Question 1

The CZ4 receptor is expressed on the surface of many cells; activation by its endogenous ligand triggers downstream signalling pathways. Overexpression of the CZ4 receptor is observed in many cancer types and this protein represents an attractive therapeutic target. In recent years, a wide range of CZ4 receptor antagonists have been developed, including small molecules and peptides.

Scenario: The therapeutics company you work for wishes to develop a next generation, orally administered CZ4 receptor antagonist. Your team has used known antagonists to build a pharmacophore model and plan to use it to screen computationally, a virtual library of compounds for ligands that contain this pharmacophore.

Answer ALL parts of this question.

a) Describe the potential advantages and limitations of this computational screening approach

(4 marks)

Answer  should  use  the  idea  of a  pharmacophore  for  full  credit  and  consider  both limitations/adv for full credit. Any reasonable adv/limitations accepted.

•   A pharmacophore is the 3D collection of steric and electronic features needed to ensure

optimal interactions with a specific target and to trigger (or block) its biological response (1 mark)

Examples of potential advantages

•   Using a pharmacophore to screen a virtual library could help identify molecules that contain these features so more likely to be active – increase efficiency of process

•   Can potentially  screen  more  compounds virtually than  in  an  assay – might  increase diversity of possible hits

Examples of potential limitations

•   Computational approach alone is not sufficient; testing still required

•   Active conformations may not be known and modelled

•   Wouldn’t necessarily broaden chemical diversity if based on existing structures/IP challenges if too close to known structures

•   (Scoring metrics can be variable)

b)  Answer ALL parts of this question.

Your team identifies a hit, 1, and conducts a structure-activity relationship (SAR) study.  Compounds 1-7 were tested in an assay testing inhibition of CZ4 receptor activation. The assay was conducted at physiological pH and 37 °C.

i)  Based on the structural and assay data for compounds 1-7 shown, provide a list of SAR features of the compounds in Series A. Provide a justification for each feature.

(4 marks)

•   A hydrogen bond donor is required at position X                                (1 mark)

•   Evidence from reduction in activity when H bond acceptor activity retained (4) as well as adding an alkyl group (2)                                                         (1 mark)

•   Potentially only a small pocket around X as a larger group has a greater drop in activity than from loss of NH alone

(1 mark)

•   Basic centre/hydrogen donor at R1 is less significant for binding as significant changes to character e.g. loss of basic centre have less impact(1 mark)

•   If not considering sterics at X then steric comment about R1 can also be given credit (1 mark)

ii)    Compound 2 was   selected   for   further   evaluation   and   some   key physiochemical  and  pharmacokinetic  characteristics  determined.  These  are summarised below:

From the data provided, identify characteristics of compound 2 that may be problematic, and explain your reasoning.

(3 marks)

Oral bioavailability of this compound is low (1 mark)

Likely origin of this is poor absorption given the high number of basic centres, leading to very low logD (1 mark)

Very flexible alkyl linker may contribute to low absorption (1 mark)

Other acceptable challenges from data e.g. logD linking to fast renal clearance (0.5), specific discussin of possible metabolism of structure (0,5)

iii)  Using the information available, suggest structures for two new analogues that might address or improve on the problematic characteristics you have identified for compound 2.

(3 marks)

Suggested compounds need to retain the features identified by SAR as important but look to reduce number of basic centres. Must be a valid chemical structure, must be justified.   Variations to tertiary amine would be accepted as no data given to show importance or     not.

0.5 mark – per valid structure

1 mark per reasonable explanation

The following are representative examples only:

N

H

N

O

HN

Amide is no longer basic. data suggeste tolerated based on 7

N

N

HN

dats suggests amine may not be important

explore removing entirely

N

N

H                                                         N                N

N               N

HO O

restrict conformational freedom

Try to retain a H bond donor but no longer basic  (also aromatic)

c) It was found that in addition to its activity against CZ4, compound 2 also inhibits CYP2D6 (IC50 = 0.3 µM). Explain how and why this property might ultimately affect the clinical use of compound 2 as a therapy?

(2 marks)

•   CYP2D6 is a Phase I enzyme involved in the metabolism of many drugs (1 mark)

•   Risk ofharmful drug-drug interactions as inhibition could prevent other drugs being metabolised as expected and lead to unexpected accumulation and/or higher drug concentrations that could exceed safe therapeutic window (1 mark)

d) In vivo metabolism studies in the rat show that compounds 2 and 3 have several common urinary metabolites. Provide an explanation for how this may occur.

(2 marks)

-            Several possibilities, so any plausible answer permitted. For example:

o Demethylation of 3 would yield compound 2; subsequent biotransformations would likely be similar (qualitatively)

o Methylation of 2 would yield compound 3; subsequent biotransformations would likely be similar (qualitatively)

o Dealkylation of both compound 2 and compound 3 would yield a common metabolite;

o Note: They may share several metabolites as it is common for there to be multiple routes of metabolism for a given compound.

e) In vivo metabolism studies reveal one of the urinary metabolites of compound 2 to be a mercapturic   acid.   What   can   be   inferred   form   this   observation   about   the biotransformation(s) of compound 2 in vivo?

