 through Sandbox Reserved 439.

MTH1/crizotinib

Introduction

MTH1 is known as 7,8-dihydro-8-oxuguanine triphosphate (2). MTH1 is a protein in the human body encoded by the NUDT1 gene. In general, MTH1 is often synonymous for the gene NUDT1.

It is a protein that belongs to the Nudix hydrolase family and is characterized by a conserved of 23 – residue sequence segment. Since, MTH1 is part of the Nudix hydrolyse family, it has the typical Nudix structure, fold, structure arranged as a mixed of with one on each side. MTH1 is a protein prevents the misincorporation of oxidized nucleotides into DNA. Hydrolysis of a triphosphate nucleotide to a monophosphate nucleotide prevents this incorporation.The hydrolysis of the triphosphate nucleotide 8-oxo-dGTP to the monophosphate nucleotide 8-oxo-dGMP will not be recognized by DNA polymerase, preventing its incorporation into DNA (1).Specifically, the hydrolysis of the triphosphate nucleotide 8-oxo-dGTP to the monophosphate nucleotide 8-oxo-dGMP will not be recognized by DNA polymerase, preventing its incorporation into DNA.Cancer cells frequently overexpress MTH1. In order to slow down cancerous tumor growth, suppressing this protein could cause an increase in DNA misincorporations and induce cell death.

MTH1 is inhibited by Crizotinib has an aminopyridine structure and functions as a protein kinase inhibitor because it binds with the ATP – binding pocket of target kinase (3). Crizotinib has a high impact in drug designer because it causes tumors to shrink or stabilize. When (S)-Crizotinib inhibits MTH1, it increases the DNA single – strand to break, it activates DNA repair in colon carcinoma cells. When (S)-crizotinib inhibits MTH1, the protein can longer function as a nucleotide ‘healer’, and impairment rate of the nucleotide pool is increased. MTH1 is broadly used in drug production because based on researched made, it is proved that it helps fighting tumors. This because a loss of function of MTH1 impaires growth of KRAS tumor cells and an overexpression of MTH1 mitigates the cancer cells(4). Reactive oxygen species (ROS) produced as byproducts from cellular metabolism can cause damage to DNA and free nucleotides. Misincorporation of oxidized nucleoside triphosphates into DNA and RNA is one cause of mutation during transcription and replication that can result in DNA damage and ultimately cell death . This misincorporation of oxidized nucleotides could be used as a tool to combat tumor growth. Also, MTH1 assists in eliminating RAS – induced oxidative damage to prevent induction of senescence, so any inhibitors of MTH1 increase the amount of RAS induced oxidative damage and make cancer cells to enter pre – mature senescence which reduces cancer growth.

Overall Structure

MTH1/S-crizotinib is a member of a protein family of phosphorohydrolases known as the Nudix family (2). Proteins in the Nudix family are used to hydrolyze nucleoside phosphates; they also act as catalysts for the same reaction. Proteins in the Nudix family consist of two unique parts:

a Nudix fold, and a Nudix motif</scene> (2). The Nudix fold is the secondary structure, or backbone, of the protein; the backbone has an α/β/α structure, meaning that the backbone consists of mixed beta sheet (gold) with an alpha helix on each end (magenta) (2).

The second component of proteins in the Nudix family, such as MTH1/S-crizotinib, is the Nudix motif, which is different for every protein. The Nudix motif in MTH1/S-crizotinib is made up of 23 amino acid sequence that orient into a small alpha helix (dark purple) (5,6). This helix attached to one of the main alpha helices in MTH1/S-crizotinib’s backbone. The amino acids that catalyze the hydrolysis reactions are located in this alpha helix (2,5,6). All of these components establish the

The polarity of MTH1/S-crizotinib (4c9x) where the magenta represents the polar aspects of the protein and gray symbolizes the non-polar parts

Drag the structure with the mouse to rotate

polarity</scene> of the protein; the outer parts of the helices and beta sheet are polar and hydrophilic (purple), while the inner parts and non polar and hydrophobic (gray).

MTH1/S-crizotinib also has an

The different components of the active site in MTH1/S-crizotinib (4c9x) are highlighted in this structure

Drag the structure with the mouse to rotate

active site pocket</scene> that is formed between the beta sheet and one of the alpha helices (2). This pocket is built out of the residues Leu9 (aqua), Phe27 (violet), Phe72 (magenta), Met81 (lime green), Val83 (brown), Trp117 (orange), Trp123 (gold), and Phe139 (salmon). Furthermore, the amino acid

Glu100</scene> is used to coordinate metal-binding in the protein and is located outside the alpha helix motif (2).

