COR388 (atuzaginstat), a novel gingipain inhibitor, decreases ApoE fragmentation in the CNS of Alzheimer’s disease patients

Debasish Raha1, Sean Broce1, Ursula Haditsch1, Leo Rodriguez1, Florian Ermini1, Michael Detke1, Shirin Arastu-Kapur1, Dave Hennings1, Mai Nguyen1, Leslie J. Holsinger1, Casey Lynch1, Stephen Dominy1

BACKGROUNDBackground

ApoE4 allele is the major risk factor for sporadic AD and has been linked to major pathological hallmarks of the disease including amyloid plaques, neurofibrillary tangles, inflammation and oxidative stress. The results presented here indicate that gingipains, which have been identified in >90% of AD brains, target ApoE proteins for proteolytic cleavage, and that the ApoE4 protein is more susceptible to gingipain fragmentation than ApoE3 or ApoE2, suggesting a plausible mechanism for how APOE4 is linked to AD, i.e., through a more rapid and extensive gingipain-induced loss of critical ApoE functions and/or the generation of toxic fragments as suggested by others. The concept of enhanced ApoE4 proteolysis being linked to AD is not new, but the evidence presented here showing that gingipains can preferentially fragment ApoE4, is novel. The physiological relevance of this mechanism is supported by the ability of the brain-penetrant gingipain inhibitor, COR388 (atuzaginstat), to decrease the level of LMW ApoE fragments in AD CSF after 28 days of treatment. In addition, 28 days of dosing with atuzaginstat was associated with significant improvements in speech from baseline and compared to placebo. A Phase 2/3 study assessing the safety, tolerability, and efficacy of atuzaginstat in subjects with mild-to-moderate AD (GAIN) (www.gaintrial.com) is now ongoing.

Conclusion

Protease virulence factors known as gingipains from the bacterial pathogen Porphyromonas gingivalis (P.g) were recently identified in Alzheimer’s disease (AD) brains and gingipain levels correlated with AD diagnosis and tau and ubiquitin pathology (1). Studies in wild-type mice demonstrated that P. gingivalis invades the brain after infection of the oral cavity, resulting in inflammation, neurodegeneration, and an Aβ1-42 response that is blocked with brain-penetrant gingipain inhibitors. The e4 allele of APOE gene is the greatest genetic risk factor for sporadic AD. Of note, proteolytic products of ApoE have been detected in AD brain and CSF. Previous research demonstrated that ApoE4 may be linked to AD by being more susceptible than ApoE3 or ApoE2 to proteolytic cleavage by an unknown protease or proteases (2,3). We interrogated whether ApoE proteins are targets of gingipain proteolytic cleavage. Here we report that gingipains preferentially cleave ApoE4 compared to ApoE3 and ApoE2 in vitro. ApoE proteolysis in the cerebrospinal fluid of 9 AD subjects was monitored in a Phase1b clinical trial where subjects were treated with a brain penetrant oral Kgp-gingipain inhibitor COR388 (atuzaginstat) for 28 days. Kgp protease is essential for survival and virulence of P. gingivalis. Treatment with COR388 significantly reduced the CSF level of low-molecular-weight ApoE fragments of ~ 15 Kd compared to placebo. COR388 treatment also demonstrated significant improvements in measures of speech, a function that is affected in a significant proportion of AD patients (4).

Poster #40578

1Cortexyme, South San Francisco, CA, USA

Fig. 1 RgpB colocalizes with neurons and astrocytes in postmortem AD hippocampusImmunofluorescent colabeling with the arginine-gingpain (RgpB)-specific CAB101 antibody reveals RgpB colocalization with MAP-2 positive neurons and glial fibrillary acidic protein (GFAP)-positive astrocytes. Scale bars, 10 um.

GFAP MAP2 RgpB

DAPI merge control merged

Fig. 2 Proteolytic products of ApoE in AD brainLow-molecular-weight (LMW) ApoE proteolytic fragments(~15kD) are detected in AD postmortem brain by immunoblot using an ApoE antibody that was raised against human ApoE4 protein (Novus) (pan-ApoE antibody). Mass spectrometry (MS) analysis of brain proteins of 10-15 kD in size extracted from gel identified ApoE peptides that mapped to the C-terminal half of the ApoE protein.

AD Brain Control Brain

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Fig. 3 Proteolytic products of ApoE in AD CSFIn a separate cohort of subject samples unrelated to subject samples in Fig. 2 above, LMW ApoE proteolytic fragments (~15 kD) are detected in the CSF by immunoblot using the pan-ApoE antibody. MS analysis of CSF proteins of 10-15 kD in size extracted from gel identified peptides that mapped to ApoE protein.

