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Design and Evaluation of Highly-Selective Human Immunoproteasome Inhibitors Reveals a Compensatory Process that Preserves Immune Cell Viability Ena Ladi, Christine Everett, Craig E. Stivala, Blake E Daniels, Matthew R Durk, Seth F. Harris, Malcolm Huestis, Hans E Purkey, Steven T. Staben, Martin Augustin, Michael Blaesse, Stefan Steinbacher, Celine Eidenschenk, Rajita Pappu, and Michael Siu J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00509 • Publication Date (Web): 08 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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Journal of Medicinal Chemistry

Design and Evaluation of Highly-Selective Human Immunoproteasome Inhibitors Reveals a Compensatory Process that Preserves Immune Cell Viability Ena Ladi,1a Christine Everett,2a Craig E. Stivala,3a Blake E. Daniels,3 Matthew R. Durk,4 Seth F. Harris,5 Malcolm P. Huestis,3 Hans E. Purkey,3 Steven T. Staben,3 Martin Augustin,6 Michael Blaesse,6 Stefan Steinbacher,6 Celine Eidenschenk,2 Rajita Pappu,1* and Michael Siu3* 1Departments 4Drug

of Immunology, 2Biochemical and Cellular Pharmacology, 3Discovery Chemistry,

Metabolism and Pharmacokinetics, and 5Structural Biology, Genentech, Inc., 1 DNA

Way, South San Francisco, California 94080, United States 6Proteros

Biostructures GmbH, Bunsenstraße 7 a, 82152 Planegg-Martinsried, Germany

aContributed

equally to this work

ABSTRACT: The pan-proteasome inhibitor bortezomib demonstrated clinical efficacy in off-label trials of Systemic Lupus Erythematosus. One potential mechanism of this clinical benefit is from the depletion of pathogenic immune cells (plasmablasts and plasmacytoid dendritic cells). However, bortezomib is cytotoxic against non-immune cells which limits its use for autoimmune diseases. An attractive alternative is to selectively inhibit the immune cell-specific immunoproteasome to deplete pathogenic immune cells and spare non-hematopoietic cells. Here we disclose the development of highly subunit-selective immunoproteasome inhibitors using insights obtained from the first bona fide human immunoproteasome co-crystal structures. Evaluation of these inhibitors revealed that immunoproteasome-specific inhibition does not lead

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to immune cell death as anticipated and that targeting viability requires inhibition of both immuno- and constitutive proteasomes. CRISPR/Cas9-mediated knock-out experiments confirmed upregulation of the constitutive proteasome upon disruption of the immunoproteasome, protecting cells from death. Thus, immunoproteasome inhibition alone is not a suitable approach to deplete immune cells.

INTRODUCTION Pathogenic autoantibodies and pro-inflammatory cytokines are key drivers of the tissue damage associated with autoimmune diseases such as systemic lupus erythematosus (SLE).1 Deposition of autoantigen-antibody immune complexes (IC) in the kidney and other tissues leads to inflammation induced injury and ultimately end-organ damage. Additionally, ICs promote the secretion of inflammatory cytokines from cells bearing activating Fc-receptors, such as monocytes and dendritic cells (DCs). Among the DCs, activation of plasmacytoid DCs (pDCs) is particularly deleterious as it leads to production of type I IFN, which amplifies the autoimmune response. Corticosteroids and other pan-immunosuppressants are the standard of care treatments; however, these agents are not sustainable for long-term use due to concomitant toxicities. A number of approaches to target either the plasmablasts (PB)/autoantibodies or type I IFN pathways in SLE patients have been evaluated, albeit with limited clinical success. In the last 50 years the only FDA-approved medication to treat SLE is belimumab, which inhibits the B cell component of the disease by neutralizing the growth factor BAFF/BlyS, and shows limited efficacy. However, emerging clinical data suggest targeting both axes may provide a substantial benefit to SLE patients.2


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Table 1a Subunit specific in vitro biochemical IC50 values of literature proteasome inhibitors β5i IC50 Compound

Structure

β1i IC50

(β5c / β5i) Bortezomib

(β5c / β1i)

3.4 ± 1.1 nM

7.9 ± 2.5 nM 1.9 ± 0.86 nM

1

(0.56x)

ONX-0914

145 ± 69 nM

32 ± 3 nM (0.24x)

790 ± 450 nM 1300 ± 100 nM

2

(9.0x)

PR-893

2100 ± 120 nM

13000 ± 2000 nM (1.6x)

690 ± 350 nM 78 ± 9 nM

3

β1c IC50

β5c IC50

(0.04x)

11000 ± 500 nM (0.11x)

Merck 8.1 ± 0.52 nM KGaA

2200 ± 1000 nM 2400 ± 890 nM

>20000 nM (1.1x)

(296x) 4

Roche

2.2 ± 2.4 nM

8.1 ± 4.2 nM 37 ± 12 nM

5 a

(17x)

1400 ± 80 nM (5.2x)

All assays results represent the mean of a minimum of three determinations with the standard

deviation reported.

