GTx011* (GBT440), an Anti-Sickling Compound, Improves SS Blood Rheology by Reduction
of HbS Polymerization via Allosteric Modulation of O2 Affinity
Mira
1
Patel ,
Pedro
2
Cabrales ,
Kobina
1
Dufu ,
Brian
1
Metcalf
1
Sinha
And Uma
1Global
Blood Therapeutics, Inc., South San Francisco, CA 94080
2Department of Bioengineering, University of California San Diego, La Jolla, CA
Introduction
Structural and Rheological Studies
Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β-globin gene
leading to formation of hemoglobin S (HbS). A primary and obligatory event in the molecular
pathogenesis of SCD is the polymerization of deoxygenated HbS and the resultant sickling of red
blood cells (RBC). Sickle cell disease is characterized by hemolytic anemia and vaso-occlusion leading
to progressive end-organ damage with a clinical course of life-long pain, disability and early death.
Though the childhood death rate of individuals with SCD has drastically fallen due to disease
management, transfusion therapy and hydroxyurea, patients with SCD continue to suffer serious
morbidity and premature mortality (1). To date, no drugs have been approved that specifically target
the underlying mechanism of SCD.
A drug that inhibits HbS polymerization in all RBCs has the potential to provide superior efficacy.
Because oxyhemoglobin is a potent inhibitor of HbS polymerization, allosteric modification of
hemoglobin to increase the proportion of oxyhemoglobin is a promising strategy to achieve
inhibition of HbS polymerization in all RBCs (2). By delaying polymerization the irreversible damage
done to the RBC membrane can be prevented and thereby alter the downstream pathophysiology of
the disease (3).
In order to fulfill this unmet need, Global Blood Therapeutics (GBT) has developed GBT440, a small
molecule allosteric modulator of hemoglobin oxygen affinity, for the treatment of SCD. Though
GBT440 binds to the N-terminus of the α chain, it allosterically affects the heme pocket as well as
the α1β1 interface. These allosteric effects allow hemoglobin to maintain a population in the
oxyhemoglobin, relaxed state, delay polymerization, reduce cytoplasmic hyperviscosity, improve
membrane elasticity and improve deformability.
Mechanism of Action
Under physiologic conditions, HbS will form polymers upon deoxygenation. The formation of
extensive polymers leads to sickling in the microcirculation. In vitro experiments show that GBT440modified HbS delays polymerization and thereby delays formation of sickled cells, allowing them to
exit the microcirculation and get reoxygenated in the lungs.
Oxy-HbS
monomers
Oxy-HbS [GBT440]
monomers
&
Oxy-HbS monomers
released to tissues
O2
released to tissues
Oxy/deoxyHbS monomers
Oxy/deoxyHbS monomers
Extensive HbSpolymers formed
Minimum HbSpolymers formed
GBT440 improves SS RBC filterability under hypoxic conditions
Structural studies, using crystallography and solution phase NMR, indicate
that GBT440 allosterically affects the heme pocket of the β chains
A.
B.
11°C
29°C
37°C
A.
C.
Thin film de-oxygenator
Low O2 gas (outlet)
Syringe Injection
HbS +
GBT440
Low O2 gas (inlet)
α
Syringe Collection
B.
β
Polycarbonate filter
HbS
Figure 1. (A) An overlay of crystallographic data from HbA and HbA bound to GBT440 shows a side chain movement (3.95 Å to
4.14 Å) in the distal valine of the β chain, which is known to play an important role in O2 binding (15). (B) 1D 1H NMR spectra
collected at varying temperatures (600 MHz instrument). Solution phase spectra at different temperatures highlight the dynamic
nature of the protein including the allosteric change in the signal for the distal valine, -1.7 and -1.8 ppm.
Figure 4. (A) Schematics of the apparatus wherein RBCs are rapidly deoxygenated (3% O2) following a 30 minute incubation with
GBT440. (B) RBCs are filtered through a polycarbonate filter (3-5 μm) and a differential pressure is measured as the RBCs pass
through. (C) Deformable RBCs, such as AA RBCs, pass through the filter with a fairly low differential pressure, while SS RBCs have
a greater differential pressure as their reduced deformability makes it more difficulty for SS RBCs to pass through small pore
sizes. SS RBCs incubated with GBT440 are more deformable and are therefore able to migrate through the filter with a smaller
differential pressure. Results are Mean ± SD and represent 5 replicates from 1 SCD blood sample
GBT440 reduces tension required to aspirate SS RBCs under hypoxic
conditions
A.
B.
GBT440 delays polymerization and reduces cytoplasmic hyperviscosity
A.
O2
Rheological Studies
B.
C.