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Fig. 4 ApoE4 protein is preferentially cleaved by purified gingipains Recombinant ApoE3 and ApoE4 (rApoE3 and rApoE4) proteins were incubated with purified gingipains Kgp and RgpB. The cleavage products were detected by immunoblot using a pan-ApoE antibody. Gingipains cleave recombinant ApoE3 and ApoE4 rapidly in vitro, indicating cleavage of ApoE by the gingipain proteases is direct. Cleavage of ApoE4 is more extensive and more rapid than ApoE3 indicating ApoE4 may be a preferred substrate for gingipains.

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Fig. 7 P.g-induced ApoE proteolysis in astrocytes is blocked by COR388 (A) Human iPSC-derived astrocytes (ApoE genotype E3/E4) were infected with P.g either in the presence or absence of individual gingipain inhibitors. (B) Proteolysis of cellular ApoE was detected in infected cells at MOI 50. Incubation of P.g prior to infection with the lysine-gingipain (Kgp) inhibitor COR388 protected ApoE protein from degradation. However, this protective effect was not seen with the RgpB inhibitor COR613. This data suggests that endogenous ApoE protein synthesized in astrocytes may have one or more Kgp cleavage sites initially exposed. The cleavage of these Kgp sites may then allow Rgp access its cleavage sites. Lipidation of ApoE in astrocytes may change the 3-dimensional structure of ApoE and mask certain gingipain cleavage sites when compared to non-lipidated recombinant proteins.

Results

Results

Fig. 8 Gingipain cleavage sites in ApoE proteins and frequency of cleavage of ApoE4 vs. ApoE3

MS analysis of gingipain digests (1 min) of recombinant hApoE3 and hApoE4 identified extensive overlap in cleavage sites between both isoforms except one site (R226) that is unique to ApoE4. However, the frequency of cleavage in hApoE4, as measured by peptide counts, was found higher for most of the sites. (A) Shows a map of gingipain cleavage sites on ApoE protein and (B) the bar graph shows the difference in frequency of cleavage for each peptide (shown by unique color) between ApoE3 and ApoE4.

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(a) LDLR binding domain (red), (b) Lipid binding domain (red), (c) RgpB-generated fragment from the LDLR domain, (d) Kgp-generatedfragment from the LDLR domain, (e) Kgp-generated fragment from the lipid binding domain.*Christchurch mutation site (R→S). **ApoE2 158 (R→C). ***R226 = RgpB cleavage site cleaved in ApoE4 but not in ApoE3.

Days of Treatment

Fig. 9 ApoE fragment found in CSF is reduced in COR388-treated subjectsApoE is proteolytically cleaved by gingipains and a similar ApoE cleavage product of approx. 15kD is detected in AD brain (Fig. 2) and CSF (Fig. 3). ApoE fragmentation was reduced in COR-388 treated subjects compared to placebo in a phase1b study . COR388 (n=6), placebo (n=2).

p=0.017

Results

Fig. 10 Effect of COR388 treatment on cognitive testing and speech variable changes in phase 1b study (A) MMSE scores showed a trend to improvement from baseline and compared to placebo; the difference was not statistically significant. (B) CANTAB memory composite of cognitive function showed a trend to benefit of COR388 treatment comparted to baseline and compared to placebo; the difference was not statistically significant. (C) Winterlight speech and cognitive

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Lysates were prepared from human iPSC derived neurons of uninfected and Pg-infected cells treated with or without gingipain inhibitors. These lysates were incubated with rApoE3 or rApoE4.Uninfected cell lysates, while containing a large number ofproteases, did not exhibit any significant ApoE cleavage activity. Infected cell lysates produced rapid cleavage of ApoEwithin 1 minute, with preferential cleavage of ApoE4. This activity is completely inhibited by gingipain inhibitors (COR388, a Kgp inhibitor and COR613 a Rgp inhibitor).

Fig. 5 ApoE4 protein is preferentially cleaved by gingipains in infected cell lysate

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(A) Preferential cleavage of ApoE4 over ApoE3 and ApoE2 protein was observed when brain extracts from human ApoE knock-in mice, produced in nonionic detergents, were incubated with a mixture of purified Kgp and RgpB gingipains. These data indicate that the native structure of human APOE found within the native environment of the brain, were also cleaved by gingipains and with the same allelic preferences for cleavage. (B) A quantitative assessment of this cleavage, normalized to cellular actin protein.

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Fig. 6 Human ApoE4 expressed in mouse brain is more sensitive to gingipains than human ApoE3 or human ApoE2

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battery was used to assess the change from baseline in 35 speech variables. Improvement in the level of detail provided during the picture description was seen in the treatment group but not in the placebo group. Three speech measures significantly increased in the treatment group including use of prepositions and subordinating conjunctions in which COR388 vs. placebo remained significant after Bonferroni correction.

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1. S Dominy et al. (2019) Science Advances 5, 1-222. Y Huang et al. (2001) PNAS 98, 8838–88433. A Mouchard et al. (2019) Scientific Reports 9, 39894. KC Fraiser et al. (2016) Journal of Alzheimer’s Disease 49, 407–422

References

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