From X-ray structure to successful calf therapy

From X-ray structure to successful calf therapy

BKIs for Cryptosporidiosis: From X-ray
Structure to Cattle Therapy
Wesley C. Van Voorhis MD PhD
Professor and Division Head
Allergy and Infectious Diseases Division
Adjunct Professor of Global Health and Microbiology
Director, CERID
University of Washington
[email protected]
CDC.gov
Cryptosporidiosis: Human Clinical
– Protozoan Parasite
• Lives inside plasma membrane, yet outside cytoplasm
of gut epithelial cells
– Prolonged diarrhea (3-14 days) in
immunocompetent individuals
• Outbreaks :poor water filtration; Milwaukee 400K
– Deadly diarrhea in HIV infection if CD4 counts
not boosted by anti-retroviral therapy
– Second-most common pathogen in severe
diarrhea of children of 6-18 months old in
resource-poor environments
• Associated with mortality & growth failure
– Only one drug licensed for Crypto: nitazoxanide
• Only 33% efficacy in malnourished children
• No efficacy in HIV infected persons
Calcium-dependent protein kinases

CDPKs: kinases of plants and
apicomplexan parasites

Calcium-activated
serine/threonine protein
kinase

Toxoplasma gondii CDPK1
required for motility and cell
entry and exit; likely also
Cryptosporidium spp. CDPK1