(2 marks)

Any two of the following awarded 1 mark (max 2 marks):

-            It is highly likely that one or more biotransformations of compound 2 have resulting in a reactive metabolite (electrophilic)

-            Conjugation of compound 2 directly (displacement of F by GSH) or with a reactive metabolite has occurred

-           A GSH conjugate of the parent or a metabolite has undergone hydrolysis and N-acetylation ot form the final mercapturate.

Q2 - High Throughput and High Content Screening

Answer ALL parts of this question

a)  Answer ALL parts of this question.

Traumatic Brain Injury (TBI) is typically diagnosed by brain imaging, however a blood test would be quicker and cheaper.

i)   To identify potential protein biomarkers an untargeted, bottom-up proteomics workflow is developed using blood samples taken from a group of patients diagnosed with TBI from brain imaging and from a second group with no signs of TBI.

Explain why this strategy is the most appropriate in this case.

(5 marks)

Bottom-up refers to proteomic methods where the proteins in the sample are converted to tryptic peptides, the masses of the peptides are used to derive a peptide mass fingerprint  (1 Mark). Additionally the peptides may be sequenced, and the proteins identified by       comparison with a genome sequence (1 mark).

Untargeted means that the proteomic analysis is not looking for pre-specified or known proteins (1 mark).

As significant differences in protein composition of the blood between the two groups are being sought and blood is a complex mixture of proteins, high resolution is needed (1       marks) and peptide separations by LC-MS have this resolution.

LC-MS quantitation based on multiple peptides is more reliable than a single protein concentration (1 mark).

ii)   Describe a possible liquid chromatography-mass spectrometry workflow that could be used to identify potential protein biomarkers. Your answer should include a description of any sample pre-treatment, the type of columns used in the liquid chromatography step, as well as the ionization mechanism and mass analyser that could yield amino-acid sequence information.

(5 marks)

Initially the blood is allowed to clot to remove cells and fibrin (fibrinogen) and the supernatant treated with trypsin to generate peptides (1 mark).

The  peptides  are  then  separated by  HPLC  using  reverse  phase  and  strong  cation  exchange chromatography (1 mark).

The eluted peptides are then ionised by electrospray and separated by a quadrupole mass analyser generating a peptide mass fingerprint (1 mark).

The peptides are then then introduced into a collision cell where the peptides are fragmented by collision induced dissociation and the fragments separated in a second mass analyser (i.e. a tandem mass spectrometer) (1mark)

and then used to construct the peptide amino acid sequence as CID fragments peptides at the amide bond (1 mark).

iii)   The results  of your untargeted proteomics  study  suggest that high blood concentrations of the protein S100B could be a biomarker for TBI and that concentrations in saliva would be similarly elevated. Describe how you could use a protein microarray to test this idea. Your answer should include  a description of the reagents needed and how the results would be interpreted. A detailed description of the array fabrication methods is NOT required.

(4 marks)

A set of samples (plasma and saliva) from a group of individuals diagnosed with TBI are spotted on the array surface such that they are irreversibly bound (1 mark).

The array is then treated with an anti-S100B antibodies, typically monoclonal ones from mouse that bind to S100B in the sample spot (1 mark).

To develop a signal a fluorescently labelled anti-mouse antibody is then added (1 mark)   and the fluorescence intensity of each spot is then proportional to the amount of S100B in the sample (1 mark).

b)  Answer ALL parts of this question.

The principal components analysis (PCA) scores plot below is ofurine metabolite signatures of samples taken from pancreatic cancer patients.  The samples have been quenched and stored under different conditions. The three different treatments of the urine samples are treatment with cold methanol, ultrafiltration and treatment with cold saline solution.

i) What do you understand by the term ‘Principal Components Analysis’ and why is it commonly used with metabolomics data?                     (2 marks)

ii) Using the PCA scores and loadings from the above plot to justify your answer, describe if and how storage temperature appears to affect the urinary metabolite profiles obtained in this study.

(2 marks)

Principal components analysis is a statistical procedure that allows you to summarize the information content in large data sets by means of a smaller set of summary indices (1     mark). Metabolomics data typically comprises very large data sets and PCA identifies the most statistically significant variables (1 mark). The closest clustering is seen in the         samples at the lowest storage temperature of -20oC (1 mark).  In all cases shows that is    this the optimal storage condition (1 mark).

iii) Using the PCA scores and loadings in the above plot to justify your answer, identify which  quenching  conditions would be most  appropriate to use to minimise the preanalytical variability  attributable to  this  aspect  of sample collection. Which would be the most detrimental quenching conditions for the urine samples and which would result in the greatest loss of metabolites over the longest time shown of 48 hrs.

(2 marks)

The combination of storage conditions of 40oC (1 mark). and quenching method of cold saline (1 mark).