Small Molecule Inhibitors of SARM1 Prevent Axonal Degeneration in vitro and in vivoRaul Krauss*, Todd Bosanac, Thomas Engber, Rajesh Devraj, Robert HughesDisarm Therapeutics, Cambridge, Massachusetts 02139, USA

3. A Proprietary Screening Platform Identified Small Molecule SARM1 Inhibitors

7. SARM1 Inhibitors Prevent Neuropathy in vivo in a Paclitaxel CIPN Model

Axonal degeneration is an early and ongoing event that causes disability anddisease progression in many neurodegenerative disorders of the central,peripheral, and ocular nervous systems, including multiple sclerosis, ALS,peripheral neuropathies, and glaucoma. SARM1 is the central driver of anevolutionarily conserved program of axonal degeneration downstream ofinflammatory, mechanical, metabolic, or chemical insults to the axon.SARM1 contains an intrinsic NADase enzymatic activity essential for its pro-degenerative functions, making it a compelling therapeutic target to treatneurodegeneration characterized by axonopathy.Disarm has developed a high-content screening platform and identifiedproprietary drug-like small molecule inhibitors of SARM1. These inhibitorsprotect rodent and human axons in vitro from mechanical,chemotherapeutic and mitochondrial damage. In sciatic nerve transection,SARM1 inhibitors prevent increases in plasma NfL, a clinically accessiblebiomarker of axonal degeneration. In a paclitaxel model of chemotherapy-induced peripheral neuropathy (CIPN) SARM1 inhibitors protect axonalstructure (plasma NfL) and function (SNAP amplitude).This is the first demonstration that pharmacological inhibition of SARM1 canreproduce the axonal protective phenotype observed in SARM1 knockoutmice. The availability of SARM1-dependent biomarkers of axonaldegeneration enables rapid translation of SARM1 inhibitors from discoveryto the clinic. SARM1 inhibitors have the potential to prevent axonaldegeneration in peripheral and central axonopathies and provide atransformational disease-modifying treatment for these disorders.

1. SARM1 Drives Rapid NAD+ Loss Leading to a Bioenergetic Crisis and Axonal Degeneration

Triggers Diseases

Axonal Degeneration Glaucoma

Multiple sclerosis

CIPN

Neurodegenerative (PD, ALS)

Peripheral neuropathies (diabetic, CMT)

CNS, Eye PNS

SARM1

Immuno/inflammatory

Metabolic / Toxins

Trauma

Mitochondrial / Other

Intraocular pressure

2. SARM1 - The Central Driver of Axonal Degeneration in Neurological Diseases

4. SARM1 Inhibitors Protect from Mechanical, Chemical and Mitochondrial InsultsDisarm’s small molecule inhibitors phenocopy SARM1 KO in primary DRG

5. SARM1 Inhibitors Protect Human AxonsHuman IPSC-derived motor neurons

Inhibitors from multiple chemical series inhibit SARM1 NADase

Disarm’s small molecule inhibitors phenocopy SARM1 KOPrevent increase of axonal structure biomarker plasma NfL and functional axonal conduction

Control Cut Cut + DSRM-XX Axonal Fragmentation

Uncut Cut Cut +Series 1

0

20

40

60

80

100

% A

xona

l Deg

ener

atio

n

********

Pharmacological SARM1 InhibitionGenetic Inhibition in SARM1 KO

** p< 0.01; **** p < 0.0001; ANOVA with Bonferroni post-hoc

Conclusions§ Disarm has advanced a proprietary R&D platform to discover

small molecule inhibitors of SARM1, the central driver of axonal degeneration

§ We are providing the first demonstration that Disarm’s novel small molecule SARM1 inhibitors reproduce the axonal protection phenotype seen in SARM1 knockout. Disarm’scompounds: • Protect axons in vitro from multiple pathological insults• Protect human iPSC-derived motor axons from traumatic injury• Prevent axonal degeneration and preserve axonal function in vivo in a

rodent model of chemotherapy-induced peripheral neuropathy (CIPN)

Disarm is developing small-molecule SARM1 inhibitors for patients with neurological diseases such as MS, ALS and CIPN

CutUntreated Cut + DSRM-XX Axonal Fragmentation

PaclitaxelUntreated Paclitaxel + DSRM-XX Axonal Fragmentation

Axonal Fragmentation NfL Biomarker

NfL Biomarker

NfL Biomarker

0

2000

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8000

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12000

NfL

(pg

/ml)