Altered RBC
morphology
Preserved RBC
morphology
Materials and Methods
Crystallography studies
Purified HbA-CO was diluted to 20 mg/ml in 20 mM Hepes buffer pH 7.4. HbA protein was mixed with an equal volume of the crystallization
buffer containing 0.1 M BisTris pH 6.0, 0.2 M MgCl2, and 23 % PEG 3350. Diffracting crystals grew within 7-10 days using the hanging drop vapor
diffusion method at 23°C. Crystals were cryoprotected by adding glycerol (10-12% (v/v) final concentration) to the crystallization buffer before
flash-freezing in liquid nitrogen. Data collection was carried out at beamline 8.3.1. at the Advanced Light Source (ALS). HbA was co-crystallized
with compound in the C21 spacegroup with two HbA tetramers per asymmetric unit. Data reduction was carried out using MOSFLM (4) and all
models were built using COOT (5) and further refinement was carried out using the latest builds of the PHENIX suite (6).
1D 1H NMR studies
The 1H-NMR experiments were performed on Bruker AVANCE DRX-600 NMR spectrometers operating at 600.13 MHz using a jump-and-return
pulse sequence with a delay time of 1.0 s. A total of 256−1024 transients were accumulated for each spectrum. GBT440 and HbS (1.5 mM) were
prepared in 0.1 M sodium phosphate (pH 7.0). HbS was liganded with CO to stabilize the protein for the extended studies (7).
In vitro HbS Polymerization
HbS was purified from SS RBC lysates through gel filtration and DE-52 anion exchange chromatography. Purified HbS was mixed with GBT440-HbS
(final [HbS]=50 µM) in 1.8 M potassium phosphate for 1 hour at 37oC. The reaction mixture was then passively de-oxygenated (99.5 % N2/ 0.5%
O2) at 4°C for 90 min and polymerization was induced via a temperature jump from 4°C to 37°C. Polymerization was quantitated by measuring
turbidity of the HbS solution at 700 nm under continued hypoxia (adapted from 8).
Figure 2. (A) GBT440-modified HbS dose-dependently delays the polymerization of HbS. (B) GBT440-modified SS blood dosedependently reduces the cytoplasmic hyperviscosity induced by enzymatic deoxygenation (representative curves). An increase in
cytoplasmic viscosity is proportional to the concentration and viscosity of HbS (3) (C) Summary of 6 SCD patient blood samples
wherein a delay in the cytoplasmic hyperviscosity is proportional to the percentage of blood that has been modified by GBT440.
Results are Mean ± SEM
GBT440 maintains RBC deformability under hypoxic conditions
A.
Conclusions
B.
Viscosity
SS Blood Hct was adjusted to 30%, and then incubated for 30 min at 37°C with GBT440 or DMSO. The blood was then stored overnight at 4°C. The
following day, isotonic ascorbic acid (final concentration, 25 mM, pH 7.4) and ascorbic oxidase (1,250 units/μL) were added to the blood and the
mixture was immediately transferred to a 37°C cone plate viscometer (9) to induce HbS polymerization and a subsequent increase in blood
viscosity. Viscosity data was collected at a shear rate of 60 s-1 every 5 seconds for 60 mins. Ascorbic oxidase is an enzyme that catalyzes the
depletion of solubilized O2 in the following reaction: 2 L-ascorbic acid + O2 ⇋ 2 dehydroascorbic acid + H20. This reaction allows for
deoxygenating the whole blood without the need of chemicals such as sodium dithionite or a hypoxic chamber (10).
RBCs
Deoxygenated PBS
Sephacryl S-500 resin
centrifuge
Gel filtration deformability assay
No compound
A 500 μL packed S-500 sephacryl (GE Healthcare) column containing deoxygenated phosphate buffered saline (PBS) was prepared. Next, a
suspension of RBCs (20% Hct) in PBS was incubated with varying concentrations of GBT440 for 1 hr at room temperature and then deoxygenated
in a humidified hypoxic chamber (98% nitrogen/2% O2) for 30 min. Six μL of the deoxygenated RBCs were loaded on top of the packed column
and the column was then centrifuged for 3 min at 3000 g. During centrifugation, deformable RBCs migrate through the sephacryl column,
whereas rigid non-deformable sickled RBCs remain on top of the sephacryl resin (adapted from 11). The relative concentration of Hb in
deformable and non-deformable RBC fractions was determined by measuring the optical density (OD) at 542 nm and 700 nm. Thus, the relative
absorbance of the two Hb fractions was used to estimate percent deformability as follows:
[OD (542 nm -700 nm) of Hb in deformable RBC fraction / OD (542 nm -700 nm) of Hb in deformable RBC + non-deformable RBC fraction] * 100
GBT440
(mM)
0.5 1
2
5
AA SS AA SS SS SS SS SS
RBC filterability
RBC suspensions were adjusted to 20% Hct and incubated with GBT440 for 30 min at RT in PBS containing 0.5% bovine serum albumin (BSA).