One approach to simultaneously perturb antibody production from PBs and cytokine secretion from pDCs is to take advantage of the highly secretory nature of these cell types. Central to functional protein synthesis and secretion is maintenance of ER homeostasis through the ubiquitin-proteasome system. Genetic and pharmacologic studies reveal disruption of ER



homeostasis impedes the function and viability of PBs and pDCs.3,4 The clinical benefit from targeting this pathway in SLE has been demonstrated in off-label trials and small case studies using bortezomib (1), a pan-proteasome inhibitor that has been approved for the treatment of multiple myeloma.5,6 Amelioration of disease activity was accompanied by reductions in both autoantibody titers and Type I IFN production. However, significant adverse events, particularly neuronal toxicity, restrict the use of this class of inhibitors for the treatment of chronic inflammatory diseases. A means to circumvent these adverse effects while maintaining suppression of pDCs and PBs is to selectively inhibit the immunoproteasome, a unique proteasome species predominantly expressed in hematopoietic cells. In immune cells, the enzymatic subunits of the constitutive proteasome (1c, 2c, 5c) are replaced by immunoproteasome specific subunits (1i, 2i, 5i).7 As bortezomib (1) blocks the chymotrypsin-like activity of both the immune (5i and 1i) and non-immune or constitutive proteasomes (5c and 1c), the therapeutic benefit seen in autoimmune indications is hypothesized to result from perturbation of immunoproteasome activity triggering immune cell apoptosis.7 Published data indicate that compounds with a greater selectivity towards the 5i confer the same level of therapeutic benefit as bortezomib (1) in pre-clinical models of various autoimmune diseases.8 However, in a subunit specific in vitro biochemical assay, these compounds such as ONX-0914 (2) only displayed 10-20 fold selectivity for 5i over 5c and thus may not provide a significant margin against inhibition of 5i over 5c in vivo.9 As part of our ongoing drug discovery efforts, we set out to develop inhibitors with greater immunoproteasome selectivity to avoid effects on non-hematopoietic cells while maintaining the


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potent cytotoxicity of bortezomib (1) toward immune cells. Notable progress in selectivity improvements have been reported. For example, acrylamide boronic acid 4 has improved selectivity for β5i;10 however, concerns exist that irreversible covalent inhibitors may obscure proteasome selectivity benefits especially in a non-oncology patient population. Accordingly, we have focused on developing reversible inhibitors that specifically targeted the 5i subunit alone and in combination with the 1i subunit of the immunoproteasome.11 To this end, we have generated a series of highly specific inhibitors against the chymotrypsinlike subunits of the immunoproteasome and found no correlation between inhibition of β5i and cell death but rather a correlation between inhibition of β5c and cell death. We report here that selective inhibition of the immunoproteasome is not a suitable strategy to induce immune cell death. Instead, in response to immunoproteasome inhibition, immune cells maintain ER homeostasis through induction of the constitutive proteasome catalytic subunits, thus subverting apoptosis. CRISPR/Cas9 mediated genetic ablation of the genes encoding the 5i and/or 5c subunits in human primary T cells revealed cell death is only seen when both proteins are knocked-out. We conclude that routinely used immunoproteasome inhibitors are only marginally selective (vide infra), and sufficiently block the constitutive proteasome to promote cell death. Since both 5i and 5c contribute to the Ubiquitin-proteasome pathway in immune cells, depletion of the pathogenic immune cells can only be achieved by inhibition of both subunits.

RESULTS AND DISCUSSION



We started by evaluating the β5 and β1 biochemical activity of literature compounds as show in Table 1. We selected -aminoboronic acid 5 from Roche for optimization given the multiple vectors available on the triazole scaffold and its initial indication of moderate 5i selectivity.12 To facilitate design and development of immunoproteasome-selective inhibitors, we determined the X-ray structure of the human immunoproteasome 20S particle bound to compound 5 at 2.8 Å resolution (Figure 1). This structure represents the first reported crystal structure of the bona fide human immunoproteasome, a unique supplement to existing analyses of the constitutive proteasome, chimeric immuno-constitutive versions engineered in yeast, and a recent electron microscopy report.13,14 Overall features of the immunoproteasome structure are consistent with prior examples, as alignment of β5i subunits gives an RMSD of 0.54 Å over 201 C-α atoms relative to a recently determined electron microscopy structure (PDB 6AVO). The structure shows a consistent overall architecture of four heptameric rings, the outer rings of alpha subunits 1-7 and the inner rings consisting of the beta subunits 1-7 (Figure 1B). Refined 2Fo-Fc density at 1 sigma contour near subunit β1i (Figure S1A) and β5i (Figure S1B) is consistent with the modeled ligand binding mode.


