Systemic Isoform-Selective Heat Shock Protein 90 Inhibitors Improve the Therapeutic Index

of Morphine
Kerry Chou, David Duron, Christopher Campbell, Parthasaradhireddy Tanguturi, Paul Bejarano, Katherin Gabriel, Jessica Bowden, Sanket Mishra,
Christopher Brackett, Deborah Barlow, Karen Houseknecht, Brian Blagg, and John Streicher
Department of Pharmacology, College of Medicine, University of Arizona

Abstract Systemic Grp94-Selective Inhibitor Recapitulates Spinal Inhibitor CRISPR Knockout Matches Isoform Inhibitor Our Model
Opioids like morphine are the benchmark for treating severe acute pain; however, severe side effects including A. KUNG65 (IV) Tail Flick 1 mg/kg B. KUNG65 (IV) Tail Flick 3.2 mg/kg C. KUNG65 (IV) Tail Flick 5.6 mg/kg Figure 8:
respiratory depression and addiction limit their viability. One approach to improve opioid therapy focuses on a dose- 10 10 Vehicle 10 Identification of
Vehicle
reduction strategy by amplifying opioid analgesia without boosting side effects, so that less opioid can be given. To this 9 9 9 ** * spinal-cord specific

Tail Flick Latency (s)

KUNG65

Tail Flick Latency (s)

KUNG65

Tail Flick Latency (s)

8 8
end, we’ve focused on Heat shock protein 90 (Hsp90), a central protein regulator in the cell; ongoing studies from our lab 8 N=9
7 ******* ******
N = 19
7 Hsp90 isoforms that
7 ****
have shown that inhibition of Hsp90 in the spinal cord improves morphine antinociceptive potency by 2-4 fold in acute 6 6 6 regulate opioid anti-
5 ** 5
and chronic pain while reducing tolerance, rescuing established tolerance, and not changing reward and constipation, 5 * 4
nociception. Male
4 ** 4
enabling a dose-reduction strategy. However, our results also showed that non-selective Hsp90 inhibitors given * 3 and female CD-1
3 3
2
systemically (intravenous, IV) have the opposite effect, blocking opioid pain relief. Seeking a means to surmount this 2 2
1
mice were treated
1 1 KUNG65 N = 10
roadblock, we determined which Hsp90 isoforms regulate opioid pain relief in brain and spinal cord in mice using 0 0
Vehicle with 0.01 nmol of
0
selective small molecule inhibitors and CRISPR/Cas9 gene editing. We found that Hsp90α alone regulated opioid 15 30 45 60 75 90 105 120 15 30 45 60 75 90 105 120 isoform-selective

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15 30 45 60 75 90 105 120

.B
L

.B
Post-Morphine Time (Min.)

.B

j.
Post-Morphine Time (Min.) inhibitor or Vehicle

hr
signaling and pain relief in the brain. However, in the spinal cord, we found that the isoforms Hsp90α, Hsp90β, and

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Post-Morphine Time (Min.)

hr
j.

In
hr

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In

24
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24

Pr
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24
control i.t. with a 24

Pr
Grp94 all regulates opioid signaling and pain relief. This led to our hypothesis that targeting spinal cord Hsp90 with

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isoform-selective inhibitors given IV could boost opioid pain relief without altering side effects, enabling a dose-reduction hr treatment time; or
strategy. We tested this hypothesis with the novel Grp94-selective inhibitor KUNG65 and the novel Hsp90β-selective D. KUNG65 Dose Response Curve Figure 4: Systemic, Grp94-Selective Inhibitor Boosts Opioid
Pain Relief in Tail Flick Pain. Male and female CD-1 mice injected with an isoform-
100
inhibitor KUNB106, given at a 1mg/kg dose IV in male and female CD-1 mice, followed by a 24hr treatment time, then Vehicle
IV with 1 mg/kg KUNG65 (selective Grp94 inhibitor) or Vehicle, 24 selective CRISPR
KUNG65 knockdown construct
analysis of opioid antinociception and side effects. We found that systemic (IV) KUNG65 treatment resulted in a 1.9 fold 80
N = 9-19 hrs, followed by 1-10 mg/kg SC morphine and a tail flick time course
increase in morphine potency to relieve tail flick pain with similar findings for KUNB106, consistent with our earlier measured (10 sec threshold, 52°C water). A-C) Individual morphine or universal negative
60