Activity dependent on Ca2+

Major structural
rearrangement

CAD ends up on opposite
surface of kinase domain

Active site now accessible to
protein & peptide substrates
Exploit the small gatekeeper structural difference:
Develop specific & potent bumped kinase inhibitors
Gly
“Gatekeeper”
residue
CDPK1 ATPbinding site:
Large
(accessible)
hydrophobic
sub-pocket
Binds bumped kinase
inhibitors (BKIs)
ATPbinding
pocket
Bumped
kinase
inhibitor
Eric Larson
Exploit the small gatekeeper structural difference:
Develop specific & potent bumped kinase inhibitors
Typical
Mammalian
Protein Kinase
ATP-binding site:
Bulky
“Gatekeeper”
residue
Does not bind
bumped kinase
inhibitors (BKIs)
ATPbinding
pocket
Small
(inaccessible)
hydrophobic
sub-pocket
CLASH
!
Bumped
kinase
inhibitor
Eric Larson
Medicinal Chemistry & SBDD
• Made 600 BKIs to date
• Optimize:
– Potency
R1
R2
• Activity against Parasites’ CDPK (SBDD) pyrazolo[2,3-d]pyrimidine (PP)
Dusty Maly
• Activity against Parasite replication in vitro
– Safety
• Lack activity against mammalian kinases (specificity)
• Lack activity against mammalian cell lines (toxicity)
• Lack rodent toxicity (whole animal dosing)
– ADME
R2
N N
NH2
R1
O
NH2
O
N
• Orally Absorbed
5-aminopyrazole-4-carboxamide (AC)
• Distributed to CNS
Erkang Fan
• Low Metabolism & Excretion for high exposure
In mammalian cell culture (RT-PCR)
1294
Cryptosporidium Quantity
1294 inhibits C. parvum proliferation in vitro
1294 Exp 1
1294 Exp 2
Alejandro Castellanos-Gonzalez and Clinton White (UTMB)
A novel Calcium Dependent Protein Kinase Inhibitor as a lead compound for treating
CONFIDENTIAL
Cryptosporidiosis. Castellanos-Gonzalez
A, et al. . J Infect Dis. 2013; 208 (8): 1342-1348.
1294 inhibits C. parvum and promotes intestinal
healing in immunocompromised mouse model
•
•
•
•
•
SCID/beige mouse
model
Infect with 1 X 10 6 C.
parvum oocysts (Iowa
strain) by oral gavage
Four days after
infection, begin 10
day therapy with 1294
@ 100mg/kg 1x/day
or vehicle alone
Monitor stool output
of oocysts for 33 days
Histology/TUNEL
assay of intestine at
33 days
Mean Stool
Parasites:
Mean Stool
Parasites: Mice (7)
Control Mice (7)
given vehicle
only on d4-13
after infection
treated with 1294
(100mg/kg po 1x/d)
on d4-13 after
infection
Outcome of Intestinal Villi
1294 Treated
Vehicle Alone
Uninfected Controls
Alejandro Castellanos-Gonzalez and Clinton White (UTMB)
A novel Calcium Dependent Protein Kinase Inhibitor as a lead compound for treating
Cryptosporidiosis. Castellanos-Gonzalez A, et al. . J Infect Dis. 2013; 208 (8): 1342-1348.
Newborn Calf – Cryptosporidium challenge Study
Mike Riggs: Univ of Arizona
• Newborn calves
• Susceptible to C. parvum infection
• Can develop severe diarrhea like humans
• Infection with 5 x 107 Oocysts/calf on day 0
• Diarrhea in both groups at onset of dosing
• Start treatment on day 2
• 1294 @ 5mg/kg every 12 hrs for 5 days
1294
Efficacy of 1294 5mg/kg PO q12hr (2x daily):
Daily Oocyst Numbers Shed
Average Total Oocysts per Day +/- SD
4.50E+08
4.00E+08
3.50E+08
Control
treated
*
*
3.00E+08
*
2.50E+08
2.00E+08
*
*
1.50E+08
*
1.00E+08
*
Newborn
CalfC. parvum
challenge
model
5.00E+07
0.00E+00
3
4
5
6
Days PI
7
8
9
10
Rx with 1294
began on day 2
after C.p.
challenge
• After 24hrs of therapy (d3 PI), oocyst excretion was significantly
reduced in calves treated with 1294
Efficacy of 1294 5mg/kg PO q12hr (2x daily):
Daily Clinical Evaluation Scores
Treated
17.0
Total Clinical Evaluation Score
Mean ± SEM
*
15.0
14.0
Control
*
16.0
*
*
*
*
*
13.0
*
12.0
11.0
10.0
9.0
8.0
Newborn
CalfC. parvum
challenge
model
7.0
6.0
5.0
3
4
5
6
7
8
9
10
Rx with 1294
began on day 2
after C.p.
challenge
Day Post Infection
• After 24hrs of therapy (D3 PI), calves were significantly
more well when given 1294
Efficacy of 1294 5mg/kg PO q12hr (2x daily) vs C. parvum
Diarrhea: Total Fecal Volume (ml)
18000.0
Fecal vol (ml) Days 3 - 10 PI
16000.0
*
Control
Treated
14000.0
12000.0
10000.0
8000.0
6000.0
Newborn
CalfC. parvum
challenge
model
4000.0
2000.0
* Significantly
different p=<0.05
0.0
• Calves had significantly less diarrhea when given with