MTH1/S-crizotinib has three different types of ligands attached to it: one chloride ligand, five sulfate ligands, and the

S-crizotinib ligand</scene>. Crizotinib is a tyrosine kinase inhibitor, and its molecular structure is C21H22Cl2FN5O. The stereochemistry of the crizotinib determines if this inhibitor prevents MTH1 from carrying out its purpose in cancer cells; the S-crizotinib stops MTH1 from working while the R-crizotinib has no effect on the protein (3,7).

1 Binding Interactions
2 Additional Features
3 Quiz Question 1
4 Quiz Question 2
5 See Also
6 Credits
7 References

Binding Interactions

Human MutT homologue, also known as MTH1, is a nucleotide pool sanitization enzyme and the target for crizotinib, a competitive inhibitor. Crizotinib is a kinase inhibitor that suppresses MTH1 activity in concentrations as low as nanomoles/liter, suggesting high levels of specificity (1). Crizotinib acts on MTH1 through competitive inhibition, meaning it displaces kinases, the usual substrates. Because the specificity of the binding pocket of MTH1 depends on the stabilization of the enol tautomer of 8-oxo-dGTP, crizotinib’s chemistry interacts with this conformation to be effective (2). The binding of crizotinib yields no major structural changes in the MTH1 protein.

The most significant interactions between crizotinib and the MTH1 active site are hydrogen bonding interactions. Hydrogen bonding occurs between the Asparagine residue 33 and the N3 of 8-oxo-dGMP and between the acidic Aspartate residues 119 and 120 and the oxygen in 8-oxo-dGMP. Both the Asp 119 residue and the Asn 33 residue are crucial to binding specificity (2). Because crizotinib has a chiral center, the (S) and (R) enantiomers act differently in the binding pocket of MTH1. After recent testing of MTH1 using both enantiomers, the (S) enantiomer has shown a higher affinity for the MTH1 binding pocket, whereas the (R) enantiomer is a better inhibitor for most other similar protein kinases. An eclipsed conformation of a methyl group at the chiral center and a chlorine attached to the benzyl ring are responsible for reducing the energetic favorability of the (R) enantiomer in the binding pocket of MTH1 (1).

Additional Features

The can be seen inside the globular protein structure. The majority of the residues appear exposed on the protein surface rather than buried within the protein. Acidic and basic residues are also prevalent within the binding pocket.

MTH1 structurally recognizes and distinguishes the 8-oxo-dGTP from 8-oxo-dGMP different from one another. In order to understand how MTH1 functions, this is essential to explore. The protein MTH1 has been co-crystallized with the substrate 8-oxo-dGTP and the structure of the crystal was observed (2). A reaction took place and the product, 8-oxo-dGMP, was bound in a pocket formed by the . This green scene representation is not exact, and a structural change may have occurred upon 8-oxo-dGTP binding. make hydrogen bonds to the 2-amino group of 8-oxo-dGMP. This amino position is central for the interaction. Nucleotide analogs were used to test MTH1 substrate recognition; results were in agreement. Removal of the 2-amino group led to a 10-fold less effective degradation (2). Mg²⁺ also appeared in the co-crystal structure, but the electron density, coordination geometry, and coordination distances showed Mg²⁺ was not bound to the complex. It was hypothesized that Mg²⁺ may coordinate with the substrate of the triphosphate nucleotide (2). Many other residues involved in specificity and recognition have been identified.

8-oxo-dGTP to 8-oxo-dGMP
Modified from: http://aci.cm.umk.pl/~karolb/activity.htm

MutT is a homolog of MTH1, shares the same nubix family, and is expressed in Escherichia coli. MutT also catalyzes the hydrolysis of 8-oxo-dGTP to 8-oxo-dGMP, but has a 21% (low) sequence identity to MTH1. Co-crystal structure produced the anti conformation of bound 8-oxo-dGMP in MTH1 while syn conformation was observed in MutT (2). From the crystal structure it was determined that the base–protein interactions are quite different and reveal no conservation of structure. Although the proteins shared a conserved catalytic region, the nucleotide recognition is not conserved. Diverging evolutionary routes have been taken, and one possibility is to allow MTH1 to evolve more non-specific substrate specificity (2). Understanding the mechanisms of non-specific substrate binding could be an important step to developing new inhibitors for similar enzymes.

Quiz Question 1

- This scene colors the protein from the N-terminus to the C terminus.

- This scene shows the active site of the protein. Individual amino acids are labelled and active site residues are colored according to charge (red = positive, blue = negative, white = uncharged). The ligand (S-crizotinib) in the center is colored by atom (Black = Carbon, Red = Oxygen, Blue = Nitrogen, Green = Halogen).

Using the green scenes provided above, answer the following:

a.) Suppose there is a point mutation causing a change at ASP-120 in MTH1. Which of the following amino acids substitutions would be least likely to affect substrate binding? Explain your reasoning.