% MPE
studies injecting inhibitors directly into the spinal cord. We also found that both KUNG65 and KUNB106 boosted doses reported. KUNG65 caused consistent elevation over Vehicle control CRISPR
morphine potency in paw incision pain. Additionally, we found that KUNG65 and KUNB106 injection could rescue 40 treatment. D) Data from A-C transformed into % Maximum Possible construct (NC) with a
established morphine tolerance in the tail flick assay, again as we found for direct spinal cord injection. These results Veh = 3.6 mg/kg Effect (MPE) and used to construct dose/response curves. Potency 10 day treatment time
20
support our hypothesis that isoform-selective Hsp90 inhibitors can selectively engage Hsp90 in the spinal cord when KUNG = 1.9 mg/kg values calculated by linear regression. A50: Vehicle = 3.6 mg/kg, (see Methods). Pre-
1.9 fold shift
given systemically, resulting in improved opioid antinociception and side effects. These results strongly suggest that 0 KUNG65 = 1.9 mg/kg, 1.9 fold shift. Same as for direct spinal and post-treatment
Hsp90 isoform-selective inhibitors could be a powerful new tool to improve opioid therapy through a dose-reduction 0 1 2
inhibition in Fig 1A. baselines were
Log[Morphine], mg/kg Figure 11: Model of Hsp90 Regulation of Opioid Anti-Nociception in the Nervous System.
strategy, and further show that this effect can be achieved through a translationally relevant dosing route. recorded using the
52°C tail flick assay The mu opioid receptor (MOR) can drive ERK MAPK phosphorylation and anti-nociception in
Spinal Cord Hsp90 & Opioid Dose-Reduction Systemic Grp94 Inhibitor Boosts Pain Relief in Paw Incision Pain (as above), as well as response to opioid drug (e.g. morphine) in both brain and spinal cord. Hsp90 has an opposite
tail flick timecourses effect in either site, promoting this process in brain and blocking it in spinal cord, so that brain
KUNG65 (IV) - Paw Incision
Figure 1: Spinal Hsp90 KUNG65 (IV) - Paw Incision KUNG65 (IV) - Paw Incision Hsp90 inhibition blocks opioid pain relief while spinal cord Hsp90 inhibition promotes opioid
Inhibition Improves the A. 1 mg/kg B. 1.8 mg/kg C. 3.2 mg/kg in response to 3.2
mg/kg morphine s.c.. pain relief. Different molecular isoforms carry out this role in brain vs. spinal cord, and spinal-
2.5 2.5 **** ****
Therapeutic Index of

Mechanical Threshold (g)

Vehicle
2.5
specific isoform inhibitors (e.g. KUNG65) can be given systemically to boost pain relief and

Mechanical Threshold (g)

Mechanical Threshold (g) Vehicle
**** KUNG65
Data are presented as
Morphine. Male and 2.0
KUNG65 2.0 **** N = 10
2.0 * the mean ± SEM with reduce side effects, enabling a translational opioid dose-reduction strategy.
N = 10 ****
female CD-1 mice used; *** ** 1.5 1.5 the sample size of
1.5
****
sample sizes noted in 1.0 mice/group noted in
* 1.0
graphs. A50 values 1.0
* each graph. Each
calculated by linear 0.5 0.5 0.5
experiment was
regression method, 0.0 0.0 0.0 completed in 2
30 60 90 120 150 180
calculating the dose at half 30 60 90 120 150 180

hr L
L
30 60 90 120 150 180 technical replicates. *, **, ***, **** = p < 0.05, 0.01, 0.001, 0.0001 vs. same time point Vehicle/NC group

hr L
L

24 j B
.B
hr L
L

24 j B
.B
Post-Morphine Time (Min.)
24 j B
.B

/In
Post-Morphine Time (Min.)
maximal activity; values Post-Morphine Time (Min.)

/In
/In

by 2 Way RM ANOVA with Sidak’s post hoc test. A) The Hsp90α isoform targeted with the selective

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Vehicle KUNG65 N = 10

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and fold shifts noted in
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inhibitor KUNA115 as well as selective CRISPR. B) The Hsp90β isoform targeted with the selective

