Download Michael Wong

Chemical, Metabolic &
Structure-Activity Relationships
to Probe Abacavir Toxicity
Michael Wong
Introduction to Abacavir
•
Antiretroviral drug used in HAART treatment of the HIV-1 virus
•
Nucleoside Reverse Transcriptase Inhibitor (NRTI)
•
Toxicity of abacavir (ABC):
– Idiosyncratic hypersensitivity reaction
– Symptoms include headaches, skin rash and gastrointestinal
problems
– Occurs in patients who bear the HLA-B*57:01 allele
•
Determination of toxicity can be used to determine mechanisms of drug
hypersensitivity reactions related to HLA alleles
Abacavir
Aims
•
To develop a synthesis for a deuterated form of ABC to probe 5’-OH
oxidative ABC metabolism
•
To undertake a series of metabolism experiments to determine
contribution of ABC metabolism to its associated toxicity
•
To synthesise a range of ABC analogues using a SAR approach to
block metabolism and/or binding within HLA-B*57:01
•
To use synthesised analogues in a range of toxicological assays to
further probe its toxicity
•
To use all chemical, metabolic and toxicology results to deduce or
validate a mechanism of ABC toxicity
Hapten Hypothesis & ABC Metabolism
Hapten Hypothesis
•
Processing of drug to form a reactive metabolite
•
Reaction with endogenous proteins
•
Covalently binds to MHC
•
Immunogenic
Aldehyde
Application to ABC
•
Major metabolites:
• Carboxylic acid metabolite
• Glucuronide metabolite
•
Oxidation results in formation of reactive
aldehyde intermediate (Hapten hypothesis)
•
Aldehydes can react with proteins
via a 1,2-Schiff base or 1,4-Michael addition
Major carboxylic
acid isomer
Abacavir
Glucuronide
Walsh et al (2002)
ABC & D2-ABC
•
Deuterium is a naturally occurring non-radioactive isotope of hydrogen (100 % heavier than
hydrogen)
•
Very similar properties to hydrogen and when incorporated into molecules deuterium is essentially
indistinguishable (pharmacologically sound – no significant toxicological problems)
•
Can be used to determine mechanisms of drug bioactivation
•
Deuterated-ABC will assist in probing the oxidative metabolism of ABC
•
Proton/deuterium extraction will be monitored
Abacavir
Deuterated-Abacavir
Kinetic Isotope Effect
•
Kinetic Isotope Effects (KIE) are evident when certain atoms in a reactant are isotopically
substituted
•
KIE is defined as the ratio of the rate constants of bond cleavage
•
A C-D bond is more stable than a C-H bond: More energy is required to break the C-D bond
therefore slower bond cleavage
•
Vmax and Km parameters need to be calculated to compare two compounds
Vmax is the maximum velocity of the reaction at saturating substrate concentrations
Km is the substrate concentration at which the rate is half of Vmax
Vmax
kH
KIE 
kD
Rate
•
•
Km
Substrate Conc [S]


KIE 
Vmax H
Vmax D
Metabolism – D2-ABC
8-step synthesis used to synthesise D2-ABC (two hydrogen atoms substituted for deuterium at 5’-carbon )
D2-ABC
Zhou et al (2008)
Metabolism
• Series of metabolism experiments conducted:
• [Product formation] vs protein concentration (0.1 – 1.5 mg/mL)
• [Product formation] vs time (1 – 24 hours)
• Comparing metabolic pathway of ABC and D2-ABC (with use of LADH and ALDH inhibitors)
• Once experiments completed incubations of [product formation] vs time over 120 minutes completed
• All analysis was completed using LC-MS/MS
Abacavir
Deuterated Abacavir
Carboxylic Acid
Major COOH
isomer
Major COOH
isomer
ABC
D2-ABC
Metabolism
ABC (Rate = 0.083 µM/min)
14
y = 0.083x + 1.1059
R² = 0.9851
10
8
6
4
2
0
0
20
40
60
80
Time (min)
100
120
140
D2- ABC (Rate = 0.017 µM/min)
2.5
[D2-ABC-COOH] (μM)
[ABC-COOH] (μM)
12
Representative graphs obtained from samples
of concentration of [product formation] vs time
at 0.5 mM substrate concentration
y = 0.0165x + 0.1499
R² = 0.9746
2.0
1.5
1.0
0.5
0.0
0
20
40
60
80
Time (min)
100
120
140