1.) Lysine (LYS)
2.) Glutamate (GLU)
3.) Glycine (GLY)

b.) If there were a point mutation at PHE-27, which of the following amino acid substitutions would likely affect ligand binding the most? Again, explain your reasoning.

1.) Tryptophan (TRP)
2.) Tyrosine (TYR)
3.) Arginine (ARG)

Quiz Question 2

The above two green scenes highlight the chiral center of the MTH1 inhibitor called crizotinib. Research has shown that the s-enantiomer binds with more affinity to it's MTH1 substrate but that it is not due to interaction with the protein itself at the binding site.

Can you see an unfavorable interaction near the chiral center in the r-enantiomer that does not exist in the s-enantiomer? Explain.

Crizotinib Structure Modified from adooq.com

Credits

Introduction - Megi Marina

Overall Structure - Cassandra Martin

Drug Binding Site - Ben Ryter

Additional Features - Eric Rice

Quiz Question 1 - Matt Tuttle

Quiz Question 2 - Colin Hannahan

References

Huber, Kilian V. M., Eidarus Salah, Branka Radic, Manuela Gridling, Jonathan M. Elkins, Alexey Stukalov, Ann-Sofie Jemth, Camilla Göktürk, Kumar Sanjiv, Kia Strömberg, Therese Pham, Ulrika Warpman Berglund, Jacques Colinge, Keiryn L. Bennett, Joanna I. Loizou, Thomas Helleday, Stefan Knapp, and Giulio Superti-Furga. "Stereospecific Targeting of MTH1 by (S)-crizotinib as an Anticancer Strategy." Nature 508 (2014): 222-27. Web.
Svensson, Linda M., Ann-Sofie Jemth, Mathhieu Desroses, Olga Loseva, Thomas Helleday, Martin Higbom, and Pal Stenmark. "Crystal Structure of Human MTH1 and the 8-oxo-dGMP Product Complex." Http://www.sciencedirect.com. N.p., 19 Aug. 2011. Web.
"Crizotinib | C21H22Cl2FN5O - PubChem." Crizotinib | C21H22Cl2FN5O - PubChem. N.p., n.d. Web. 03 Apr. 2015.
Fricker, Janet. "Two Studies Show MTH1 Offers Promising New Target for Cancer Treatment." . Ecancer. Cancer Intelligence, 02 Apr. 2014. Web. 03 Apr. 2015.
M. Mishima,; Y. Sakai,; N. Itoh,; H. Kamiya,; M. Furuichi,; M. Takahashi, Y. Yamagata, S. Iwai,; Y. Nakabeppu,; and M. Shirakawa. “Structure of the Human MTH1, a Nudix Family Hydrolase that Selectively Degrades Oxidized Purine Nucleoside Triphosphates”, J Biol Chem., 279, no. 32, 33806-33815, 2004.
S.B. Gabelli,; M.A. Bianchet,; M.J. Bessman,; L.M. Amzel “The structure of ADP-ribose pyrophosphates reveals the structural basis for the versatility of the Nudix family”, Nat Struct Biol, 8, 467-472, 2001.
"Crizotinib, (S)-." (S)-Crizotinib. N.p., n.d. Web. 03 Apr. 2015.