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Pr

graphs. Vehicle or 0.01 inhibitor KUNB106 as well as selective CRISPR. C) The Grp94 isoform targeted with the selective
nmol KU-32 injected it with D. KUNG65 Dose-Response Curve Figure 5: Systemic, Grp94-Selective Inhibitor Boosts Opioid Pain
inhibitor KUNG65 as well as selective CRISPR. Both methods caused significant anti-nociceptive
100 Vehicle Relief in Paw Incision Pain. Male and female CD-1 mice injected IV
24 hour recovery, and elevation for all 3 isoforms, confirming that all 3 isoforms regulate opioid anti-nociception in the spinal
KUNG65 with 1 mg/kg KUNG65 (selective Grp94 inhibitor) or Vehicle, along with
morphine injected sc for all 80 N = 10 cord.
paw incision surgery, then 24 hrs recovery, followed by 1-3.2 mg/kg SC
noted doses. A) Acute
morphine and a mechanical allodynia time course measured. A-C)
Systemic Grp94 and Hsp90β inhibition rescues established
60
% MPE

52°C tail flick pain;

Individual morphine doses reported. KUNG65 caused consistent
threshold of 10 seconds 40

(100% MPE). KU-32 20

A50 Values:
Veh = 2.0 mg/kg
elevation over Vehicle treatment. *, **, ***, **** = p < 0.05, 0.01, 0.001, tolerance, no change to opioid-induced respiratory depression
KUNG = 0.9 mg/kg 0.0001 vs. same time point Vehicle group by 2 Way ANOVA with
caused 1.9 fold shift. B) Paw incision surgery with 24 hour recovery, measured by Von Frey. KU-32 caused 2.3 fold shift. 2.2 fold shift
Sidak’s post hoc test. D) Data from A-C transformed into % Maximum
0
C) HIV neuropathy induced by 3 daily injections of gp120 protein, with KU-32 injection on day 20 and testing on day 21 0 1 2 Possible Effect (MPE) and used to construct dose/response curves.
using Von Frey. KU-32 caused 3.3 fold shift in potency. D) Tolerance induced by twice daily injection of noted morphine Log[Morphine], mg/kg
Potency values calculated by linear regression. A50: Vehicle = 2.0
dose (1-10 mg/kg, sc). Tail flick latencies recorded after morning injection. Day 1 and Day 4 curves created, with A50 mg/kg, KUNG65 = 0.9 mg/kg, 2.2 fold shift. Similar to direct spinal
values calculated for each. The fold shift between Day 1 and Day 4 (tolerance) is noted; KU-32 reduced tolerance from inhibition in Fig 1B.
21 fold to 2.9 fold. E) Conditioned place preference (CPP) performed over 4 conditioning days with morning injection of
noted morphine dose, and saline injection in opposite chamber in the afternoon. Preference recorded with free access Systemic Hsp90β-Selective Inhibitor Recapitulates Spinal Inhibitor
for 15 minutes on day 5 with % Difference Score for paired chamber noted. KU-32 did not alter the CPP A50. F)
Constipation recorded by weighing feces in 1 hour bins for 6 hours after morphine injection, normalized to saline A. KunB106 (IV) Tail Flick 1.0 mg/kg B.10 KunB106 (IV) Tail Flick 3.2 mg/kg C.10 KunB106 (IV) Tail Flick 5.6 mg/kg
controls. KU-32 did not alter constipation potency. 10
9
Tail Flick Latency (s)

Tail Flick Latency (s)

Tail Flick Delay (s)

8 8 8
Can We Target Spinal Cord Hsp90 6
7
6 6
Paw Incision - ip 17-AAG 5
Vehicle
Figure 2: Systemic Non-Selective Hsp90 Inhibitor Blocks Opioid Pain 4 4 4
2.5
Relief. Male and female CD-1 mice had paw incision surgery performed as in
Mechanical Threshold (g)

17-AAG 3
2.0 N = 10 2 2 2
1.5 ****
**** Figure 1B, along with 50 mg/kg 17-AAG or Vehicle IP. 17-AAG is a non- KUNB-106 Vehicle
1 KunB106 Vehicle KunB106 Vehicle
0 0
1.0
* selective Hsp90 inhibitor. 24 hrs later, 3.2 mg/kg morphine was injected SC and 0
15 30 45 60 75 90 105 120 15 30 45 60 75 90 105 120 Figure 12: Model of Hsp90 isoform inhibition in the brain vs. spinal cord. In the brain,
-50 0 50 100 150
24 BL

24 BL

pain relief measured. Systemic, non-selective inhibitor blocked anti-nociception

.B

.B

Post-Morphine Time (Min.) Post-Morphine Time (Min.) Hsp90α alone regulates opioid signaling, while in the spinal cord, Hsp90α, Hsp90β, and Grp94
j.

