Characterisation of the Binding Modes of Inhibitors and Cofactors in the KMO Enzyme

4036 words (16 pages) Essay in Chemistry

23/09/19 Chemistry Reference this

Disclaimer: This work has been submitted by a student. This is not an example of the work produced by our Essay Writing Service. You can view samples of our professional work here.

Any opinions, findings, conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of UK Essays.

Characterisation of the binding modes of inhibitors and cofactors in the KMO enzyme

Strategic Alignment

Briefly outline how the research proposal is aligned to the selected Research Council Objectives [up to 1000 chars inc. spaces].

This research aligns with the strategic priorities of the Medical Research Council (MRC) as it aims to investigate the gaps in knowledge and infrastructure needs to deliver new insights and benefits to human health. As stated in their strategic plan one research priority is “living a long and healthy lifestyle” and in doing this is understanding molecular datasets and diseases by using biological indicators and being able to target treatments. The Molecular, Cellular and Medicine Board (MCMB) is keen to understand dynamic biological systems and this research proposal requires understanding the dynamics of the KMO enzyme and application of this allows development of inhibitors to match the drug site and gaining an understanding of  their mechanism of action. This research uses analytical tools and systems approaches to understand complex and dynamic biology different scales. These approaches will be used to develop cell therapies and cell engineering systems biology for drug target discovery and development. This will help us understand the mechanisms of the enzyme and allow us to develop new drugs that target the enzyme.

Proposal Objectives

List the main objectives of the proposed research in order of priority [up to 2000 chars inc. spaces].

Inhibition of KMO has been identified as a potentially beneficial therapeutic strategy in diseases including Huntington’s disease, Alzheimer’s disease and acute pancreatitis. There is therefore a need to develop potent inhibitors of KMO that do not act as effectors. In this work we aim to detect the flavin movement during inhibition by selected inhibitors. This will allow us to investigate the effects of the substituents. Crystallisation of the KMO enzyme with the inhibitors will allow us to understand their binding and important interactions between the inhibitor and the enzyme which will give us insight for developing more potent inhibitors which could be used clinically.

Crystal structures of KMO have been obtained but there is not thorough characterisation of residues in the active site has been completed. We aim to investigate the binding of NADPH to KMO and will try to trap it and characterise the binding site. The information in this work should complement previously obtained structures and by the use of binding simulations we should be able to characterise the binding site.

 

Lay Summary

Describe the proposed research in simple terms in a way that could be publicised to a general audience [up to 2000 chars inc. spaces].

Tryptophan is an essential amino acid in mammals. 95% of tryptophan metabolism occurs via the kynurenine pathway (KP). The KP uses the enzyme kynurenine monooxygenase (KMO) to catalyse the conversion of L-kynurenine (L-Kyn) to 3-hydrokynurenine (3-HK) in the presence of nicotinamide adenosine dinucleotide phosphate (NADPH) and molecular oxygen. Further conversion of 3-HK produces quinolinic acid (QUIN). The metabolites 3-HK and QUIN have biological activity and imbalances in their levels are associated with neurological disorders such as Alzheimer’s and Huntington’s disease. 3-HK generates unstable, toxic molecules that stimulate neuronal cell death. QUIN also generates unstable, toxic molecules that cause neuronal damage in the central nervous system by activating the N-methyl-D-aspartate (NMDA) receptors. The KP has also been associated in the development of other brain disorders such as schizophrenia, bipolar disease, cancers and autoimmune disorders. The activity of KMO has significant influence over the resultant ratio of neurotoxic to neuroprotective metabolites therefore its inhibition is a leading strategy for normalising the KP pathway and a shifting it towards neuroprotection in neurodegenerative diseases.

KMO undergoes a conformation change when L-Kyn binds. This conformational change frees space in the enzyme for NADPH to bind. The flavin gets changed and 3-HK is produced alongside H2O2. It is proposed that conformational changes via interactions between L-Kyn and the loop of the flavin trigger flavin reduction in KMO. These interactions do not occur in some inhibitors and NADPH cannot bind which means the flavin is not reduced and no H2O2 is produced. The investigation of the binding site of NADPH and of flavin dynamics would be useful for structure-based drug design to stop the production of harmful metabolites and by-products.