Metabolism
All rates from linear graphs were obtained and plotted against substrate concentration
Concentration
(mM)
Mean rate for
ABC-COOH
(μM min-1) ± SD
Mean rate for
D2-ABC-COOH
(μM min-1) ± SD
0.5
0.815 ± 0.02
0.023 ± 0
1
1.815 ± 0.304
0.42 ± 0.028
2.5
3.985 ± 0.954
1.485 ± 0.181
5
6.227 ± 0.078
2.88 ± 0.526
7.5
7.52 ± 0.484
3.265 ± 0.31
10
7.362 ± 1.245
3.618 ± 0.578
20
7.545 ± 1.823
3.69 ± 0.495
Metabolism
Michaelis-Menten graph from which Vmax and Km values can be calculated
Rate vs substrate concentration (rates obtained from linear graphs of [product formation] vs time)
10
V e lo c ity [  M /m in ]
A b a c a v ir
D 2 - A b a c a v ir
8
n=2
6
4
2
0
0
5
10
15
20
25
S u b s t r a t e C o n c e n t r a t io n [ m M ]
Compound
Abacavir
Vmax Value ± SD
Km Value ± SD
9.564 ± 0.875
3.186 ± 0.903
KIE 
Vmax H
Vmax D
 1.88
D2-Abacavir
5.093 ± 0.62
5.076 ± 1.603
 
D2-ABC – Pharmacology & Toxicology
Toxicology
• T cell clones from ABC responsive
HLA-B*57:01 positive patients
• ELISpot assay (cytokine IFNγ release)
• Comparable toxicology results
compared to ABC
Pharmacology
•
Cytotoxicity determined using MTT assay
in MT4 cells, anti-viral activity determined
using HIVIIIB propagated in MT4 cells
•
Comparable anti-viral activity to ABC
Cytotoxicity
IC50 (µM)
Cytotoxicity
IC50 (µM)
Efficacy EC50 (µM)
Sample
24 hours
5 days
5 days
ABC
370.4±10.8
240.2±16.4
7.45±0.73
D2-ABC
397.2±6.71
255.7±6.36
13.43±1.46
Metabolism Summary
•
D2-ABC was successfully synthesised via an 8-step route
•
Series of metabolism experiments conducted to determine similarity of D2-ABC compared
to ABC with respect to 5’-OH metabolism
•
Rate of oxidative metabolism was monitored over 120 minutes and Michaelis-Menten
parameters Vmax and Km were determined
•
A KIE value of 1.88 obtained
•
Comparable toxicity of D2-ABC in cytokine (ELISpot) and proliferation assays
•
D2-ABC maintained anti-viral activity
•
Can deduce that D2-ABC has no effect on reducing ABC hypersensitivity
D2-ABC
Altered Repetoire Mechanism
• Proposed by Ostrov et al and Illing et al as a mechanism of ABC toxicity
• Parent drug responsible for T cell stimulation
• Binding of ABC within HLA-B*57:01 in turn results in altered peptides binding (pep-V)
• Significant Van der Waals & H-bonding interactions between protein amino acids and ABC
Amino acid interactions with
Asp114, Ser116, Tyr123 and
Ile124
Cyclopropyl moiety situated
in F pocket
ABC docked (blue) within HLA-B*57:01
Pep-V (red)
Key amino acid residues (yellow)
Ostrov et al (PNAS, 2012)
Illing et al (Nature, 2012)
SAR – 6-position analogues
Range of analogues synthesised at the 6-position
SAR – 6-position analogues
Compound
Yield (%)
Compound
Yield (%)
Compound
Yield (%)
1
62
5
68
9
81
2
85
6
71
10
70
3
71
7
74
11
73
4
73
8
70
27 analogues synthesised
SAR – Cytokine ELISpot assay
Compound
1
2
3
4
5
6
7
T cell
response?
8
No
9
No
10
No
11
No
No
• Quantitative results shown
• Cytokine release assay – ABC
responsive HLA-B*57:01
positive T cell clones
No
• Indicative of T cell response
No
10
Yes
Abacavir
Abacavir
Yes
N-isopropyl 6
6
CH3-N-propyl 7
7
No
No
Significant IFNγ release for
compounds 4 and 5
N-propyl 5
5
No drug
25
50
100
250μM
SAR – Docking results
Compound
1
2
3
4
5
6
7
T cell
GOLDScore
response?
No
34.4
No
46.0
No
31.4
Yes
55.7
Yes
64.3
No
48.4
No
24.1
Compound
8
9
10
11
T cell
GOLDScore
response?
No
-175.1
No
28.1
No
29.0
Yes
45.2
•
Compounds docked into HLA-B*57:01 and their
binding within the protein is quantified (Van der
Waals, H-bonding)
•
Used to support T cell assay results & for future
direction in 6-position analogue synthesis
•
Any values above 50 = stimulate T cells
SAR – Docking results
Compound
1
2
3
4
5
6
7
T cell
GOLDScore
response?
No
34.4
No
46.0
No
31.4
Yes
55.7
Yes
64.3
No
48.4
No
24.1
Compound
8
9
10
11
T cell
GOLDScore
response?