j.
hr

hr

Time (min)
In

In

0.5
e-

e-

much like brain inhibition in our earlier work. *, **** = p < 0.05, 0.0001 vs. same
Pr

Pr

0.0 all do so. When Hsp90α is inhibited systemically with non-selective inhibitor or Hsp90α-
D.
30 60 90 120 150 180
time point 17-AAG group by 2 Way ANOVA with Sidak’s post hoc test. Figure 6: Systemic, Hsp90β-Selective Inhibitor Boosts Opioid selective inhibitor, opioid anti-nociception is blocked, since brain Hsp90 is inhibited and a
ry
t-S ry
ge

ge

Post-Morphine Time (Min.)

ur

ur

Pain Relief in Tail Flick Pain. Male and female CD-1 mice injected brain-like response is evoked. In contrast, when Hsp90β or Grp94 are inhibited systemically
hr e-S
os
Pr

Figure 9: Systemic Grp94 and Hsp90β inhibition rescues established tolerance without worsening
.P

IV with 1 mg/kg KUNB106 (selective Hsp90β inhibitor) or Vehicle, with selective inhibitors, brain inhibition of Hsp90α is avoided, and a spinal cord-like response
24

24 hrs, followed by 1-5.6 mg/kg SC morphine and a tail flick time opioid-induced respiratory depression. Male and female CD-1 mice used for every experiment, with is evoked – increased anti-nociception and decreased side effects. This model suggests that
course measured (10 sec threshold, 52°C water). Individual data presented as the mean ± SEM and the sample size of mice/group noted in each graph. The tolerance
Hsp90 Isoform Differs in Brain Vs. Spinal Cord morphine doses reported. KUNB106 caused consistent elevation experiments were performed in 2 technical replicates, and the respiratory depression experiments in 1
Hsp90β and Grp94 inhibitors could be given by translationally-relevant routes to improve the
therapeutic index of opioids and enable a dose-reduction strategy. Figure created using
Figure 3: Identification over Vehicle treatment. Data transformed into % Maximum technical replicate. *, **, ***, **** = p < 0.05, 0.01, 0.001, 0.0001 vs. same time point Vehicle group by biorender.com.
of Hsp90 Isoforms that Possible Effect (MPE) and used to construct dose/response 2 Way RM ANOVA with Sidak’s post hoc test. A) Tolerance induced in all mice over 3 days with twice
Regulate Opioid Anti- curves. Potency values calculated by linear regression. A50: daily injection of 10 mg/kg morphine s.c. (as in Figure 4). On day 3, mice injected with 1 mg/kg Summary
Nociception in Brain Vehicle = 6.5 mg/kg, KUNB106 = 1.7 mg/kg, 3.9 fold shift. KUNG65 or Vehicle i.v., 24 hrs, followed by 10 mg/kg morphine s.c. and another tail flick time course.
Specific Hsp90 isoforms regulate brain vs. spinal cord opioid anti-nociception.
vs. Spinal Cord. Male KUNG65 rescued established tolerance much like intrathecal injection of KU-32 above. B) Mice
Delivering a systemic inhibitor specific to a spinal isoform (e.g. KUNG65 for Grp94, and
and female CD-1 mice
injected icv (A-C) or it
Systemic Hsp90β Inhibitor Boosts Pain Relief in Paw Incision Pain injected with 1 mg/kg KUNG65 or Vehicle i.v., 24 hrs, then habituated and baselined in a whole body
plethysmography chamber for 30 minutes (see Methods). All mice were then injected with 7.5 mg/kg
KunB106 for Hsp90β) enhances opioid pain relief and reduces side effects. This is
verified with CRISPR knockout which demonstrates similar antinociceptive effects
(D-F) with Vehicle or 0.1 A. KUNB106 (IV) - Paw Incision 1mg/kg
B. KUNB106 (IV) - Paw Incision 1.8mg/kg
C. KUNB106 (IV) - Paw Incision 3.2 mg/kg morphine i.v., and respiratory activity recorded for another hour. KUNG65 had no effect on respiration
through removing the proteins of interest.
nmol of isoform 2.5 2.5 2.5 before or after morphine injection (p > 0.05). C) Tolerance induced as above, and on day 3, 1 mg/kg
Mechanical Threshold (g)

KunB106
Mechanical Threshold (g)

Mechanical Threshold (g)

