S1

Supporting Information

A substrate-free activity-based protein profiling screen

for the discovery of selective PREPL inhibitors

Anna Mari Lone1, Daniel A. Bachovchin

2, David Westwood

1, Anna E. Speers

2, Timothy P. Spicer

3,

Virneliz Fernandez-Vega3, Peter Chase

3, Peter S. Hodder

3,4, Hugh Rosen

2,5, Benjamin F. Cravatt

2, and

Alan Saghatelian1*

1Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge,

Massachusetts 02138, USA

2The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps

Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037

3Scripps Research Institute Molecular Screening Center, Lead Identification Division, Translational

Research Institute, 130 Scripps Way, Jupiter, FL 33458

4Department of Molecular Therapeutics, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458

5The Scripps Research Institute Molecular Screening Center, The Scripps Research Institute, 10550

North Torrey Pines Road, La Jolla, CA 92037

S2

List of abbreviations used: Fluopol-ABPP, fluorescence polarization activity-based protein profiling;

HCS, hypotonia-cystinuria syndrome; HTS, high-throughput screening; FP-Rh, fluorophosphonate-

rhodamine; FP-PEG-Rh, fluorophosphonate-polyethylene glycol-rhodamine.

S3

Figure S1. Expression, purification and activity assessment of recombinant PREPL. 6His-tagged

PREPL expressed well (A) in Rosetta cells with high-salt media and was purified using standard

immobilized metal affinity chromatography (B). The activity of the enzyme was assessed (C) by

reacting with FP-Rh and monitoring fluorescence on an SDS-PAGE gel.

S4

Figure S2. Comparison of labeling kinetics with FP-Rh and FP-PEG-Rh at different PREPL

concentrations. With FP-Rh, complete labeling was observed by 5 minutes for every concentration of

PREPL tested.

Figure S3. Structures of the ABPP probes used in this study.

S5

Figure S4. 4-methylumbelliferyl p-guanidinobenzoate (MUGB) assay to confirm hits. MUGB is a

substrate of PREPL which is unsuitable for high-throughput screening but could be used to confirm

some hits in a non-FP-Rh dependent manner. Briefly, 3 μM PREPL was preincubated with 20 μM

inhibitor for 20 minutes, whereupon 0.5 mM MUGB was added and cleavage of this substrate monitored

(excitation at 320 nm, emission at 460 nm). The rate of cleavage was assessed for three replicates each

of the no inhibitor and 20 μM inhibitor 8 reactions. Inhibitor 8 significantly reduced PREPL cleavage of

this substrate.

S6

Figure S5. Other serine hydrolases in the Neuro2a cell line are not affected by the addition of PREPL

inhibitors in cell culture. The entire gel is shown to demonstrate that no other bands change in response

to these compounds. The arrows indicate the band corresponding to PREPL.

S7

Figure S6. Mass spectrometric characterization of compound 8. The MS/MS spectrum of compound 8

(top) was used to determine two characteristic transitions (217-to-173 and 217-to-155) of this inhibitor.

When 100 pmol of compound 8 was analyzed by this MRM method (bottom), the following

chromatograms were observed. The two characteristic transitions of the inhibitor are shown, along with

the TIC (the sum of the two transitions).

S8

Figure S7. Assessment of inhibitor 8 levels in the brains of injected mice. Different amounts of inhibitor

were spiked in with half a brain and extracted as for the other samples. The samples were analyzed by

our MRM method and the peak areas for the two different transitions were used to construct a standard

curve. We then ran an injected brain sample (1 mg/kg intraperitoneal injection) and by comparing the

peak areas for the two transitions in this sample to the standard curve, we calculate that 2.7 pmol of

inhibitor is present in half the brain, and 5.4 pmol are expected to be present in the entire brain.

S9

Maybridge Hitfinder

Collection (Maybridge Code) NIH Validation Set

(PubChem SID)

BTB 05109 856266

MWP 00566 24785182

RJC 03760 11533034

CD 07229 864885

CD 04390 856836

HTS 12294 7977524

HTS 08756 855827

S 15315 855766

KM 06421 855801

HTS 01199 24831819

RJC 01813 14742700

BTB 08467

BTB 10320

S 11327

CD 11594

KM 00040

DFP 00003

SB 01037

HTS 04880

RDR 00907

SEW 05700

BTB 13553

HTS 09265

HTS 05843

HTS 01790

HTS 13007

BTB 03038

MBE 00222

Table S1. List of hits from the initial screen against the Maybridge HitFinder Collection (Maybridge

Code is listed) and the NIH Validation Set (PubChem Substance ID is listed). These compounds

inhibited PREPL fluorescence polarization by more than 50%, and PME-1 and Cgi67 fluorescence

polarization by less than 20% in two previous screens.

