Parameter Visualizations as Metadata to Facilitate Discovery
How structure can facilitate GPCR drug discovery
48
How structure can facilitate GPCR drug discovery
Transcript of How structure can facilitate GPCR drug discovery
Slide 1OUTLINE
• Or: a tale of two hallucinogen receptors
Team 2B
5-HT1
5-HT2
5-HT3
5-HT4
5-HT5
5-HT6
5-HT7
5-HT1
5-HT2
5-HT3
5-HT4
5-HT5
5-HT6
5-HT7
LSD- like Hallucinogens
Hallucinogens mediate their effects on consciousness via agonism at 5-HT2A receptors (LSD, psilocybin, mescaline)
Science 2009
Ki for 1 receptor ~14 M
Using the Similarity Ensemble Approach (SEA) genome-wide predictions of drug off-target actions made (Keiser et al, Nature Biotech 2007; Keiser et al, Nature, in press)
SEA predicts 5-HT receptors as target (Keiser et al, Nature, 2009)
Activity Class E-value
knockout (Keiser et al, Nature, 2009)
B. Roth et al, unpub
5-HT1
5-HT2
5-HT3
5-HT4
5-HT5
5-HT6
5-HT7
Valvular heart disease
Appetite
The „valvulopathy receptor
1992: Study shows that phentermine and fenfluramine, when taken together, are effective appetite suppressants with fewer side effects than either drug alone. The combination of phentermine and fenfluramine becomes known as “fen/phen”
1997: In July, Connolly et al reported 24 cases of valvular heart disease in women treated with fenfluramine and phentermine. Soon after, similar reports accumulate in patients taking either fenfluramine or fenfluramine with phentermine
2010: Single largest pharmaceutical product liability suit ($26 Billion and counting) http://www.gwc.maricopa.edu/class/
bio202/cyberheart/hartint0.htm
(-) Norfenfluramine
(+) Norfenfluramine
(+/-) Norfenfluramine
Norfluoxetine
Fluoxetine
Phentermine
5-HT2B
Ki
(nM)
Rothman et al. Circulation. 2000;102:2836; O’Connor and Roth, Nature Drug Discovery 2005
Rothman et al. Circulation. 2000;102:2836.
Agonist Activity Of Fenfluramine At Cloned Human 5-HT2B Receptors
5-HT (EC50 1 nM)
Fenfluramine (EC50 = 600 nM)
Norfenfluramine (EC50 = 80 nM)
5-HT (EC50 1 nM)
Fenfluramine (EC50 = 600 nM)
Norfenfluramine (EC50 = 80 nM)
PI Accumulation (% max 5-HT
PI Accumulation (% max 5-HT
response)
Rothman et al, Circulation, 2000; Setola et al, Mol Pharmacol 2003; Roth NEJM 2007
Roth, NEJM, 2007
• Non-conserved residues near the putative binding pocket identified
• Each mutated to corresponding 5- HT2A or 5-HT2C sequence
• Binding affinity of S-norfenfluramine measured
The $26 billion methyl
Psilocybin—orally bioavailable, nontoxic prodrug
Psilocin—5-HT2A/2B/2C agonist: anorexia and anti-OCD actions
“The most fruitful basis for the discovery of a new drug is to start with an old drug” Sir James Black
“SAR of Psilocybin Analogs: Discovery of a Selective 5-HT2C Agonist” H. Sard, G. Kumaran,
C. Morency, B. Roth, B. Toth, P. He, and L. Shuster Bioorg. Med. Chem. Lett. 2005, 15, 4555.
Compound 5-HT2A 5-HT2B 5-HT2C
O-4284 Inactive Inactive 16±3.7 (80±2%)
O-3944 520±275 (38±8%) Inactive 6.9±0.7 (88±5%)
WAY 161503
+ 2 additional, novel chemotypes
Orally-active 5-HT2C-selective agonists have anti-OCD and anorectic actions in vivo Sard et al, Bioorg Med Chem Lett, 2005 U.S. and World-wide Patents Licensed to Galenea Pharmaceuticals 2008
NHBoc
(-)-trans-PAT (7)
HTS -> tranylcypromine
Antipsychotic drug with anorectic and antidepressant actions (Cho et al, J Med Chem 2009; Kozikowski et al, revision)
Team Sally D
Sheffler and Roth. Trends Pharmacol Sci. 2003;24:107.
A B
Effects Of Salvinorin A • Effects are nearly instantaneous (ie, before smoked
material is exhaled)
• Memory of having ingested a “drug” is frequently lost (ie, anterograde amnesia)
• Users transported to “alternative/parallel universe”
• Effects wear off quickly (eg, rapid pharmacokinetics)
• Unlike effects of traditional LSD-like hallucinogens
Roth et al. Proc Natl Acad Sci USA. 2002;99:11934.