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Figure 1. (A) X-ray crystal structure of the 28-subunit human immunoproteasome 20S particle bound to α-aminoboronic acid inhibitor 5 (2.8 Å, 6E5B). Four 7-membered rings comprise the barrel, with outer -subunit rings colored grey and the two inner β rings distinguished by individual subunit colors. (B) Isolated view of the upper catalytic β ring, extracted from the particle and rotated by 90 for a bottom-up view. Ligand molecules shown in bright green are observed bound to the interior proteasome cavity-facing surfaces of subunits β1i and β5i.



As shown in Figure 2A, boronic acid 5 forms a covalent tetrahedral boronate adduct with the catalytic threonine residues (Thr1) while the P1 iso-butyl moiety of the inhibitor extends into the S1 pocket. The adjacent amide forms hydrogen bonds to the backbones of residues Gly47 and Ser21. The triazole is proximal to Cys48, a substitution unique to the 5i subunit (Gly in 5c). A significant portion of the S3 pocket is formed by the adjacent 6 subunit, though somewhat unexpectedly the distal tri-methoxybenzamide tail of the ligand has more ambiguous density and does not occupy this S3 cleft. Rather, some residual density in that area can be interpreted as a thiocyanate molecule from the crystallographic solvent, while the bulk of the ligand tail appears oriented toward the solvent-exposed inner cavity of the proteasome (S4 pocket). Combined with varying conformations in the different subunits, the partial density is strongly indicative of some degree of mobility or disorder to the feature. This additionally suggests that the benzamide tail group may not be contributing strongly to the binding affinity for the 5i subunit.


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Figure 2. Binding mode of compound 5 to 5i and chemistry strategy. (A) The crystal structure indicates compound 5 forming a covalent boronate adduct to catalytic Thr1. Additional hydrogen bond interactions are shown by thinner dashed lines. Spheres at protein C-α atoms mark residues that differ between the 5i and 5c subunits (constitutive sequence identity shown in parentheses). The tri-methoxybenzamide tail at left is swung forward into a solvent-exposed region and has more modest density (supplemental figure S2). (B) Overarching structure-based design strategy to achieve immunoproteasome-selective inhibitors.



Based on these key structural insights, we devised a flexible chemistry strategy focused on three key areas of investigation (Figure 2B). In addition to identifying a 5i-selective inhibitors, recent literature reports have suggested that dual 5i/1i-selective inhibitors could be advantageous to 5i-selective inhibitors alone.15 Since the 1i subunit of the immunoproteasome also has chymotrypsin-like activity, it is possible that compensatory degradation by 1i may occur if the 5i subunit is inhibited. Our pursuit to identify immunoproteasome-selective inhibitors began by interrogating our structural hypothesis. To further understand the potency and selectivity contributions of the trimethoxybenzamide tail we truncated compound 5 to generate methyltriazole 6 (Table 2) which improved selectivity (5c/5i = 174x) and maintained appreciable levels of immunoproteasome potency (5i IC50 = 55 nM and 1i IC50 = 9.0 nM). With this more selective starting point, we reintroduced substitution into the S3 pocket. Incorporating cyclohexyl, phenyl or benzyl substituents (Table 2, Entries 7-9) restored potency (5i IC50 = 1.4–4.1 nM) and uniformly improved immunoproteasome selectivity (5c/5i = 220x–710x and 5c/1i = 1300x–3400x). To the best of our knowledge, compounds 7, 8 and 9 are three of the most selective 5i/1i dual inhibitors reported to date.

Table 2a Structure activity relationship of proteasome inhibitors


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O MeO MeO

OMe HN

N

N N

O N H

B OH

N

R2

O

OH

N H

N N

OH

R1

B OH

Core

N H

OR' B OR"

O

5

6

R',R" = H or pinanediol 7-21

PBMC Compound

R1

Core

R2

β5i IC50

β5c IC50

β1i IC50

β1c IC50

(nM)

(nM)

(nM)

(nM)

Chymotrypsin IC50 (nM)