Impact Summary

Describe the academic benefit and potential economic and societal impact of this research. Who might benefit from this research and how?  How will the results of this research be disseminated? [up to 2000 chars inc. spaces].

The research aims to deliver improved drugs for neurodegenerative disorders (NDs) such as Alzheimer’s disease (AD). Currently there are around 30 million people worldwide suffering from AD; which is also the main cause of dementia. Drugs are available for the management of the symptoms of AD, however no treatments that halt the disease’s progression or onset are available.

AD is estimated to be the disease with the biggest cost of lost years to disability (11%). Dementia costs the UK £26.3 billion a year and AD costs the UK £17 billion a year. The total number of people with dementia in the UK is predicted to double in the next 25 years; increasing costs to £50 billion. Studies show that ND costs the NHS more than heart diseases and cancer combined. The identification of an effective treatment for AD and other NDs could cut these expenditures worldwide.

Biotechnology and pharmaceutical companies with research and development and/or commercial interests also stand to benefit. It will have important implications for the potential development of a new therapeutic strategy in AD. The work will form the basis of developing treatments for those who have developed or are likely to develop the disease. Significant breakthroughs in KMO-based therapies will encourage participation in the clinical trials.

Understanding the mechanistic underpinning of the disease will assist in the development of treatments. The discovery of underling therapeutic targets in one ND offers hope for researchers and patients alike. This is particularly the case with the kynurenine pathway as it has been implicated in the pathogenesis of several NDs. The neuronal loss during neurodegeneration is irreversible therefore therapies need to target the pre-degenerative disease state. Research in Alzheimer’s, for which there is an enzyme that will inhibit the production of neurotoxic metabolites may form the basis of the development of preventative treatments in other NDs.

 

Finance

Staff

Description (Job title, % effort)

Cost £

PDRA, 100 % effort

37,813

PI 5% effort

3,565

Total £

21,283

 

Consumables

Description (supplier, quantity etc.)

Cost £

15 ml Screw top glass vials (500 units, Sigma)

228

Screw top vial caps (500 units, Sigma)

80

NADPH (5 g, Sigma)

445

Horse Radish Peroxidase (5 KU 50-150 units/mg, Sigma)

34

o-Dianisidine dihydrochloride (5 g, Sigma)

50.70

L-Kynurenine (400 mg, Sigma)

257.2

2 ml Eppendorf (400 units, Sigma)

84.40

Crystallography Mounted CryoLoop – 10 micron (25 units, Hampton Research)

63.15

Crystallography Double/Bubble Epoxy (5 packets, Hampton Research)

8.40

IPTG (5 g, Sigma)

249

LB Broth with agar (Lennox) (1 kg, Sigma)

285

X-Gal (100 mg, Sigma)

98.80

Escherichia coli, Strain K12 (5g, Sigma)

558

Total £

2,441.65

 

Research Facilities/School Services

Description

Cost £

UV/Vis

0

X Ray Crystallography

0

Mass Spec (20 samples)

488.20

EaSTCHEM Research Computing Facility (structural analysis and binding simulation)

4,398

4,886.20

 

Research Council Facilities (details of any proposed usage of national facilities)

Name of Facility

Units

Cost £

Access to Diamond synchtron via the UoE BAG

0

Total £

0

 

Travel and Subsistence

Destination and purpose

Cost £

International MinD Conference 2019, London

1,300

18th International Conference of Structural Biology, Germany

1,700

Total £

 3,000

Project Partner(s) (where applicable):details of partners in the project and their contributions to the research.  These contributions are in addition to resources identified above.

Name of partner organisation

Division or Department

Name of contact

GSK

Discovery Partnerships with Academia

John Liddle

Contribution to project

Description

Value £

Staff/staff time

 

Materials

KMO inhibitor molecules

0

Facilities/equipment

Other

Total £

0

Summary of Financial Resources Required for Project

Funding heading

Cost  £

Staff Costs

21,283

Consumables

2,441.65

Equipment and Facilities

4,886.20

Travel and Subsistence

3,000

Total requested from RC

31,610.85

Justification of Resources

Briefly justify each of the resources requested [up to 2000 chars inc. spaces].

Staff
The proposed research will provide an excellent project for a PDRA student at the early stage of their research career. It requires a student with structural biology skills and they will gain experience in modelling using standard software packages such as Pymol and will be using the PDB to download crystallographic structures. Supervisor time at 2 h per week has been included (5% effort) to allow discussion of the ongoing results.