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@@ Line 20: / Line 20: @@
 ==Overall Structure==
-<Structure load='4c9x' size='300' frame='true' align='right' caption='Basic Structure of MTH1/S-crizotinib' scene='48/483888/Basic/1' />
+<Structure load='4c9x' size='300' frame='true' align='right' caption='Basic Structure of MTH1/S-crizotinib 4c9x' scene='48/483888/Basic/1' />
-<br> MTH1/S-crizotinib is a member of a protein family of phosphorohydrolases known as the Nudix family (2). Proteins in the Nudix family are used to hydrolyze nucleoside phosphates; they also act as catalysts for the same reaction. Proteins in the Nudix family consist of two unique parts: <scene name='48/483888/Different_backbone_components/1'>a Nudix fold, and a Nudix motif</scene> (2). The Nudix fold is the secondary structure, or backbone, of the protein; the backbone has an α/β/α structure, meaning that the backbone consists of mixed beta sheet- gold with an alpha helix on each end- magenta (2).
+<br> MTH1/S-crizotinib is a member of a protein family of phosphorohydrolases known as the Nudix family (2). Proteins in the Nudix family are used to hydrolyze nucleoside phosphates; they also act as catalysts for the same reaction. Proteins in the Nudix family consist of two unique parts: <Structure load='4c9x' size='300' frame='true' align='right' caption='The Nudix fold and Nudix motif of the MTH1/S-crizotinib protein 4c9x' scene name='48/483888/Different_backbone_components/1'>a Nudix fold, and a Nudix motif</scene> (2). The Nudix fold is the secondary structure, or backbone, of the protein; the backbone has an α/β/α structure, meaning that the backbone consists of mixed beta sheet (gold) with an alpha helix on each end (magenta) (2).
-	The second component of proteins in the Nudix family, such as MTH1/S-crizotinib, is the Nudix motif, which is different for every protein. The Nudix motif in MTH1/S-crizotinib is made up of 23 amino acid sequence that orient into a small alpha helix- dark purple (5,6). This helix attached to one of the main alpha helices in MTH1/S-crizotinib’s backbone. The amino acids that catalyze the hydrolysis reactions are located in this alpha helix (2,5,6). All of these components establish the <scene name='48/483888/Polarity_of_mth1-s-crizotinib/1'>polarity</scene> of the protein; the outer parts of the helices and beta sheet are polar and hydrophilic (purple), while the inner parts and non polar and hydrophobic (gray).
+	The second component of proteins in the Nudix family, such as MTH1/S-crizotinib, is the Nudix motif, which is different for every protein. The Nudix motif in MTH1/S-crizotinib is made up of 23 amino acid sequence that orient into a small alpha helix (dark purple) (5,6). This helix attached to one of the main alpha helices in MTH1/S-crizotinib’s backbone. The amino acids that catalyze the hydrolysis reactions are located in this alpha helix (2,5,6). All of these components establish the <Structure load='4c9x' size='300' frame='true' align='right' caption='The polarity of MTH1/S-crizotinib (4c9x) where the magenta represents the polar aspects of the protein and gray symbolizes the non-polar parts' scene name='48/483888/Polarity_of_mth1-s-crizotinib/1'>polarity</scene> of the protein; the outer parts of the helices and beta sheet are polar and hydrophilic (purple), while the inner parts and non polar and hydrophobic (gray).
-	MTH1/S-crizotinib also has an <scene name='48/483888/Active_site_of_mth1/1'>active site pocket</scene> that is formed between the beta sheet and one of the alpha helices (2). This pocket is built out of the residues Leu9- aqua, Phe27- violet, Phe72- magenta, Met81- lime green, Val83- brown, Trp117- orange, Trp123- gold, and Phe139- salmon. Furthermore, the amino acid <scene name='48/483888/4c9x-_the_glu_100_amino_acid/1'>Glu100</scene> is used to coordinate metal-binding in the protein and is located outside the alpha helix motif (2).
+	MTH1/S-crizotinib also has an <Structure load='4c9x' size='300' frame='true' align='right' caption='The different components of the active site in MTH1/S-crizotinib (4c9x) are highlighted in this structure' scene name='48/483888/Active_site_of_mth1/1'>active site pocket</scene> that is formed between the beta sheet and one of the alpha helices (2). This pocket is built out of the residues Leu9 (aqua), Phe27 (violet), Phe72 (magenta), Met81 (lime green), Val83 (brown), Trp117 (orange), Trp123 (gold), and Phe139 (salmon). Furthermore, the amino acid <Structure load='4c9x' size='300' frame='true' align='right' caption='The Glu100 amino acid in MTH1/S-crizotinib (4c9x)' scene name='48/483888/4c9x-_the_glu_100_amino_acid/1'>Glu100</scene> is used to coordinate metal-binding in the protein and is located outside the alpha helix motif (2).
-	MTH1/S-crizotinib has three different types of ligands attached to it: one chloride ligand, five sulfate ligands, and the <scene name='48/483888/S-crizotinib_ligand/1'>S-crizotinib ligand</scene>. Crizotinib is a tyrosine kinase inhibitor, and its molecular structure is C21H22Cl2FN5O. The stereochemistry of the crizotinib determines if this inhibitor prevents MTH1 from carrying out its purpose in cancer cells; the S-crizotinib stops MTH1 from working while the R-crizotinib has no effect on the protein (3,7).
+	MTH1/S-crizotinib has three different types of ligands attached to it: one chloride ligand, five sulfate ligands, and the <Structure load='4c9x' size='300' frame='true' align='right' caption='The S-crizotinib ligand in MTH1/S-crizotinib (4c9x)' scene name='48/483888/S-crizotinib_ligand/1'>S-crizotinib ligand</scene>. Crizotinib is a tyrosine kinase inhibitor, and its molecular structure is C21H22Cl2FN5O. The stereochemistry of the crizotinib determines if this inhibitor prevents MTH1 from carrying out its purpose in cancer cells; the S-crizotinib stops MTH1 from working while the R-crizotinib has no effect on the protein (3,7).
-Val83- brown
 <br>

Sandbox Reserved 431

From Proteopedia

Revision as of 12:52, 5 April 2015

MTH1/crizotinib

Introduction

Overall Structure

Contents

Binding Interactions

Additional Features

Quiz Question 1

Quiz Question 2

See Also

Credits

References

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