5

-

-

-

2.2 ± 2.4

37 ± 12

8.1 ± 4.2

1400 ± 80

34 ± 17

6

-

-

-

55 ± 22

9600 ± 570

9.0 ± 2.3

>20000

250 ± 48

7

4.1 ± 4.6

2900 ± 550

0.85 ± 0.14

480 ± 80

60 ± 33

8

1.4 ± 0.85

760 ± 170

0.58 ± 0.068

4200 ± 900

25 ± 6

9

4.0 ± 0.22

890 ± 220

0.55 ± 0.065

71 ± 6

45 ± 15

10

360 ± 140

>10000

19 ± 5.9

>20000

8900 ± 2100

11

19 ± 1.1

350 ± 39

6.3 ± 4.2

61 ± 8

200 ± 43

12

23 ± 5.6

3000 ± 950

9.4 ± 3.5

1400 ± 100

85± 19

13

40 ± 21

7300 ± 770

15 ± 3.4

>20000

290 ± 218

14

130 ± 32

>10000

41 ± 3.2

18000 ± 310 ± 133 2000

15

0.91 ± 0.10

64 ± 4.0


15 ± 2.6

5500 ± 600

3.2 ± 1.7


a

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16

6.8 ± 7.3

3200 ± 680

12 ± 8

>20000

130 ± 39

17

0.54 ± 0.047

46 ± 6.4

3.9 ± 2.8

530 ± 40

2.3 ± 0.8

18

87 ± 25

7700 ± 1100

110 ± 14

>20000

470 ± 110

19

4.1 ± 1.8

1300 ± 560

92 ± 21

>20000

80

20

0.68 ± 0.18

270 ± 74

50 ± 34

>20000

6.9

21

1.2 ± 0.59

420 ± 62

17 ± 2.2

>20000

11 ± 5

22

4.1 ± 0.50

9100 ± 770

8500± 2700

>20000

79 ± 18

All assays results represent the mean of a minimum of three determinations with the standard

deviation reported.

Having quickly identified dual 5i/1i-inhibitors we continued our chemistry efforts with a focus on identifying 5i-selective chemical matter. We shifted our attention to the S2 pocket by examining various heterocyclic replacements (Table 2 Entries 10-12). While most heterocyclic cores sustained high levels of 5c/5i selectivity, incorporating a methyl substituent onto the 1,2,3-triazole core (13) was one of the few structural modifications that enhanced 1i/5i selectivity (13 vs. 9, 0.4x vs. 0.14x respectively). The S1 pocket offered several opportunities to bias selectivity in favor of 5i as there are a number of larger residues situated in the S1 pocket of 1i. These residues include Val20 (Ala in


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5i), Phe31 (Val in 5i), and Leu45 (Met in 5i). Indeed, incorporation of larger P1 residues, (i.e. 4-chlorobenzyl or 2,4-dimethylbenzyl) into the S1 pocket generally enhanced 5i potency and insulted binding to the 1i subunit (15-22). Incorporation of the 2,4-dimethylbenzyl P1 substituent onto the 1i/5i selective core delivered an exquisitely selective 5i inhibitor 22 (2200x selective against 5c, 2100x selective against 1i, and >4800x selective against 1c).

Table 3. PBMC, pDC, and PB viability IC50 values for selected proteasome inhibitors. Compound

1

5

22

7

4

β5c / β5i

0.5x

17x

2200x

710x

283x

β1i / β5i

2.3x

5.2x

2100x

0.2x

247x

β1c / β5i

9.4x

640x

>4800x

120x

>2400x

PBMC IC50

2.7 nM

100 nM

>10000 nM

7300 nM

5700 nM

pDC IC50

5.2 nM

22 nM

>10000 nM

3700 nM

6000 nM

PB IC50

3.9 nM

54 nM

9300 nM

2900 nM

670 nM

With a set of potent and highly selective immunoproteasome inhibitors in hand, compounds were assessed for their effects on the viability of bulk PBMCs, purified pDCs, and plasmablasts (Table 3, Figure 3). Much to our surprise, 5i selective inhibitors did not affect cell viability to the same extent as the pan-inhibitor.16 On the contrary, we discovered that effective immune cell cytotoxicity correlated strongly with 5c inhibition (Figure 3 and Table S1). Selective 1i inhibition or dual inhibitors of both chymotrypsin subunits 5i and 1i (5c sparing) also failed to be potent inducers of cell death (Table S1).17 Further examination of other reported



immunoproteasome selective inhibitors, such as compound 4 from Merck KGaA, also failed to show effects on cell viability (Table 3). Despite similar biochemical β5i inhibition as bortezomib, cell death was only observed at concentrations that also inhibit the constitutive proteasome for compound 2. These selective inhibitors are able to inhibit the immunoproteasome in a cellular context as demonstrated by the inhibition of chymotrypsin-like activity in PBMC lysates (Table 2). To unravel the underlying mechanisms responsible for the inability of immunoproteasomeselective inhibitors to affect cell viability, we used compound 4 in subsequent experiments. Containing both reversible (boronic acid) and irreversible (acrylamide) covalent functional groups, compound 4 provided the ability to generate a “chemical knock out” of the 5i subunit. A central feature of dysregulated proteasome activity is the accumulation of ubiquitinated proteins followed by Caspase 3/7 activation to signal apoptosis. When PBMCs were treated with compound 4, neither ubiquitin accumulation or apoptosis were observed (Figure 4). This observation is consistent with the absence of an effect on proteasome function following 5i inhibition. While the correlation plots clearly demonstrate a compulsory role for 5c on immune cell viability, we were unable to detect significant expression of this subunit in untreated PBMCs or purified immune cell populations (Figure 5A). Previous literature reports have noted that treatment of rat vascular smooth muscle cells with the proteasome inhibitor MG132 caused a transient and concerted induction of all the genes comprising the 26 S constitutive proteasome, with apoptosis being a subsequent effect of continuous compound blockade of the enzymatic subunits.18


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Figure 3. Correlation plots between biochemical potency of inhibitors and cell viability (A) PBMC IC50 vs. β5i biochemical IC50 or β5c biochemical IC50 (B) pDC IC50 vs. β5i biochemical IC50 or β5c biochemical IC50 (C) Plasmablast IC50 vs β5i biochemical IC50 or β5c biochemical IC50.