KunB106 KunB106 KunB106 and KunG65 have also been demonstrated to not change dangerous side
selective inhibitor, as 2.0 Vehicle 2.0 Vehicle 2.0 Vehicle KUNB106 or Vehicle injected i.v., 24 hrs, followed by 10 mg/kg morphine s.c. and another tail flick
effects like respiratory depression which enables a translational opioid dose-reduction
indicated. Paw incision timecourse. KUNB106 also rescued established tolerance like systemic KUNG65 or intrathecal KU-32.
1.5 1.5 1.5 strategy where less opioids need to be administered to achieve desired pain relief, but
mice (A-C) also had D) Mice injected with 1 mg/kg KUNB106 or Vehicle i.v., 24 hrs, then respiratory activity measured as
with less overall side effects.
paw incision surgery. 1.0 1.0 1.0
above (including 7.5 mg/kg morphine i.v. challenge). KUNB106 had no impact on respiratory activity
Recovered for 24 hours,
followed by 3.2 mg/kg
0.5 0.5 0.5 before or after morphine (p > 0.05). Future Studies
morphine SC and Von
0.0
30 60 90 120 150 180
0.0
30 60 90 120 150 180
0.0
30 60 90 120 150 180
Systemic Hsp90α-selective Inhibitor Matches Non- The focus will now be on developing and testing oral formulations of the isoform
hr L
L
hr L
L
24 BL

24 Inj B
.B
24 Inj B
.B

Selective Inhibitor inhibitors for clinical use surrounding efficacy, side effects, toxicity, etc.
.B

Post-Morphine Time (Min)

Frey for Paw Incision Post-Morphine Time (Min.) Post-Morphine Time (Min.)
k
hr
/In

/
/

rg
rg
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We will also delve into the mechanisms by which these isoforms regulation opioid
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mice (A-C) or 52°C tail

Su
Su

e-
e-

KUNA177 (IV) - Paw Incision 3.2mg/kg

e-

Pr
Pr

signaling (e.g. signal transduction cascades – candidates like Src, PKC, and GAT-2 from
Pr

flick for Tail Flick mice Figure 7: Systemic Hsp90β inhibition enhances morphine anti- Figure 10: Systemic, Hsp90α-Selective Inhibitor
2.5
(D-F). Mice/group noted D. Blocks Opioid Pain Relief. Male and female CD-1 proteomic studies.
Mechanical Threshold (g)

KunA115 Vehicle
nociception in paw incision pain. Male and female CD-1 mice treated
in graphs. *, **, ***, **** 2.0 mice injected IV with 1 mg/kg KUNA177 (selective In addition more research can help expose other novel mechanisms that modulate the
with paw incision surgery and 1 mg/kg KUNB106 or Vehicle control i.v.,
= p < 0.05, 0.01, 0.001, Hsp90α inhibitor) or Vehicle, along with paw incision opioid pathway, providing for more systems of interest that can be manipulated for the
24 hrs, followed by 1 – 3.2 mg/kg s.c. morphine and a Von Frey 1.5
0.0001 vs. same time surgery, then 24 hrs recovery, followed by 3.2 mg/kg clinical use.
mechanical allodynia timecourse performed. Data presented as the 1.0
point Vehicle group by 2 SC morphine and a mechanical allodynia time course
Way ANOVA with
mean ± SEM, with sample sizes of mice/group noted in each graph;
experiments performed in 2 technical replicates per dose. Data 0.5 measured. Acknowledgement
Sidak’s post hoc test. A-C) Inhibitor injection into brain with paw incision pain model revealed that KUNA115 (Hsp90α) transformed into %MPE and used to construct dose/response curves. Systemic, selective inhibitor blocked anti- This work was supported by an Arizona Biomedical Research Commission New Investigator
0.0
but not KUNB106 (Hsp90β) or KUNG65 (Grp94) blocked opioid anti-nociception, suggesting Hsp90α is the active A50: Vehicle = 2.5 (2.0 – ∞) mg/kg, KUNB106 = 0.99 (∞ – ∞) mg/kg; 2.5 30 60 90 120 150 180 nociception much like brain inhibition in our earlier Award #ADHS18-198875, NIH R01DA052340, and institutional funds from the University of
hr L
L
24 nj B
.B

isoform in brain. D-F) Inhibitor injection into spinal cord with tail flick pain model revealed that all inhibitors enhanced Post-Morphine Time (Min) work.
fold improvement in morphine potency. Arizona. JMS has an equity stake in Teleport Pharmaceuticals, LLC and Botanical Results, LLC;
/I
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opioid anti-nociception, suggesting that all 3 isoforms regulate opioid anti-nociception.

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no company products or interests were tested in this work.

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