Consumables
KMO and NADPH are the subjects of this research. Chemical indicators are required to determine the presence of H2O2. Labware is required to prepare, dissolve and store the proteins. For cultivation of E.coli and production of proteins the LB broth with agar is required as it includes the agar, tryptone, yeast extract, NaCl. IPTG and X-Gal are required for blue-white screening which allows rapid efficient identification of bacteria able to produce recombinant protein.

Research Facilities/School Services
X-ray diffraction is required to solve the structure of the NADH binding site.
EastCHEM RCF time is required for the prediction of the binding modes of NADH to KMO. It will also be used to do docking and energy minimisation studies which would require more power. It will be required to interpret the results using binding simulations and by modelling onto previously solved crystal structures. Access charges cover the use of software, machine, running and staff support.

Travel and Subsistence
Funds have been requested to send the PDRA and PI to the International MinD conference in September 2019 and the 18th International Conference of Structural Biology in October 2019. Here they will be able to present the project, aims and findings to a wide academic audience.

Research Proposal (maximum 2 pages which may include schems and figures. Minimum font size Times New Roman 11 point / Arial 10 point).

Background

Figure 1: KMO monomer domain

Kynurenine-3-monooxygense (KMO) is central to tryptophan metabolism in the kynurenine catabolic pathway [1]. KMO is a dimer with asymmetric subunits. The KMO monomer (Figure 1) consists of three domains: the α+ β FAD binding domain, an α+ β domain containing a six-stranded mostly antiparallel β sheet and a C-terminal four-helix bundle domain [2]. The C-terminal is hydrophobic and acts as the mitochondrial anchoring domain and participates in enzymatic activity.

 


The overall reaction is shown in Scheme 1. KMO catalyses L-kynurenine (L-Kyn) transformation to 3-hydroxy-L-kynurenine (3-HK) [1]. When L-Kyn and NADPH bind the NADPH is oxidised and FAD is reduced. This releases NADP+. Dioxygen binds and forms a L-Kynurenine-FAD-hydroperoxide intermediate [3]. This acts as an electron acceptor for L-Kyn oxidation which gives the C4a-hydroxy-FAD. Structure rearrangement forms 3-HK which dissociates and releases water and the free enzyme [2].

Scheme 1: KMO Reaction

When L-Kyn binds to KMO it undergoes a conformational change which allows NADPH to bind. The flavin gets reduced and 3-HK is produced alongside H2O2. Some inhibitors such as non-substrate effectors can stimulate the flavin reduction and produce H2O2 by uncoupling NADPH oxidation from the substrate hydroxylation. Other inhibitors bind and do not cause flavin movement. This due to structure stabilisation inhibits NADPH binding and H2O2 production. Another type of inhibitor acts by tilting the flavin. It is suggested that these prevent productive association with NADPH, prevent substrate binding and they push the FAD into an unproductive position [4].

KMO inhibitors include structures similar to L-Kyn (Figure 2). They are based off the structure of 4-aryl-4-oxobutanoic acid (Figure 2). The carbonyl is essential for inhibition, but not the amine. The amine can be replaced by thioethers which suggests the binding pocket is hydrophobic as the inhibition increases with increasing size [3]. 

Figure 2: L-Kynurenine and 4-Aryl-4oxobutanoic acid Structure

Proposed Work

 

So far there has been no characterisation of the KMO enzyme’s NADPH binding site. In this work we will attempt to characterise this binding site in order to create drugs which will bind to this site.

The KMO enzyme will be expressed in E.coli cells. In order for successful crystallisation the enzyme must be of high purity which would be achieved by using size exclusion purification. We will attempt to crystallise the structure of the KMO enzyme with and without NADPH bound. Because H2O2 production in response to KMO inhibitors is closely linked to the reduction of the flavin, the rate of NADPH oxidation and H2O2 by KMO in the presence of the inhibitors will be investigated using a coupled reaction using o-dianisidine and horseradish peroxidase. The oxidation of o-dianisidine by hydrogen peroxide which is catalysed by HRP will be monitored at 500 nm.. The NADPH analogues without the nicotinamide moiety and not redox-active so should not reduce the flavin and produce peroxide. Therefore, analogues that do not include the nicotinamide moiety should be investigated – ATP. The enzymatic activity of KMO will be determined by measuring the decrease in NADPH using UV/Vis spectrometry and by measuring 3-HK production using a modified liquid chromatography-mass spectrometry method.