We hypothesized that although absent during homeostatic conditions, pharmacological inhibition of 5i could lead to a similar upregulation of 5c preventing immune cell death. To test this hypothesis, we treated PBMCs with compound 4 or DMSO for 24, 48, or 72 hours and analyzed 5c protein expression by Western Blot. In contrast to the DMSO treated controls, addition of compound 4 led to a rapid and stable induction of 5c, supporting the idea that inhibition of 5i promotes 5c upregulation at the protein level (Figure 5B). Furthermore, we observed gene induction of all three catalytic subunits of the conventional proteasome following 5i inhibition. (Figure 5C).

Figure 4. (A) Western blot of ubiquitin (top) in lysates from PBMCs treated overnight with DMSO, 10nM 1, 100nM 2, 100nM 5, 100nM 3, or 100nM 4. Bottom panel is Gapdh loading control. (B) PBMCs were treated for 12 h with DMSO or 10 point titration of 3-fold serially


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diluted compounds with a final top concentration of 10 µM. Caspase3,7-Glo data was normalized to no drug control. Data shown is representative of 3-5 independent experiments.

Figure 5. (A) Western Blot of β5c in various cell lines. (B) Western Blot of β5c (top) and β-actin loading control (bottom) from PBMCs treated with DMSO or 100nM compound 4 for 24h, 48h, and 72h. (C) PSMB5-7 expression upon treatment with 100nM of indicated compounds for specified time.



Similar effects were seen following treatment of PBMCs with bortezomib (1), ONX-0914 (2) and compound 5. These compounds fully (1) or partially (2) inhibit 5i at the tested concentration of 100nM. On the other hand, mRNA levels of the conventional subunits were only slightly higher upon treatment of PBMCs with PR-893 (3), which is more potent for 5c than 5i.

To confirm if the regulation of the constitutive proteasome by the immunoproteasome occurs in vitro and is not a phenomenon of compound treatment, we used CRISPR/Cas9 methodology to knock-out (KO) the genes for 5c, 5i or both in primary human CD4+ T cells.19 This approach allowed us to compare the effects of 5i deletion with 5i compound inhibition, as well as 5c deficiency, where a highly selective 5c inhibitor is lacking.


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Figure 6. (A) Western Blot of β5c and β5i expression with β-actin loading control. (B) Viability curves of with KO CD4+ T cells with Bortezomib. (C) Viability curves of with KO CD4+ T cells with compound 4.

Cells were transfected with Cas9/RNP, rested for 5 days and re-stimulated with anti-CD3 and anti-CD28 for three additional days. Western blotting demonstrated the high KO efficiency for each subunit. (Figure 6A). Single KO cells were viable whereas 80% of the 5c/5i doubledeficient cells were dead after TCR stimulation (data not shown). Quantification of the WB shows that the β5iKO had a 94% reduction in β5i and a 2.6-fold increase in β5c expression. Additionally, both the pro- and mature forms of 5c were upregulated in β5iKO CD4+ T cells, confirming that inhibition or KO of 5i results in an active induction of 5c expression. The deletion of either 5c or 5i increased sensitivity of CD4+ T cells to bortezomib treatment by 3-fold. Treatment of 5c-deficient cells with compound 4 promoted cell death, again demonstrating that in leukocytes, proteostasis can only be perturbed when both the immune- and conventional proteasomes are inhibited (Figure 6C). Taken together, these results further corroborate the studies with the chemical inhibitors and demonstrate that immune cells lacking 5c are viable.