1294, as is evident in this graph comparing fecal volume
Efficacy of 1294 5mg/kg PO q12hr (2x daily) vs C. parvum Diarrhea:
Fecal Consistency Score
4.50
*
*
*
*
Treated
*
4.00
Control
*
Fecal Score ± sem
3.50
*
Newborn
CalfC. parvum
challenge
model
3.00
2.50
2.00
1.50
Rx with 1294 1.00
began on day 2
after C.p.
challenge
3
4
5
Days PI
6
7
8
9
10
* Significantly
different p=<0.05
• By the second day of therapy, d4 PI, the fecal consistency score was
significantly improved in the 6 1294 treated calves vs. the 6 control
calves, and virtually normal by 5 days after therapy
Summary of Newborn Calf C. parvum
challenge Rx w/ 1294
• Twice daily Rx (5mg/kg Q12h)
• Significantly reduced C.p. oocyst excretion on and after
the first day of therapy
– Led to outstanding clinical outcomes,
measureable by 24hrs after therapy starts
• Reduced diarrhea, improved fecal consistency scores
• Reduced stool volumes
• Better overall clinical scores
– Stool levels 0.45 µM
– Plasma levels 0.2 – 0.7 µM
• Efficacy
BKI 1294 Properties
– 2 nM IC50 CpCDPK1 enzyme & <100 nM EC50 C. parvum growth in vitro
– Proof of concept: effective treatment of newborn and SCID/bg rodent
models of C. parvum & two calf studies of C. parvum challenge
– Normalization of intestinal villi in SCID/bg rodent model after 1294 Rx
• Safety
–
–
–
–
No activity: human kinases and off-target liabilities
No activity 40 µM against 4 human cell lines
No toxicity in rodents
hERG activity: IC50 300nM
• PK/ADME
– Oral Bioavailability: ~90%
– Fecal levels measureable and >10X EC50 Crypto
– 24 hrs >EC50 Crypto: 100 mg/kg oral dose
1294
Alternative BKI Compounds
• 1517: with alternative AC scaffold to 1294
– 1.4 nM CDPK1 IC50, 50-100 nM Crypto cell EC50
– Oral absorption excellent
• 10µM peak with 10mg/kg dose, AUC 2450µM-min
• Rat oral bioavailability: 78%
• Mouse stool levels 0.45µM: 25mpk x 1
– Clean safety profile in vitro & rodents
• Single and multiple doses, up to 300 mg/kg
• No AMES or micronucleus signal
• No hERG, no off target liabilities
Mean stool C. parvum oocysts
Immunocompromised SCID/Bg Mouse C. parvum Challenge Model:
1517 20mg/kg po AM & 40 mg/kg po PM or Vehicle control
1000
900
800
700
600
500
400
300
200
100
0
1517 Rx: d4-13
Vehicle alone
1517
0
5
10
15
20
25
30
Day after infection (mice treated d 5-14) N =7 each group
• 1517 also effective in newborn mouse – C. parvum challenge model: 25mg/kg bid
Newborn Calf – Cryptosporidium challenge Study
Mike Riggs: Univ of Arizona
• Infection with 5 x 107 Oocysts/calf on day 0
• Start treatment on day 2
• 1517 @ 10mg/kg every 12 hrs for 10 doses
• Doubled dose 1517 compared with 1294 given 1517
has more rapid clearance
• Diarrhea at onset of dosing
Comparative Fecal volume: 1517 treated and control Newborn
Calves challenged with C. parvum
Mean Fecal Volume (ml) +/- SD
Efficacy of BKI 1517 vs C. parvum Diarrhea: Average Daily
Fecal Volume
5000
*
4500
*
control
1517
Treated 1517
4000
*
3500
Rx with
1517
began
on day 2
after C.p.
challenge
3000
2500
2000
1500
1000
500
0
3
4
5
6
Day PI
7
8
9
10
* Significantly
different p=<0.05
• Significantly less fecal volume in 1517-treated calves
compared with control calves on days 4, 5, & 6 post
infection demonstrates clinical benefit of 1517
Comparative Fecal Consistency: 1517 treated and control
Newborn Calves challenged with C. parvum
Fecal Consistency
4.50
4.00
Fecal Score ± sd
3.50
*
3.00
Treated
2.50
*
*
2.00
Control
*
1.50
*
Rx with
1517
began
on day 2
after C.p.
challenge
1.00
2
3
4
5
6
7
Days Post Infection
8
9
10
* Significantly
different p=<0.05
• Significantly lower fecal consistency score observed in the
1517-treated calves comparted with control calves on days 4 8 post infection demonstrates decreased diarrhea with 1517
therapy
Weight loss: 1517 treated and control Newborn Calves
challenged with C. parvum
Percent Weight Loss – Group Means
0.0
-1.0
% Weight Gain
-2.0
-3.0
-4.0
-5.0
-6.0
-7.0
Control
Group
Treated
1517
• Reduced weight loss in 1517-treated calves suggests 1517treated calves will have superior growth than control calves
1517 Treated and Control Newborn Calves Challenged with C.