LSD
In Vivo Effects Of Salvinorin A Abolished In -Opioid Receptor
Knock-Out Mice
Tail Flick
Chavkin et al,.JPET 2004.
Roth et al. Proc Natl Acad Su USA. 2002;99:11934; Yan et al, Biochem 2005; Vortherms et al, JBC 2008; Yan et al, Biochem 2009
A B
The C-2 Position of Salvinorin A Interacts with Y313 in the Binding Pocket, and C315 is Nearby
Yan, et al. Biochemistry, 2009
Y313
C315
C-2
35S]GTPγS binding)
RB-48 2.10 0.84 nM 0.19 0.01 nM
RB-64 0.59 0.21 nM 0.077 0.016 nM
RB-64 is Extraordinarily Potent
Yan, et al. Biochemistry, 2009
isothiocyanate
Applied Biosystems
Mass Spec of Chymotryptic Peptides by MALDI TOF/TOF REVEALS SITE OF ADDUCT FORMATION
Prepulse Inhibition (PPI) study in mice
Background 62 dB
Prepulse +4 dB +8 dB +12 dB +16 dB /20ms
Pulse 120 dB/100 ms
en.wikipedia.orgwww.med-associates.com/startle/
RB-64 is 20-fold more potent than salvinorin A for disrupting PPI
2 mg/kg Salvinorin A 0.1 mg/kg RB-64
GPCRs exist in multiple conformations which can be differentially stabilized by
G-protein, ligands and scaffolding proteins (Urban et al, JPET 2007)
cAMP Internalization GIRK activation
Gβγ Gαi βArr Gβγ
R + L
R* + L
SPECIFIC CONFORMATIONAL CHANGES?
R*G16-L
Ca++ Mobilization cAMPChavkin et al, JPET 2004; Yan et al, Biochem 2005
Over-expressed G proteins Force Distinct Patterns of G - Protein Coupling
Yan, et al. Biochemistry, 2008
G protein-Coupling Selectively Modulates Salvinorin A Binding Affinity
Yan, et al. Biochemistry, 2008
-F ol
Pseudo-1st order rate constants for reactivity derived
Conformation
Side View Top View
Mapping of 1st order rate constants reveals residues differentially altered by G protein environment
Conclusions
• Rational medicinal chemistry and SEA (cheminformatics/computational) essential
• Compounds developed have demonstrated efficacy in vivo
•NIMH Psychoactive Drug Screening Program (NO180002) •NIMH-National Collaborative Drug Discovery Group (U19MH82114; UNC-Duke-Wyeth) •NIDA RO1DA017204 •Michael Hooker Chair
• Or: a tale of two hallucinogen receptors
Team 2B
5-HT1
5-HT2
5-HT3
5-HT4
5-HT5
5-HT6
5-HT7
5-HT1
5-HT2
5-HT3
5-HT4
5-HT5
5-HT6
5-HT7
LSD- like Hallucinogens
Hallucinogens mediate their effects on consciousness via agonism at 5-HT2A receptors (LSD, psilocybin, mescaline)
Science 2009
Ki for 1 receptor ~14 M
Using the Similarity Ensemble Approach (SEA) genome-wide predictions of drug off-target actions made (Keiser et al, Nature Biotech 2007; Keiser et al, Nature, in press)
SEA predicts 5-HT receptors as target (Keiser et al, Nature, 2009)
Activity Class E-value
knockout (Keiser et al, Nature, 2009)
B. Roth et al, unpub
5-HT1
5-HT2
5-HT3
5-HT4
5-HT5
5-HT6
5-HT7
Valvular heart disease
Appetite
The „valvulopathy receptor
1992: Study shows that phentermine and fenfluramine, when taken together, are effective appetite suppressants with fewer side effects than either drug alone. The combination of phentermine and fenfluramine becomes known as “fen/phen”
1997: In July, Connolly et al reported 24 cases of valvular heart disease in women treated with fenfluramine and phentermine. Soon after, similar reports accumulate in patients taking either fenfluramine or fenfluramine with phentermine
2010: Single largest pharmaceutical product liability suit ($26 Billion and counting) http://www.gwc.maricopa.edu/class/
bio202/cyberheart/hartint0.htm
(-) Norfenfluramine
(+) Norfenfluramine
(+/-) Norfenfluramine
Norfluoxetine
Fluoxetine
Phentermine
5-HT2B
Ki
(nM)
Rothman et al. Circulation. 2000;102:2836; O’Connor and Roth, Nature Drug Discovery 2005
Rothman et al. Circulation. 2000;102:2836.