CONCLUSION



Using both structure and ligand-based approaches, we identified a series of highly specific inhibitors against the chymotrypsin-like subunits of the immunoproteasome. Key structural insights were gained by obtaining the first reported human proteasome co-crystal structure. Analysis of the ligand 5 to protein binding interactions provided strategies to target specific regions for selective and efficient binding. Modification of 5 afforded a class of highly selective β5i and β5i/β1i inhibitors. Ultimately, we found that selective β5i or dual β5i/β1i inhibition were insufficient to perturb leukocyte viability. In fact, by testing inhibitors with different selectivity for β5i over β5c suggested immune cell viability correlate with β5c inhibition, and not with β5i inhibition. In the absence of a selective β5c inhibitor, it was difficult to assess the exact contribution of this subunit. Immune cells express almost exclusively the immunoproteasome. We report here that, in response to immunoproteasome inhibition, human immune cells maintain ER homeostasis through induction of the constitutive proteasome catalytic subunits thus subverting apoptosis. Recently, it has been reported that, B and CD4+T lymphocytes from lmp7-deficient mice express much higher levels of 5c compare to WT cells. In line with our observations, human and mouse CD4+ T cells did not die upon treatment with ONX-0914 by analysis of PARP cleavage.20 To circumvent the lack of selective β5c inhibitors and rule out the possible off-target effects of our immunoproteasome inhibitors, we took a genetic approach. CRISPR/Cas9 mediated genetic ablation of the genes encoding the 5i and/or 5c subunits in primary human T cells revealed cell death is only seen when expression of both proteins is obstructed, indicating that both 5i and 5c contribute to the ubiquitin-proteasome pathway in immune cells. These results are reminiscent of the individual and combinatorial immunoproteasome subunit deficient mouse strains that also display broad compensation by constitutive proteasome subunits.7,18


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Furthermore, mechanisms for cancer cells to robustly recover from the loss of proteasome subunit function have been shown to involve the NRF1-dependent upregulation of constitutive proteasomal subunits.21,22,23 Taken together, data suggest that protective mechanisms are in place to prevent the disruption of this fundamental cellular process and that cell death can only be achieved by inhibiting both immune- and constitutive proteasomes. Therefore, selectively targeting the immunoproteasome is unlikely to confer clinical benefit in SLE via the mechanism of depleting PBs and pDCs, a central property of bortezomib activity. Proteasome inhibitors such as bortezomib (1) display potent inhibition towards 5c and β5i. As a result, immune cells treated with these compounds are vulnerable to ER stress induced apoptosis. Routinely used “immunoproteasome-selective” inhibitors, such as ONX-0194 (2) are only marginally selective, which is key for their ability to deplete immune cells; cell death should only be observed once the inhibitors concentration is sufficient to inhibit the conventional proteasome. Although this unprecedented feedback mechanism may limit the use of immunoproteasome selective inhibitors for autoimmune diseases where depletion of pathogenic immune cells is necessary to induce clinical response, our study provides the scientific community with highly selective immunoproteasome inhibitors which can be used as tools to further investigate immunoproteasome biology.

EXPERIMENTAL SECTION Purity of Tested Compounds. All tested compounds possess a purity of at least 95% as determined by the following Thermo QE 7-min LCMS method: Sample was analyzed on a Dionex Ultimate 3000 coupled with Thermo Scientific Q Exactive HRMS using ESI as



ionization source. The LC separation was done on an Acquity UPLC BEH C18, 1.7m, 2.1×50mm column at a flow rate of 0.5 ml / minute. MPA (mobile phase A) was water with 0.1% FA and MPB (mobile phase B) was acetonitrile with 0.1% FA. The gradient started at 2 % MPB and ended at 98% MPB over 6.7 min and held at 98% MPB for 0.7 min following an equilibration for 0.7 min. LC column temperature was 40 °C. UV absorbance was collected by a DAD detector and mass spec full scan was applied to all experiments. Biochemical assays. Compounds were tested for their ability to inhibit the 5i, 5c and 1c subunits of the 20S proteasome by monitoring the cleavage of subunit-specific fluorogenic peptides as described previously (Blackburn). Briefly for evaluating 5i and 5c inhibition, test compounds were pre-incubated for 1 hour at room temperature with 20s proteasome derived from PBMCs (Boston Biochem) in the presence of PA28a (Boston Biochem) or 20S proteasome derived from erythrocytes (Enzo) in the presence of SDS. Reactions were started by the addition of the 5i-specific substrate, Ac-(ANW)2-R110 (AAT Bioquest) or the 5c-specific substrate, Ac-(WLA)2-R110 (AAT Bioquest). Final assay conditions were as followed: 8ul reactions in black 384-well proxiplates (Perkin Elmer), 0.5nM 20S proteasome, 12nM PA28a or 0.03% SDS, 10-point, 3-fold serially diluted test compounds with a top concentration of 10uM, 0.5% DMSO, 15uM peptide in 20 mM HEPES, 0.5 mM EDTA, 0.01% BSA for 15 minutes at room temperature. Fluorescence intensity read on an Infinite M1000 (Tecan) at Ex/Em = 494/521 nm. For β1c, test compounds were pre-incubated for 1 hour at room temperature with 20S proteasome derived from erythrocytes (Enzo) with no SDS. After pre-incubation, an equal volume of the Proteasome Glo Caspase-Like substrate (Promega) was added to wells. Luminescence was read on an EnVision multimode reader (Perkin Elmer). Final assay conditions were as followed: 8ul reactions in white 384-well proxiplates (Perkin Elmer), 0.5nM 20S