parvum
Efficacy of 1517 vs C. parvum: Daily Oocyst Numbers Shed
1.80E+10
Average Total Oocysts per Day +/- SD
1.60E+10
control
1.40E+10
treated
1.20E+10
1.00E+10
8.00E+09
6.00E+09
4.00E+09
Rx with
PTT-148
began
on day 2
after C.p.
challenge
2.00E+09
0.00E+00
3
4
5
6
7
8
9
10
Days PI
• Significantly less C. parvum oocysts shed on
Days 3-5
Summary of Calf – C. p. challenge
and Rx with 1517
• Diarrhea was significantly reduced and fecal volumes
normalized by d2 after Rx with 1517 compared with controls
– Controls: diarrhea until d7 after Rx, elevated fecal volume until d6
after Rx
• Multiple Clinical outcomes were significantly better after Rx
with 1517 compared with controls
– Better urine output, fever, weight gain, overall clinical scores
• C. p. Oocyst output significantly reduced on d1 after therapy
• Simultaneous PK:
– Stool values 1.2 µM
– Plasma peak values variable, mean 3.5 µM -5 µM over course of expt
PK-PD:
What’s Needed for Effective Crypto Diarrhea Therapy?
• Don’t really understand PK/ADME necessary for anti-Crypto
efficacy
• Experiments suggest 1553 is less efficacious in mouse
Cryptosporidium model compared with 1294 & 1517 despite
better 1553 plasma levels.
• Preliminary data in calves suggest slower clinical response of 1553.
• What about stool levels and levels in enterocytes?
– May be critical determinants to treat cryptosporidium
– Mouse Stool levels of 1517 and 1553 similar (25 mg/kg dose PO: 0.5uM in stool)
Neonatal Mouse C. p. infection Results:
Poor Correlation of Stool levels, Plasma Exposure (AUC), & C.p. activity
• Neonatal mice infected with C. parvum x 2 days, then begin q12
hr dosing with 25mg/kg BKIs, measure % reduction in total GI
tract by immunostaining
• Stool levels measured after 25 mg/kg oral dose
• AUC (plasma exposure) measured after 10 mg/kg oral dose
16000
14000
6
12000
5
10000
4
AUC
STOOL CONCENTRATION (UM)
7
3
8000
6000
2
4000
1
2000
0
0
0
10
20
30
40
% C PARVUM INHIBITION
50
60
70
0
10
20
30
40
50
60
70
% C parvum Inhibition
No clear correlation of C. parvum efficacy with Cmax or Plasma Protein (not shown)
CDPK1 Inhibitors
Sumiti
Vinayak & Boris Striepen UGA
C D P K in h ib it o r s ; T K - K O ; P 1 0 ; G a in s e t t in g = 1 5 0
Luciferase-tagged (TK KO) C. parvum in δ-IFN-KO mice
400000
300000
C o n tro l (v e h ic le tre a te d )
1 2 6 6 ( -v e c o n tr o l)
1 2 9 4 ( + v e c o n tr o l)
100000
Insol. cmpd, ↓ syst. dist, ? stool
1 5 5 0 Mod sol, ↑ syst. dist, ↓ stool
1 6 0 8 ↑ soluble, ↑ syst. dist., ↑ stool
1534
100000
80000
60000
40000
20000
3
4
5
6
7
8
9
1
0
1
4
3
4
5
6
7
8
9
1
0
1
4
3
4
5
6
7
8
9
1
0
1
4
3
4
5
6
7
8
9
1
0
1
4
3
4
5
6
7
8
9
1
0
0
3
4
5
6
7
8
9
1
0
1
4
R e la tiv e lu m in e s c e n c e u n its
200000
D a y s p .i
Treat with 60mpk/d x 5 days, begin treating on day 3, luciferase = C. parvum
Ongoing Studies: BKIs for Crypto
• Calf Cryptosporidium Challenge studies:
– 1517: Working towards <3d Rx (TPP) and dose finding in calf model
– 1553: Beginning to study in calf model but initial expts underdosed
and a delayed effect was seen
• Further Tox Studies: 1517 and 1553
– Safety Index in mice: therapeutic levels vs. toxic systemic levels
– Acute CV tox studies in rats and non-GLP rat studies
• Backup molecules:
– Active in neonatal mouse C.p. & δ-KO Cp mouse models
– 1608: 1517-like (AC) compound with less systemic exposure
– 1534: 1553-like (PP) compound with much less systemic exposure
Future Studies: BKIs for Crypto
• Late pre-clinical studies
– Metabolite identification
– API Chemistry
– GLP package
• Safety (rodents/dogs)
• PK/ADME (dogs)
– Formulation
– GMP manufacture
• Clinical studies
– Phase I/IIa in human challenge
– Pediatric safety package
– Phase IIb/III in target population
Lynn Barrett
Kayode Ojo
Ryan Choi
Thanks to:
Steve N Hewitt
Kasey Rivas
Dusty Maly
Erkang Fan
Not Shown
Matt Hulverson
Nina Isoheranan
A. Clinton White
Mike Riggs
Jenni Zambriski
Marilyn Parsons
Stone Doggett
Matthias Lendner
Andrew Hemphill
Luis Ortega Mora
Dale Kempf
Geno de Hostos
Robert Choi
Boris Striepen
Sumiti Vinayak
And their groups
Ethan Merritt
Wim Hol