Agonist Activity Of Fenfluramine At Cloned Human 5-HT2B Receptors
5-HT (EC50 1 nM)
Fenfluramine (EC50 = 600 nM)
Norfenfluramine (EC50 = 80 nM)
5-HT (EC50 1 nM)
Fenfluramine (EC50 = 600 nM)
Norfenfluramine (EC50 = 80 nM)
PI Accumulation (% max 5-HT
PI Accumulation (% max 5-HT
response)
Rothman et al, Circulation, 2000; Setola et al, Mol Pharmacol 2003; Roth NEJM 2007
Roth, NEJM, 2007
• Non-conserved residues near the putative binding pocket identified
• Each mutated to corresponding 5- HT2A or 5-HT2C sequence
• Binding affinity of S-norfenfluramine measured
The $26 billion methyl
Psilocybin—orally bioavailable, nontoxic prodrug
Psilocin—5-HT2A/2B/2C agonist: anorexia and anti-OCD actions
“The most fruitful basis for the discovery of a new drug is to start with an old drug” Sir James Black
“SAR of Psilocybin Analogs: Discovery of a Selective 5-HT2C Agonist” H. Sard, G. Kumaran,
C. Morency, B. Roth, B. Toth, P. He, and L. Shuster Bioorg. Med. Chem. Lett. 2005, 15, 4555.
Compound 5-HT2A 5-HT2B 5-HT2C
O-4284 Inactive Inactive 16±3.7 (80±2%)
O-3944 520±275 (38±8%) Inactive 6.9±0.7 (88±5%)
WAY 161503
+ 2 additional, novel chemotypes
Orally-active 5-HT2C-selective agonists have anti-OCD and anorectic actions in vivo Sard et al, Bioorg Med Chem Lett, 2005 U.S. and World-wide Patents Licensed to Galenea Pharmaceuticals 2008
NHBoc
(-)-trans-PAT (7)
HTS -> tranylcypromine
Antipsychotic drug with anorectic and antidepressant actions (Cho et al, J Med Chem 2009; Kozikowski et al, revision)
Team Sally D
Sheffler and Roth. Trends Pharmacol Sci. 2003;24:107.
A B
Effects Of Salvinorin A • Effects are nearly instantaneous (ie, before smoked
material is exhaled)
• Memory of having ingested a “drug” is frequently lost (ie, anterograde amnesia)
• Users transported to “alternative/parallel universe”
• Effects wear off quickly (eg, rapid pharmacokinetics)
• Unlike effects of traditional LSD-like hallucinogens
Roth et al. Proc Natl Acad Sci USA. 2002;99:11934.
LSD
In Vivo Effects Of Salvinorin A Abolished In -Opioid Receptor
Knock-Out Mice
Tail Flick
Chavkin et al,.JPET 2004.
Roth et al. Proc Natl Acad Su USA. 2002;99:11934; Yan et al, Biochem 2005; Vortherms et al, JBC 2008; Yan et al, Biochem 2009
A B
The C-2 Position of Salvinorin A Interacts with Y313 in the Binding Pocket, and C315 is Nearby
Yan, et al. Biochemistry, 2009
Y313
C315
C-2
35S]GTPγS binding)
RB-48 2.10 0.84 nM 0.19 0.01 nM
RB-64 0.59 0.21 nM 0.077 0.016 nM
RB-64 is Extraordinarily Potent
Yan, et al. Biochemistry, 2009
isothiocyanate
Applied Biosystems
Mass Spec of Chymotryptic Peptides by MALDI TOF/TOF REVEALS SITE OF ADDUCT FORMATION
Prepulse Inhibition (PPI) study in mice
Background 62 dB
Prepulse +4 dB +8 dB +12 dB +16 dB /20ms
Pulse 120 dB/100 ms
en.wikipedia.orgwww.med-associates.com/startle/
RB-64 is 20-fold more potent than salvinorin A for disrupting PPI
2 mg/kg Salvinorin A 0.1 mg/kg RB-64
GPCRs exist in multiple conformations which can be differentially stabilized by
G-protein, ligands and scaffolding proteins (Urban et al, JPET 2007)
cAMP Internalization GIRK activation
Gβγ Gαi βArr Gβγ
R + L
R* + L
SPECIFIC CONFORMATIONAL CHANGES?
R*G16-L
Ca++ Mobilization cAMPChavkin et al, JPET 2004; Yan et al, Biochem 2005
Over-expressed G proteins Force Distinct Patterns of G - Protein Coupling
Yan, et al. Biochemistry, 2008
G protein-Coupling Selectively Modulates Salvinorin A Binding Affinity
Yan, et al. Biochemistry, 2008
-F ol
Pseudo-1st order rate constants for reactivity derived
Conformation
Side View Top View
Mapping of 1st order rate constants reveals residues differentially altered by G protein environment
Conclusions
• Rational medicinal chemistry and SEA (cheminformatics/computational) essential
• Compounds developed have demonstrated efficacy in vivo
•NIMH Psychoactive Drug Screening Program (NO180002) •NIMH-National Collaborative Drug Discovery Group (U19MH82114; UNC-Duke-Wyeth) •NIDA RO1DA017204 •Michael Hooker Chair