No
-175.1
No
28.1
No
29.0
Yes
45.2
•
Compounds docked into HLA-B*57:01 and their
binding within the protein is quantified (Van der
Waals, H-bonding)
•
Used to support T cell assay results & for future
direction in 6-position analogue synthesis
•
Any values above 50 = stimulate T cells
SAR – Docking results
ABC (blue) & Compound 8
(green) docked within
HLA-B*57:01
Pep-V (red)
Key amino acid residues
(yellow)
Morpholine part of molecule protrudes into F-pocket and leads to steric hindrance
and therefore disrupts binding of pep-V. No Van der Waals interaction with Val9 &
Tyr123
Result = No T cell stimulation
GOLDScore -175.1
SAR – Docking results
ABC (blue) & Compound 6
(green) docked within
HLA-B*57:01
Pep-V (red)
Key amino acid residues
(yellow)
CH3 from isopropyl undergoes steric interactions – Tyr 123 (disrupts pep-V binding)
Result = No T cell stimulation
GOLDScore 48.4
SAR – Anti-viral results
Cytotoxicity
IC50 (µM)
Cytotoxicity
IC50 (µM)
Efficacy
EC50 (µM)
Selectivity Index
IC50/EC50
Sample
24 hours
5 days
5 days
5 days
ABC
301.8±17.0
199.2±11.8
3.79±0.19
52.56
30.2±1.21
11.5±0.75
12.6±0.17
44.5±3.71
12.2±0.74
11.6±0.51
12.0±2.36
9.9±0.66
15.0±0.33
233.3±9.0
171.2±5.2
6.8±0.89
295.4±28.3
197.9±7.46
5.3±0.77
126.4±12.2
77.7±3.63
2.7±0.03
95.8±8.88
65.3±5.18
2.4±0.02
11.7±0.45
2.8±0.14
8.5±0.86
34.6±3.26
11.7±0.39
17.9±3.31
12.7±0.43
3.0±0.09
6.4±0.48
10.2±0.34
2.4±0.07
5.9±0.42
1
2
3
4
5
6
7
8
9
10
11
0.91
0.10
0.66
25.18
37.34
28.7
27.2
0.33
0.65
0.47
0.41
SAR – Anti-viral results
Cytotoxicity
IC50 (µM)
Cytotoxicity
IC50 (µM)
Efficacy
EC50 (µM)
Selectivity Index
IC50/EC50
Sample
24 hours
5 days
5 days
5 days
ABC
301.8±17.0
199.2±11.8
3.79±0.19
52.56
30.2±1.21
11.5±0.75
12.6±0.17
44.5±3.71
12.2±0.74
11.6±0.51
12.0±2.36
9.9±0.66
15.0±0.33
233.3±9.0
171.2±5.2
6.8±0.89
295.4±28.3
197.9±7.46
5.3±0.77
126.4±12.2
77.7±3.63
2.7±0.03
95.8±8.88
65.3±5.18
2.4±0.02
11.7±0.45
2.8±0.14
8.5±0.86
34.6±3.26
11.7±0.39
17.9±3.31
12.7±0.43
3.0±0.09
6.4±0.48
10.2±0.34
2.4±0.07
5.9±0.42
1
2
3
4
5
6
7
8
9
10
11
0.91
0.10
0.66
25.18
37.34
28.7
27.2
0.33
0.65
0.47
0.41
SAR – Summary
•
27 analogues (6-position) were synthesised
•
Cytokine IFNγ and proliferation assays were conducted on 11 of these analogues with two analogues
(4 & 5) showing comparable toxicity to ABC
•
Docking ABC & analogues within HLA-B*57:01 complimented T cell assay results with high
GOLDScore values showing stimulation of T cell cytokine release (comparable to ABC)
•
Further analysis of docking shows those 6-position groups with low GOLDScore values and no T cell
response exhibit steric interactions within the protein
•
Most of the 11 compounds exhibited low anti-viral activity with compounds 4 – 7 showing comparable
efficacy to ABC
•
From this, compounds 6 and 7 show promise with lower cytotoxicity in MT4 cells compared to the other
analogues, reduced T cell response and fair anti-viral activity. Compound 6 gives comparable PK
parameter values to ABC
•
It has been shown that modification of the 6-position of ABC can reduce ABC related toxicity
ABC
4
5
6
7
Future Work
• Completion of further T cell assay and anti-viral work for the other 16 analogues (6-position)
• Further synthesis of 6-position analogues, with interest in those that bear a modified
cyclopropylamine moiety
• Completion of PK parameters for compound 7 & other analogues that display low T cell
stimulation but good anti-viral activity
• Further studies to complete isotopic metabolism work including determining rate constants of
D2-ABC and ABC
Target Identification, Validation and De-Risking
Amodiaquine (Isoquine,
Phase 1, 2008, GSK
MMV)
Artemisinin (RKA 182,
E209, Candidate
Selected)
Abacavir (NIH, USA)
Primaquine (MMV)
Rational Drug Re-design