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proteasome in 20 mM HEPES, 0.5 mM EDTA, 0.01% BSA, 10-point, 3-fold serially diluted test compounds with a top concentration of 20uM, 0.5% DMSO, 1x Z-nLPnLD-Glo™ peptide. Data was normalized relative to DMSO control wells and no proteasome control wells. Isolation, in vitro stimulation and culturing of Plasmacytoid Dendritic Cell. Buffy coats acquired from the Blood Centers of the Pacific were depleted of CD3 cells using RosetteSep Human CD3 Depletion Cocktail (StemCell 15661). Plasmacytoid Dendritic Cells (pDCs) were then isolated from buffy coats using EasySep Human pDC Enrichment kit (StemCell 19062), and rested overnight in complete RPMI, containing 10% FBS (hyclone), 1x Glutamate (Gibco), 1x Pen Strep (Gibco), as well as rhuIL3 (R&D Systems, 0.01ug/ml). The cells were plated at 3x104 pDCs per well in CpG (Invivogen, 0.5uM ODN2216) and rhuIL3, and treated with DMSO or titrated compounds. Isolation and culturing of PBMCs. Using Ficoll-Hypaque density gradient centrifugation, PBMCs were isolated from heparinized human whole blood, and residual RBCs were removed using ACK lysis. For permeability assays, PBMCs in PBS + 25 mM HEPES were plated at 2x104 cells per well in a 384-well plate. For apoptosis assays, PBMCs were plated at 5x104 cells per well in complete RPMI with 10% FBS (hyclone), 1x Glutamax (Gibco), 1x Pen Strep (Gibco), and treated with DMSO or titrated compounds for 6 hrs or 12 hrs. For viability studies, 2.5x105 cells/well in white/clear 384-well plates (Greiner) in complete RPMI for 72 hrs. For ubiquitination studies, 2x106 PBMCs were treated with DMSO or compound for 6 hours. For gene expression studies, PBMCs were plated at 106 cells/mL in complete RPMI with 10% FBS (Hyclone), 1x Glutamax (Gibco), 1x PenStrep (Gibco), 1x Non-essential amino-acids (Gibco), 1x Sodium pyruvate (Gibco), 55uM b-mercaptoethanol (Invitrogen) and 10mM Hepes in a 96well flat bottom plate (Costar). Compounds were added to plates and cells were incubated for 3h,



6h, 24h, 48h or 72h. Bortezomib, ONX-0914, PR893 and compound 1 were added at 100nM. 5 concentrations of compound 2 were tested: 10uM top concentration, 10-fold dilution. Cell Permeability assays. To ensure compounds were cell permeable, the activity of the chymotrypsin-like activity of the proteasome in PBMCs was determined as previously described by Basler, et al.15 Briefly, cells were treated with DMSO or a titration of compounds (10 point, 3-fold serially diluted compounds with a final top concentration of 10uM) for 30 minutes at 37 °C. Then 40 uM of the cell permeable fluorescent substrate Meo-Suc-GLF-AM was added to the wells and incubated for an additional 30 minutes at 37 °C. Plates were then read for fluorescence (360 nm/465 nm) on a Multi Detection Microplate Reader (Tecan Infinite M1000). Viability and apoptosis assays. To determine viability or apoptosis, cells treated with DMSO or titration of compounds (10 point, 3-fold serially diluted compounds with a final top concentration of 10uM), CellTiter Glo or Caspase 3,7 Glo, respectively, were used following manufacturer’s instructions. Briefly, substrate is added and plates are read on Perkin Elmer EnVision 2104 Multi Detection Microplate Reader. Western Blot. Cells were collected, washed twice with ice cold PBS, and dry pellets were frozen at -80 degrees. Pellets were lysed in RIPA Buffer (0.1% SDS 1% NP40 25mM 150mM NaCl Tris pH7.4) with 1x HALT protease and phosphatase inhibitors (Thermo) and 1uM PMSF (CST) for 15 minutes on ice. Lysate was quantitated by BCA (ThermoSci 23227) and 10-50ug protein was loaded on PAGE (4-12% Bis-Tris gel with Mops buffer). iBlot was used to transfer blot to a nitrocellulose membrane, which was subsequently blocked in 2% BSA in TBS-T, Odyssey blocking buffer (Li-Cor 927-40000) or 5% nonfat dry milk in TBS-T, and probed with primary antibodies anti-Ubi (Enzo clone FK1), anti-β5c (Cell Signaling clone D1H6B), anti-beta-actin (Cell Signaling clone 8H10D10), or anti-gapdh (Genetex GTX100118). Bands were detected