Untreated Rotenone Rotenone +Series 1

**** ****

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2000

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NfL

pg/

ml

**

Uncut Cut Cut + Series 1

***

0

10000

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40000

NfL

(pg

/ml)

**

Untreated Paclitaxel Paclitaxel +Series 1

****

0

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% A

xona

l Deg

ener

atio

n

Untreated Paclitaxel Paclitaxel +Series 1

********

0

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% A

xona

l Deg

ener

atio

n

Rotenone Rotenone +Series 1

Untreated

*****

0

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% A

xona

l Deg

ener

atio

n

Uncut Cut Cut +Series 1

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In vitro Assay in Primary DRG Neurons

** p< 0.01; *** p <0.001; **** p < 0.001; ANOVA with Bonferroni post-hoc

Rotenone Rotenone + DSRM-XXUntreated

Axonal cut

Imaging field Traumatic injury

Non-traumatic injury

Trau

mat

icCh

emot

hera

peut

icM

itoch

ondr

ial

Axon

al S

truc

ture

NfL

Bio

mar

ker

*p<0.05; ** p< 0.01; *** p <0.001; **** p < 0.0001; ANOVA with Bonferroni post-hoc

Pharmacological SARM1 InhibitionGenetic Inhibition in SARM1 KO

Axon

al S

truc

ture

NfL

Bio

mar

ker

Axon

al F

unct

ion

SNAP

Am

plitu

de

0 1 2 3 4 5 6 7 8 90

10

20

30

0

2

4

6

8

Time (h)

NAD

(ng

/ 100

,000

cel

ls) cADPRNAD

cADPR

(ng / 100,000 cells)

ADPR

0 .1 1 1 01 0 0

-2 0-1 0

01 02 03 04 05 06 07 08 09 0

1 0 01 1 01 2 0

T im e d e p e n d e n t in h ib it io n o f '1 2 9 65 µ g /m L N R K 1 -H E K 2 9 3 T + E V 5 4 6 7

5 µ M N A D + , 0 - 2 h p r e in c u b a t io n , 1 2 0 m in a t R T

c (to o l c m p d ) [µ M ]

% S

AR

M1

ac

tiv

ity

1 % D

MS O

w /o ly

sa te

S u b s tra te c o m p e t it io n o f '1 2 9 65 µ g /m L N R K 1 -H E K 2 9 3 T + E V 5 4 6 7

7 5 µ M N A D + , 2 h p r e in c u b a t io n , 1 2 0 m in a t R T

DMSO 0.03 X 0.3 X 3 X 30 X BlankDSRM-XX

Biochemical SARM1 Assay Cell-based DRG Axotomy AssaySARM1 produces cADPR after axotomy SARM1 Inhibitors Prevent cADPR IncreaseDisarm’s Small Molecules Inhibit SARM1

0

5

10

DSRM-Series 1

cAD

PR (n

g / 1

00,0

00 c

ells

)

1 X 10 X0.1 X0

Cut

Uncut

SARM1 Pathway

NAM

NMN

NAD+

NAM+ADPR

cADPR

NAMPT

NMNAT

SARM1

?

NAM + ADPR/cADPR

NAD+ cleaves into ADPR/cADPR

6. SARM1 Inhibitor Protects Axons in vivo After Sciatic Nerve Transection Disarm’s small molecule inhibitors phenocopy SARM1 KO

DSRM-XX

DSRM-XX DSRM-XX

DSRM-XX DSRM-XX

DSRM-XXDSRM-XX

DSRM-XX

0

1000

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4000

Plas

ma

NfL

(pg/

ml)

Naive Vehicle DSRM-XXLow

DSRM-XXHigh

**

******

**

0

2000

4000

6000

Plas

ma

NfL

pg/

ml

WT WT SARM1HET

SARM1KO

PaclitaxelVehicle

******

****

0

50

100

150

200

250

SNA

P am

plitu

de (µ

V)

WT WT SARM1HET

SARM1KO

PaclitaxelVehicle

*****

*******

0

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6000

Plas

ma

NfL

(pg/

ml)

** **

Vehicle Vehicle DSRM-XXLow

DSRM-XXHigh

Paclitaxel

0

50

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SNA

P A

mpl

itude

(µV) ** **

Vehicle Vehicle DSRM-XXLow

DSRM-XXHigh

Paclitaxel

0

200

400

600

1800

2000

Plas

ma

NfL

pg/

ml

WT SARM1KO

****

Naïve SARM1HET

**

**