RBCs were then deoxygenated by direct exposure to (3% O2), as a thin film of blood on an inclined glass plate was exposed to air in a cylindrical
container (Figure 4A). The equilibrated RBC suspension was passed through a polycarbonate filter at various flow rates (0.5, 0.6, 0.7, 0.8, 0.9 and
1.0 mL/min) and the differential pressure drop across the filter was measured (12). The stretching factor (β factor) was calculated, to quantify the
resistance of a single RBC to pass through a single pore independent of cell concentration and cell to pore volume (13).
Deformable RBCs
migrate to the bottom
of resin
Normoxia
Rigid non-deformable RBCs
remain on top
of resin
Hypoxia
Micropipette aspiration
RBC suspensions were adjusted to 20% Hct and incubated with GBT440 for 30 min at RT in PBS containing 0.5% BSA. RBCs were then diluted to
0.5% Hct and deoxygenated by direct exposure (4% O2), as a thin film of blood on an inclined glass plate was exposed to air in a cylindrical
container (Figure 4A). The equilibrated RBC suspension was transferred to a glass chamber attached to an isothermal stage regulated to 37°C
(Sensortec, Inc.) and (4% O2) was continually passed on top of the samples to prevent re-oxygenation. Micropipettes (A.M. Systems) were pulled
(P-97, Sutter Instruments) to an internal diameter of ~2 micron and opening angle of ~9°. The micropipette was attached on a pneumatic
micromanipulator (Narishige, Japan) and secured to an inverted microscope (Olympus IMT-2, Tokyo, Japan) Aspiration pressure was induced
hydrostatically and kept constant. Measurements were done with a high magnification 100X LUMPFL (Olympus) objective and a video system
(Cohu, Inc.). Subsequent aspiration length was measured off line from recorded images. Cell membrane elastic module and cell volume were
calculated based on the relationship between pressure and aspiration length as previously reported (14).
©2014 Global Blood Therapeutics
Figure 5. (A) Using a micropipette and a high magnification objective with a video system, the cell membrane elastic moduli
(tension) and cell volume are calculated based on the parameters described in Fig. 5A. (B) AA RBCs, SS RBCs and GBT440modified SS RBCs have different membrane elasticity, especially under deoxygenated conditions. SS RBCs have a high shear
elastic modulus when compared to AA RBCs. RBCs are incubated with GBT440 and then diluted and deoxygenated (4% O2) using
the same apparatus described in Fig. 4A. In the presence of GBT440, SS RBC deformability is improved under hypoxic conditions
as shown by the reduced elastic modulus compared with that of untreated SS RBCs.
Figure 3. (A) A schematic of the method used to measure SS RBCs deformability. Oxygenated (20% O2) or deoxygenated (3% O2)
RBCs are loaded onto a Sephacryl S-500 resin bed. (B, inset) During centrifugation only deformable RBCs are capable of migrating
to the bottom of the bed. AA cells under all conditions and normoxic SS cells are able to migrate through the sephacryl resin.
Addition of GBT440 to the SS RBCs improves the deformability of deoxygenated SS RBCs. The graph shows a spectroscopic
quantification of the RBCs that migrated to the bottom of the bed and indicates that in presence of GBT440, deoxygenated SS
RBCs are able to migrate to the bottom similar to oxygenated SS RBCs. Results are Mean ± SD
For a digital copy of the poster please use the QR code
GBT440, an antisickling compound, has been shown to
• Allosterically affect the heme pocket of hemoglobin
• Induce a delay in polymerization and reduce cytoplasmic hyperviscosity in a dose dependent manner
• Improve SS RBC deformability under hypoxic conditions
Based on these findings, GBT440 is expected to inhibit the polymerization of deoxygenated HbS, improve RBC
deformability and reduce whole blood viscosity in SCD patients.
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2.
3.
4.
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8.
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Adams et al., Acta Crystallogr D Biol Crystallogr 2010; 66: 213
Yuan et al., Biochemistry 2010; 49: 10606
Adachi and Asukara, Blood Cells 1982; 8:213
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Wells/Brookfield (Lubin et al., PNAS 1975; 72:43)
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Pais E etal., J Biomol Screen 2009; 14:330
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Evans, Biophys J 1983; 43: 27
Perutz, Ann Rev Biochem 1979; 48: 327
Acknowledgements
Blood from SCD patients was kindly provided by:
UNC Comprehensive Sickle Cell Program, Chapel Hill, NC
Children Hospital and Research Center Oakland, CA
Professor Chien Ho at Carnegie Mellon University, Pittsburgh, PA
for 1D 1H NMR data
James Partridge at GBT for the X-ray Crystallography data
Global Blood Therapeutics Project Team
*GTx011 is now being referred to as GBT440
**For more information on the effect of GBT440 on Townes SS Mice Model please attend our Oral presentation on Monday (#217)