S10

Hits from Full-Deck Screen

PubChem Substance IDs

24831400 56422265 17403555 17514859 17414285 7970525

49821024 50086477 14729760 842406 24781451 26664932

17432766 49640598 17515898 22415591 26671183 24823154

17510341 49716586 22403695 22410705 49641394 855568

49730895 49818172 17516071 17509606 14739316 17409786

49678255 57266226 7978018 856840 17402120 17507779

49641946 24799289 17432407 22409323 24829174 17504175

56317589 49730839 17517252 858538 14733368 22404094

51087549 24812979 4250669 7977057 49824790 22406683

85146214 49678228 4263993 4242973 85199272 49647737

85146061 49815989 22402768 24786852 14734023 24790560

17513115 26664000 17510864 24781030 864125 24783166

56322243 24793672 22400933 4247368 844645 47196424

858863 7967860 22411754 24823970 17409013 17517026

85272425 7966516 844418 14732635 49821136 56436672

85146215 7966400 22410782 858151 85149087 56436666

49731406 7965699 17509576 49715301 17385788 852615

85272415 85198370 7964858 24814366 49716901 864349

22406132 49678276 845329 24816559 14745083 49647816

49715302 26666621 24810218 24791890 85201723 4243730

7970989 14729757 7965067 49673347 85147263 24833978

56422892 56316028 7969309 49723066 74374084 856350

49735218 85148685 7965935 26665655 863475 50085995

51089164 85272429 24812207 49730011 861882 51087851

49646659 85272428 24810717 49736397 22408243 24833220

49647708 85272416 7968358 14740715 17386252 24810214

85146243 85272385 24829403 49670628 22413647 51089734

49718694 85201553 49677683 14721022 22405393 22404156

56463615 85147356 24816594 850965 17508397 7966121

51088129 85147294 49647887 24779747 49724360 49724728

49678113 56324470 24810592 17387076 17401315 26664474

24840761 49731608 24805326 24834023 51089910 56422267

49678254 26659978 24780631 50085659 49819947 26665472

864166 57265387 7964780 16953455 51086454 49716340

853126 57256898 24820089 24838247 24783351 24825251

56463074 51090191 24786145 24833741 24786443 17508425

85272427 7964986 861877 17403588 49667019 49670559

85201685 14740992 24799873 4243551 24830866 24832390

S11

85147355 24829525 49648873 26724749 47203389 17512177

49715622 49723582 49676381 861732 57264321 74373406

24841513 26661674 49667537 7970414 24833743 4248779

57265372 57261282 49715586 4241958 49717826 24830337

24841281 57261275 49668534 22403517 14739104 26728304

49670560 85147437 24779772 7966497 85147265 57266640

24824735 49816430 49731053 24831819 842953 56436680

49677827 4241901 24824947 56463492 17414113 49715905

49727202 851386 49666526 24810571 863314 49669868

865895 864635 49648074 14738689 17509318 26663610

85272431 862951 49824791 26724584 856765 49641429

85147352 7965250 49737198 24833649 24833822 49676421

81066385 848747 49678141 7969084 22414381 49642517

49734999 7966692 14741387 7968138 85272424 17431710

49677680 7966119 863754 24791059 49642423 56436779

49671681 7965799 4249559 17409356 24840681 24782097

24779468 14726025 4265802 7969026 17411241 56436777

51088938 14741636 4258037 24833985 49715909 17508583

7969556 14730666 14725165 24833982 49819822 49681580

24840824 17414774 863775 7969942 49679475 56436780

846116 17515798 851425 860932 22408244 56424031

56424135 22408346 863062 14743314 17508153 51089876

85272430 17506992 17413869 851902 4244225 56463066

85201277 22416155 17387625 14732084 4246262

56317566 17513897 859825 14743702 17509520

49817392 22410844 17410001 7967928 49727199

49717158 17431500 17415573 24835178 24824961

49717170 17431717 860219 49669337 17510013

24831357 24810246 4244537 24785600 24833725

24816015 24825343 26663394 24838207 14742663

24785163 24834725 26661405 24824241 17509419

24780052 24784464 22408555 49731851 14729464

56463500 24780762 22407630 49715004 26724840

49729971 24804836 24796318 17512781 24833996

85199239 24824903 24791332 49644868 26664175

24803933 24808205 3716478 49670432 26657610

49826817 24779743 46500451 57265383 49667815

49732281 24824647 24779722 862898 24797250

26663373 24824679 24779757 864398 24815558

24802372 7968125 24810821 7969131 17512796

57266028 4242762 24779782 865574 49716683

S12

26725271 4243593 864231 49648516 14732918

85272432 4261303 864214 50085658 24833928

85201391 863597 863513 863758 860397

85149116 7967716 14728961 14730241 26667076

85147354 7966677 4247166 3715530 49644438

26663432 851378 4249317 7971844 49716148

57261524 4246050 14726732 26730839 4245336

24779762 7968387 14739647 26730840 49667767

24779729 26665223 14740402 24810253 49648699

49672229 4250950 7968681 17409498 22413865

24815536 26658015 14732863 24816460 26660076

49715166 26663512 14736054 864643 22412394

49723477 24813031 7968340 24824159 4241310

49718069 26663048 17415105 17512665 49644683

4255904 4246090 17408888 851580 855827

17386896 7970321 17414369 49726580 56436670

26669095 4251745 7967826 7970071 26670820

85201547 14741051 22412370 22401110 85201278

85149085 14742363 4242811 17408398 49727556

85147362 17401169 47195494 862910 17509522

Table S2. List of hits from the full-deck screen. These compounds inhibited PREPL>50% in AID2803

and showed less than 20% inhibition in AID 1515 (RBBP9), AID1974 (GSTO1), AID2130(PME-1),

AID2177(LYPLA2). Compounds containing esters have been filtered out.

S13

S14

Table S3. Overview of the compounds used in this work and their numbering. Compounds 4, 6-8 and

10-18 were found to be selective in the proteome inhibition and PEP assays, and carried forward into the

cell-based screen.

Structure SID CID Outcome Inhibition [%]

858863 660169 Active 73.51

14741636 3773502 Active 63.71

14740992 4365944 Active 55.55

857461 658812 Inactive 29.84

Table S4. List of carbonitrile derivatives screened in the full-deck FluoPol-ABPP screen, along with

their PubChem Substance and Compound ID. The percent inhibition (for the conditions in this assay, 30

nM PREPL and 5.9 μM inhibitor) is shown on the right.

S15

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