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with goat anti-rabbit IgG (Li-Cor 925-68071) or IRDye 800CW anti-mouse IgG (Li-Cor 92532210), and visualized using a Li-Cor Odyssey (scan intensity 2.0, sensitivity 5). Alternatively, membranes probed with HRP-conjugated secondary antibodies (Cell Signaling 7074S; Bethyl Labs A90-101P) were visualized using SuperSignal West Pico chemiluminescence substrate (ThermoSci 34580). RNA isolation and quantitative RT-PCR. Total RNA was isolated from cultured PBMCs using the mRNA catcher PLUS Purification kit (ThermoSci K157003). cDNA synthesis was performed directly in the mRNA catcher plate using the Maxima First Strand cDNA synthesis kit (ThermoSci K1642). cDNA was pre-amplified using the Taqman Pre-amp Master Mix (ABI 4384266). Gene expression was measured by Fluidigm following the 192.24 Gene expression workflow. Results were normalized to those of the control housekeeping gene Rpl19 (encoding ribosomal protein L19) and are reported as 2ΔCT. Taqman probes used included Hs00605652_m1 (PSMB5), Hs00382586_m1 (PSMB6), Hs00160607_m1 (PSMB7), Hs00544760_g1 (PSMB8), Hs00160610_m1 (PSMB9), Hs00988194_g1 (PSMB10), and Hs02338565_gH (RPL19). In vitro CRISPR knockouts. CD4 T cells were isolated from PBMCs using the CD4+ T cell isolation kit (Miltenyi 130-096-533). Cas9/RNP transfection was done as described in Seki A. (JEM Feb 2018). After transfection, cells were incubated for 5 days in complete media. On day 5, dead cells were removed using the Dead Cell Removal Kit (Miltenyi 130-090-101). 3-5 million cells were harvested for Western-blot analysis of KO efficiency. Remaining cells were restimulated with 10ug/mL plate-bound anti-CD3 (clone OKT3, eBiosciences 16-0037-85) and 1ug/mL soluble anti-CD28 (clone CD28.2, BD Biosciences 555725) for 3 days, in presence or absence of Bortezomib or Compound 2 (top concentration of 10uM, 10 points, 3-fold dilution).



On day 8, cell survival after compound treatment was measured by CellTiter Glo. KO efficiency was again assessed on day 8 by Western blot from cell stimulated by anti-CD3/CD28, in the absence of compound treatment. Statistics: The results for all cellular studies with compound treatment were statistically analyzed using nonlinear regression with Prism software. Results are shown as "log(inhibitor) vs. normalized response -- Variable slope" with the least squares (ordinary) fitting method.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Included are Compound preparation and characterization; Biochemical and cellular potency for all compounds in Figure 3; Matched pair analysis of boronic acids and esters; Cell viability vs β1i plots; Human cell isolation method; Crystallography methods, data collection and refinement information; and Molecular Formula Strings. Coordinates and structure factors are deposited in the PDB under accession code 6E5B. Authors will release the atomic coordinates and experimental data upon article publication.

AUTHOR INFORMATION Corresponding Author * M.S. Phone: 650-467-7764. E-mail: [email protected]


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* R.P. Phone: 650-467-1829. E-mail: [email protected] Author Contributions EL, CE, CE and RP designed, performed and analyzed the cellular and biochemical experiments. MA did the protein production for the crystallography, and MB and SS did the crystallization and structure determination and refinement. BD, MPH, HP, STS, CES and MS designed and synthesized the chemical compounds and contributed to the analysis of experiments using the inhibitors. MD evaluated the pharmokinetic/pharmocodynamic properties of the chemical compounds. SFH performed structural analysis and interpretation of the crystallography. CE, CES, SFH, MS and RP wrote the manuscript. All authors approve the manuscript. Disclosure of Conflicts of Interests EL, CE, BED, MD, SFH, MPH, HEP, STS, CES, CE, MS and RP are employees of Genentech Inc. MA, MB, and SS are employees of Proteros Biostructures GmbH.

ACKNOWLEDGMENT The authors would like to thank Werner Neidhart (Roche) for information regarding Compound 5 and WuXi Apptec for synthesis support. The authors would like to thank Kathila Rajapaksa and Edward Dere for their assistance with evaluating the effects of the compounds on nonhematopoietic cells (data not shown). We would also like to thank Kewei Xu and Yanzhou Liu for their assistance in obtaining analytical data. We acknowledge the Paul Scherrer Institut, Villigen, Switzerland for provision of synchrotron radiation beamtime at beamline PXII/X10SA



of the SLS and would like to thank Dr. Anuschka Pauluhn and Dr. Florian Dworkowski for assistance. ABBREVIATIONS ER, endoplasmic reticulum; DC, dendritic cell; IC, immune complex; IFN, interferon; KO, knockout; PB, plasmablast; PBMC, peripheral blood mononuclear cell; pDC, plasmacytoid dendritic cell; RMSD, root mean square deviation; SLE, systemic lupus erythematosus; TCR, Tcell receptor; WB. Western blot. FOR TABLE OF CONTENTS ONLY

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Design and Evaluation of Highly Selective Human ... - ACS Publications

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