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SMALL MOLECULE INHIBITORS OF ANTHRAX LETHAL FACTOR. Phase I SBIR Project Period 7/01/05-6/30/07 P.I. Norton Peet John D. Williams. SPECIFIC AIMS. Optimize the in vitro LF inhibitory activity of the validated hit series Lead compound SAR Modeling/X-ray crystallography - PowerPoint PPT Presentation
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Sept 21, 2006
SMALL MOLECULE INHIBITORS OF ANTHRAX LETHAL FACTOR
Phase I SBIR
Project Period 7/01/05-6/30/07
P.I. Norton Peet
John D. Williams
Sept 21, 2006
SPECIFIC AIMS1. Optimize the in vitro LF inhibitory activity of the
validated hit seriesa) Lead compound SARb) Modeling/X-ray crystallographyc) Biochemical activity against LF—Fluorescence-based/HPLCd) Continued screening of the MBX compound library against LF
2. Demonstrate potent selective inhibitory activity in cellular models of LF action
a) Medicinal chemistryb) Cellular protection and toxicity assays
3. Identify LF inhibitors with in vitro ADME properties suitable for oral dosing
4. Identify lead LF inhibitors that are active in an in vivo model
Sept 21, 2006
LF Inhibitor NSC 12155
• Compound NSC 12155 emerged as a lead compound with: – Ki of 0.5 M in the LF assay
– EC50 < 25 M in a cell-based assay.
• NSC 12155 has also been used as a starting point for the SAR studies.
N N
NH2
NH
NH
O
NH2
Sept 21, 2006
Quinoline Core
N
NH2 HN
HN
ON
NH2
CoreLinker
Variable
Sept 21, 2006
ANALYSIS OF LF INHIBITOR MEDICINAL CHEMISTRY
Sept 21, 2006
LF ASSAY REAGENT COMPOSITION AND GENERAL PROCEDURE
1) Combine the following reagents. Component [Stock] [Final]1 μL DMSO or Compound 100 % 1%64 μL Water* 55M N/A10 μL HEPES pH 8.2* 200mM 20 mM10 μL Tween 20* 0.5% 0.05%5 μL diluted LF* ¥ 10 μg/mL 0.5 μg/mL 10 μL Peptide Substrate 200 μM 20 μM 2) Incubate Above Mix For 15 Minutes at 30 °C
3) Add stop solution [Stock] [Final]10 μL Acetic Acid 5% 0.45%
3) Read excitation 324 nm and emission 395 nm
*These reagents are combined proportionately into an assay mix, with 90 µL of mix added per microplate well. 10 µL of peptide substrate is added to initiate the reaction. ¥ Enzyme dilution buffer contains 5 mM HEPES, pH 7.4, 50 mM NaCl, 100 mM Trehalose
Sept 21, 2006
12155 Activity & Specificity
N
N H 2 HN
ON
N H 2HN
L F IC 50
(M ) M M P -1 IC 50
(M ) M M P -2 IC 50
(M ) M M P -9 IC 50
(M ) H eL a C C 50
(M )
1 .28 >100 31 >100 26
Sept 21, 2006
SAR: Ureas
N
NH2HN
HN
O
R
MBX# R= IC50 (M) MBX# R= IC50 (M)
1020 O
O
>100 1025
>100
1021
79 1030 F
95
1022 COOEt
87 1034 O
26
1023 OMe
>100 1035
>100
1024 O
55 1036
83
Sept 21, 2006
SAR: Heteroaryl Amides
N
N H 2HN R
O
M B X # R = IC 50 (M ) M B X # R = IC 50 (M )
1007 N
> 100 1029 H N O M e
33
1008 O
> 100 1032 O
90
1016 N
> 100 1040 N
52
1017 N
> 100 1042 N
N
58
1019 N
N
> 100 1043
NO
> 100
1027 N
81
Sept 21, 2006
SAR: Substituted Phenyl Amides
N
NH2HN R
O
MBX# R= IC50 (M) MBX# R= IC50 (M)
1006 O
72 1045
NMe2
8.8
1014 O
73 1048
N
1.6
1015
48 1046 N
43
1041 N
N
49 1051
N
4.6
Sept 21, 2006
SAR: Extended Tether
N
NH2HN R
O
MBX# R= IC50 (M) MBX# R= IC50 (M)
1009 O
O
36 1033 MeO
3.0
1010 F
65 1037
O
O
>100
1026 OMe
>100 1038
>100
1031 N
>100 1039 O
>100
Sept 21, 2006
SAR: Phenylpropionamides
N
NH 2HN R
O
M B X # R = IC 50 (M ) M B X # R = IC 50 (M )
1033 M eO
3 .0 1067
>100
1056 O Me
>100 1070
Cl
>100
1065 O M e
>100 1072 BzO
>100
1055 M eO
O M e >100 1068
>100
1059 MeO
O M e
>100 1057
F
>100
Sept 21, 2006
SAR: Cinnanamides
N
NH2HN R
O
MBX# R= IC50 (M) MBX# R= IC50 (M)
1033 MeO
3.0 1038
>100
1071 MeO
46 1010
F
65
1058 Cl
20
1060 F3C
14
Sept 21, 2006
SAR: Tether Length
N
NH2HN R
O
MBX# R= IC50 (M) MBX# R= IC50 (M)
1033 MeO
3.0 1064
MeO >100
1071 1073 MeO
>100
Sept 21, 2006
SAR: General Trends
• Simple aromatic compounds inactive
• Polyaromatics generally more active
• Tether of ~4 atoms seems to have highest activity
• Phenylpropionamides need 2-substituent
• Cinnanamides have lower requirements, lower activity
Sept 21, 2006
SAR: What Next?
N
HN
O
NH2 R
Substituent EffectsDouble Bond?
E or Z?Cyclopropyl?
Linker = 1,2,3
Heterocyclic?Polyaromatic?
Inhibitor-Bound X-Ray structure may provide more ideasand/or refinements
Sept 21, 2006
Computer Model
Sept 21, 2006
Proposed Compounds
N
HN
O
MeONH2
X = F, Cl, Br
X
N
HN
O
MeONH2
X
Sept 21, 2006
Hybrid Compounds
N
HN
O
NH2
MeO
N
N
HN
O
NH2MeO
N
N
HN
O
NH2MeO Br