Using the University of Edinburgh’s EastCHEM research computing facility, binding simulations will be carried out. The x-ray crystallographic structures of the KMO enzyme with/without NADPH and substrate will be modelled onto each other to allow comparison between the position of the favin and to help us characterise the NADPH binding site.

Figure 3: m-NBA and BA structure monomer Structure

An assay combining KMO and non-substrate effectors will be prepared such as m-NBA (Figure 3) and BA (Figure 3) [5]. The non-substrate effectors have π-π interactions with the enzyme and cause flavin movement and H2O2 production. Co-crystallisation will be tried with NADPH and its analogues. The peroxide production of the enzyme will be investigated using UV/Vis spectroscopy. This will be investigated with NADPH present and with NADPH and the non-substrate effectors present.

Figure 4: GSK180 and GSK428 Structure

Inhibitors that don’t cause flavin movement will be investigated with the KMO enzyme. These inhibitors include GSK180 (Figure 4) and GSK428 (Figure 4) [5]. Co-crystallisation will be attempted in the presence of small molecules like GSK180 in the presence of NADPH. The peroxide production of the KMO enzyme in the presence of these inhibitors will be investigation using UV/Vis spectroscopy.

Figure 5: GSK065 and GSK4 Structure

Co-crystallisation will be attempted with the KMO enzyme and inhibitors that do cause flavin movement such as GSK065 (Figure 5) and GSK366 (Figure 5) [5]. This will be attempted with NADPH and some analogues.

References

  1. Wilson, K., Mole, D., Binnie, M., Homer, N., Zheng, X., Yard, B., Iredale, J., Auer, M. and Webster, S. (2014). Bacterial expression of human kynurenine 3-monooxygenase: Solubility, activity, purification. Protein Expression and Purification , 95, pp.96-103.
  2. Amaral, M., Levy, C., Heyes, D., Lafite, P., Outeiro, T., Giorgini, F., Leys, D. and Scrutton, N. (2013). Structural basis of kynurenine 3-monooxygenase inhibition. , 496(7445), pp.382-385.
  3. Smith, J., Jamie, J. and Guillemin, G. (2016). Kynurenine-3-monooxygenase: a review of structure, mechanism, and inhibitors. Drug Discovery Today , 21(2), pp.315-324
  4. Kim, H., Na, B., Chung, J., Kim, S., Kwon, S., Cha, H., Son, J., Cho, J. and Hwang, K. (2018). Structural Basis for Inhibitor-Induced Hydrogen Peroxide Production by Kynurenine 3- Monooxygenase.
  5. Hutchinson, J., Rowland, P., Taylor, M., Christodoulou, E., Haslam, C., Hobbs, C., Holmes, D., Homes, P., Liddle, J., Mole, D., Uings, I., Walker, A., Webster, S., Mowat, C. and Chung, C. (2017). Structural and mechanistic basis of differentiated inhibitors of the acute pancreatitis target kynurenine-3-monooxygenase. Nature Communications, 8, p.15827.

Project Time Plan over 12 month period: Based on September 2019 Start Date

Months of Project

1

2

3

M1

4

5

M2

6

7

8

M3

9

10

M4

11

M5

12

Training

Training in use of EastCHEM RCF

Research

Preparation of KMO enzyme

Initial characterisation of KMO enzyme crystals

Preparation of KMO and inhibitor co-crystals

Characterisation of crystals – X-ray data collection, structure solution and refinement

Preparation of KMO and inhibitor and NADPH crystals

Analysis and interpretation of crystal structures using EastCHEM RCF

Conference Attendance

Paper writing

 

 

Milestones

 

M1

Crystal structures of KMO

M2

Crystal structures of KMO and different inhibitors

M3

Crystal structures of KMO, inhibitors and NADPH

M4

Characterisation of binding modes

M5

Characterisation of NADPH binding site

Cite This Work

To export a reference to this article please select a referencing stye below:

Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.

Related Services

View all

DMCA / Removal Request

If you are the original writer of this essay and no longer wish to have the essay published on the UK Essays website then please: