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Reviewing hit discovery literature for difficult targets: glutathione transferase omega-1 as an example Yiyue Xie, Jayme L. Dahlin, Aaron J Oakley, Marco G. Casarotto, Philip G. Board, and Jonathan B. Baell J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00318 • Publication Date (Web): 13 Apr 2018 Downloaded from http://pubs.acs.org on April 15, 2018

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Journal of Medicinal Chemistry

Reviewing hit discovery literature for difficult targets: glutathione transferase omega-1 as an example Yiyue Xie1, Jayme L. Dahlin3, Aaron J. Oakley4, Marco G. Casarotto5, Philip G. Board*,5 and Jonathan B. Baell*,1,2

1

Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria

3052, Australia 2

School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, 211816,

People’s Republic of China 3

Department of Pathology, Brigham and Women’s Hospital, Boston, MA 02135, USA

4

School of Chemistry, University of Wollongong, Wollongong, NSW 2522, Australia

5

John Curtin School of Medical Research, Australian National University, Canberra,

ACT 2600, Australia

Abstract Early-stage drug discovery reporting on relatively new or difficult targets is often associated with insufficient hit triage. Literature reviews of such targets seldom delve into the detail required to critically analyze the associated screening hits reported. Here we take the enzyme glutathione transferase omega-1 (GSTO1-1) as an example of a relatively difficult target and review the associated literature involving small-molecule inhibitors. As part of this process we deliberately play closer-than-usual attention to assay interference and hit quality aspects. We believe 1 ACS Paragon Plus Environment

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this Perspective will be a useful guide for future development of GSTO1-1 inhibitors, as well serving as a template for future review formats of new or difficult targets.

Introduction There are many reasons why a biological target may be considered hard to purposefully modulate with small molecules. Some difficulties include targets that are relatively large and flat (such as many protein-protein interactions), feature high-affinity interactions, or contain multiple charges1. Compounding factors can also include competition with endogenous substrates that are high in concentration, the need to substantially modulate the target site before meaningful functional response is induced, and complex allosteric regulations. A well-defined topography in enzyme active sites can impart greater druggability, but their substrate binding sites have evolved to be reactive and those that are accompanied by reduced concavity, as in the case for glutathione transferase omega-1 (GSTO1-1), can cause such targets to fall into the category of being difficult to drug.

Compared with well-established and druggable biological targets, those that are difficult or new are typically associated with a preponderance of medicinal chemistry at early stages of prosecution. These targets merit particularly close and critical analysis, yet reviews in such cases very often comprise relatively dry recounting of any reported modulators.

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In this Perspective, we suggest an alternative approach in reviewing hit discovery and early-stage medicinal chemistry for difficult targets, where a particular emphasis is placed on the rigor applied to both compound screening as well as hit triage. This allows for clarification of how advanced or otherwise associated chemical matter actually is, as well as more accurate elucidation of those compounds that comprise the better starting points for tool compounds or drug development. We use the enzyme, GSTO1-1, by way of example.

The cytosolic glutathione transferases (GSTs) comprise a superfamily of proteins with diverse biological functions and are found in all cellular life forms2. Traditionally referred to as glutathione S-transferases, the “S” denoting the sulfur atom of the glutathione thiol group, the International Union of Biochemistry and Molecular Biology now recommends3 omission of the “S” and instead, use of the simplified term “glutathione transferase”, which we correspondingly adopt throughout this manuscript. In humans and other mammals the various members of the superfamily have been categorized on the basis of their primary sequences3. Both mice and rats contain GSTO1-1 orthologues, though in general they have not been as rigorously characterized as human GSTO1-1. Many GSTs feature tyrosine or serine residues in their active site and catalyze the conjugation of electrophilic compounds to glutathione (GSH). Consequently, their role in the detoxification of xenobiotics and the metabolism of xenobiotics has been the main focus of investigation over the last 50 years4-7. However, there is an increasing realization that GSTs are involved in 3 ACS Paragon Plus Environment


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diverse enzymatic and non-enzymatic biological processes2. For example, glutathione transferase pi-1 (GSTP1-1) was found to interact with c-Jun N-terminal kinase 1 (JNK1), and the active form of the pro-drug Telintra® can block this interaction8. GSTO1-1 levels, but not expression, appear to increase in response to thermal stress9.

GSTO1-1 is one of two members of the omega-class of GSTs, the other being glutathione transferase omega-2 (GSTO2-2). Unlike most other GSTs, the omega class GSTs feature a distinctive active site cysteine, with associated activity as a dehydroascorbate (DHA) reductase, monomethylarsonate (MMA) (V) reductase, thioltransferase, and specifically in the case of GSTO1-1, S-(phenacyl)glutathione reductase and deglutathionylase activity (Figure 1).

Figure 1. GSTO1-1 features a catalytic cysteine in its active site and functions as a DHA reductase, MMA (V) reductase, thioltransferase, and S-(phenacyl)glutathione reductase.


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Some substrates have lower micromolar binding affinities, though several kinetic parameters have yet to be rigorously characterized10-13 (Table 1). The optimal pH for enzymatic activity varies depending on the substrate, while several optimal pH values remain to be determined12, 14 (Table 1). These parameters are important considerations for designing enzymatic assays, as the choice of substrate concentrations can bias the selection of competitive inhibitors, while the selection of assay buffer pH can have important implications for compound protonation and enzymatic activity rates. Table 1. Reported kinetic parameters for GSTO1-1. Specifi c Enzymatic activity

Substrate

activity (µmol min-1

kcat -1

Km

(S

(µM

)

)

Optima

Ref

l pH

s

9

10, 14

mg-1) S-(Phenacyl)-glutathion

S-(4-Nitrophenacyl)glutathion

168.6 ±

e reductase

e (4-NPG)

6.3

MMA (V) reductase

MMA (V)

DHA reductase

DHA

Thioltransferase

Hydroxyethyl disulfide

Deglutathionylase

Glutathione transferase

0.32 ± 0.035 0.22 ± 0.016 2.8 ± 0.042

glutathionylated (SG)

83.4 ±

synthetic peptide

4.4

SQLWC-SGLSN 1-Chloro-2,4-dinitrobenzene

0.18 ±

(CDNB)

0.006

84.1 ± 3.6

20.9 ± 3.9

NR

NR

NR

11

NR

NR

NR

11

NR

NR

NR

11

7

12

NR

13

2.8 ± 0.28 NR

7.9 ± 0.97 NR

NR, not reported

The upregulation of GSTO1-1 has been observed in diverse human cancers, including transitional cell carcinoma15, esophageal squamous cell carcinoma16, pancreatic 5 ACS Paragon Plus Environment


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cancers17, breast cancers18, and colorectal cancers19. Its overexpression has also been identified in chemoresistant cancer cell lines, which has been attributed to induction of its expression during chemotherapy and a role in preventing apoptosis20, 21. The effect of its depletion recently observed in a human erythroleukemia cell line also suggests it as a potential target for treatment of drug-resistant chronic myelogenous leukemia22. In addition, signaling events involving GSTO1-1 protein-protein interactions have been implicated in a pathway that stimulates breast cancer stem cell enrichment during chemotherapy18. Moreover, GSTO1-1 has recently been identified as a crucial protein in the Toll-like receptor 4 (TLR4)-mediated pro-inflammatory pathway, such that its inhibition results in the relief of lipopolysaccharide (LPS)-stimulated inflammatory response. This includes attenuating the generation of reactive oxygen species, the release of pro-inflammatory cytokines including interleukin-1β (IL-1β), interleukin-6, and tumor necrosis factor (TNF) as well as the suppression of antioxidant enzymes, all of which significantly contribute to the pathology associated with inflammation in a wide range of acute and chronic disorders23-25. It has also been reported that GSTO1-1 modifies age-at-onset of Alzheimer disease (AD) and Parkinson’s disease26, and Gsto1 variants were reported to be implicated in AD pathogenesis27. This was thought to be mediated via the enzyme’s impact on inflammation, considering the possible role of inflammation in these two neurodegenerative disorders. Consequently, therapeutic application of GSTO1-1 inhibitors could offer considerable benefit to those suffering from these afflictions. 6 ACS Paragon Plus Environment

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A human breast cancer cell line (T47-D) has been shown to be deficient in GSTO1-1 as the result of the expression of an unstable variant with a Glu155 deletion28, 29. In addition, a mouse macrophage cell line in which GSTO1-1 has been knocked down by short hairpin RNA (shRNA) expression has been reported23. These cell lines have been of use in evaluating the physiological roles of GSTO1-1 and demonstrating the high specificity of the S-(4-Nitrophenacyl)glutathione (4-NPG) substrate. Two mice strains where the Gsto1 gene has been knocked out have been described25, 30. Under normal conditions, Gsto1-/- mice grow and breed normally and have a generally unremarkable phenotype with the exception that Gsto1-/- mice have lower levels of circulating monocytes and neutrophils25. GSTO1-1-deficient mice show a diminished inflammatory response to LPS and a diminished inflammatory response to a high fat diet that causes insulin resistance25. These studies suggest that inhibitors of GSTO1-1 may be clinically useful to suppress some inflammatory reactions and the observation that Gsto1-/- mice are phenotypically normal suggests that pharmacologically-induced GSTO1-1 deficiency may not have significant adverse effects. No adverse effects arising from GSTO1-1 deficiency are observed in humans either25, which indicates that the response of mice and humans may be similar. Thus, GSTO1-1 is identified as a drug target for inflammatory diseases and the application of GSTO1-1 inhibitors in humans should not cause a problem.

In the last few years, a diverse array of small molecules have been reported as 7 ACS Paragon Plus Environment


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GSTO1-1 inhibitors, including some marketed drugs that had been developed without apparent knowledge of GSTO1-1 activity. However, to-date no compound has been progressed as a selective GSTO1-1 inhibitor from discovery to the clinic. Indeed, most of these aforementioned compounds are still in need of substantial medicinal chemistry optimization before acquiring the qualities required to entering preclinical trials. In this Perspective, we analyze these compounds by the strategies adopted for their discovery as hits or leads, briefly evaluate their drug- and lead-likenesses, and comment on how these properties may be associated with these diverse discovery strategies. This analysis should provide useful information to those who are interested in GSTO1-1 inhibitor identification and those who are interested in some current approaches typical to enzyme inhibitor discovery. It should also highlight the importance of assay conditions in interpreting apparent bioactivity.

GSTO1-1 structure and function Human GSTO1-1 is a dimer composed of identical 27.6 kD subunits13. While important for regulation in certain proteins, there are no known post-translational modifications (apart from adducts formed during enzymatic activities) or chaperone proteins of GSTO1-1. Crystal structures reveal a GSH-binding site (G-site) adjacent to a relatively hydrophobic and open pocket that could conceivably accommodate structurally diverse substrates13, which is defined as hydrophobic binding site (H-site). The H-site of GSTO1-1 is not entirely hydrophobic, however, and the subunit interface of GSTO1-1 is more open than that of any other GST dimers. This suggests 8 ACS Paragon Plus Environment

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that the substrate or other binding partner for GSTO1-1 could be a large and amphipathic molecule, which is consistent with the recent discovery of the capacity of GSTO1-1 to specifically deglutathionylate some proteins but not others12.

The S-glutathionylation of proteins is a reversible process that can influence their structure and function and has been implicated as a regulatory device that can modulate cellular metabolism and cell signaling pathways by adjusting redox balance in different cellular compartments31,

32

. The capacity of GSTO1-1 to specifically

deglutathionylate proteins12 appears to be its primary physiological function and suggests a mechanism by which GSTO1-1 could potentially regulate cellular metabolism and signaling pathways that influence the growth and survival of cancer cells and the development and course of inflammation. Although there is evidence that GSTO1-1 plays a significant role in the regulation of the pro-inflammatory release of IL-1β from macrophages via the glutathionylation or deglutathionylation of a specific protein, the target protein has yet to be identified23-25.

GSTO1-1 deglutathionylates proteins by forming a mixed disulfide at its active-site Cys32 with the GSH formerly attached to the protein thiol, and then the enzyme is regenerated with the concurrent oxidization of a reduced GSH to form oxidized glutathione (GSSG) (Figure 2). This specific deglutathionylation by GSTO1-1 leads to the potential on/off regulation of protein function, while the polarity of the on/off switch is likely to be protein-specific. 9 ACS Paragon Plus Environment


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Figure 2. The potential on/off regulation of protein function resulting from specific deglutathionylation by GSTO1-1.

Assays to monitor GSTO1-1 modulation It is important to partner any discussion on GSTO1-1 inhibitor discovery with a consideration of assay technology platforms because it informs potential mechanism(s) of compound action and compound-mediated assay interference. Such knowledge can facilitate the design of appropriate counter-screens, mechanistic experiments, and compound design.

Fluorescence polarization technology based competitive activity-based protein profiling (Fluopol-ABPP). ABPP utilizing specific and reactive chemical probes is exploited to identify small-molecules capable of blocking labelling of probe target(s). In the absence of a competitive agent, a robust, time-dependent increase in the fluopol signal can be detected as the probe labels the target enzyme, resulting in slower tumbling and an increased fluorescence polarization/anisotropy signal33. The ability of a test compound to attenuate fluorescence polarization reflects its ability to block probe labelling, but such a readout does not necessarily reflect enzymatic modulation


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(though in principle covalently modifying Cys32 should attenuate enzymatic activity). Follow-up enzymatic activity assays can in principle address this point. A fluorescent activity-based probe, phenyl sulfonate-rhodamine (SE-Rh) (Figure 3), has been used to label GSTO1-1 due to its high affinity towards the enzyme within the human proteome34, and adapted for use in a high-throughput screening (HTS) campaign for inhibitors of GSTO1-135. It is a member of a probe library bearing a reactive sulfonate ester coupled to a variable alkyl/aryl-binding group. The library was originally designed for seeking a common chemotype with proteomic reactivity based on mechanistic studies of distinct enzyme classes. SE-Rh was incidentally found to label GSTO1-1 with high affinity when screening this library against a human breast cancer cell line, though this GSTO1-1 affinity is not specific. N

O

N

O HO HN

O

HN

O O S O

O

Figure 3. The structure of phenyl sulfonate-rhodamine (SE-Rh).

Gel-based competitive ABPP. This assay is supplementary to fluopol-ABPP. Compounds and GSTO1-1 probes are incubated with a biological sample, and the reaction mixture is separated by gel electrophoresis and analyzed by in-gel fluorescence densitometry. Gel-based analysis can assess compound selectivity amongst a complex biological matrix such as cell or tissue lysates if labelled proteins



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produce sufficient fluorescence for detection. Gel separation clearly limits assay throughput and is generally more suited to secondary screening. Besides SE-Rh, alternative

fluorescent

labels

may

also

be

suitable.

For

example,

5-chloromethylfluorescein diacetate (CMFDA) has also been used in a gel-based ABPP assay for GSTO1-1 inhibition36. One notable advantage of the gel-based ABPP assays is that electrophoresis can separate interfering compounds from many of the analyzed proteins.

4-NPG substrate assay. GSTO1-1 is the sole member of the GST family that can catalyze the reduction of S-(phenacyl)glutathiones to corresponding acetophenones. Among S-(phenacyl)glutathiones, 4-NPG is the substrate of choice due to its relatively high turnover by GSTO1-110. In the proposed chemical mechanism, the thiolate on Cys32 of GSTO1-1 acts as a biological nucleophile, forming a covalent bond with the electrophilic cysteinyl sulfur atom on 4-NPG, yielding a disulfide and a carbanion

stabilized

by

enolization.

Subsequent

protonation

yields

4-nitroacetophenone (Figure 4). GSTO1-1 can be regenerated via reducing agents such as 2-mercaptoethanol (BME), tris(2-carboxyethyl)phosphine, or dithiothreitol (DTT). Enzyme activity is measured by monitoring the reduction of 4-NPG to 4-nitroacetophenone via light absorbance at 305 nm, although the difference in molar absorptivity between 4-NPG and 4-nitroacetophenone is relatively small in magnitude (1.1-fold).


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O+ O SG

-

GSTO1-1

S

S GS

GSTO1-1

O 2N O

O 2N H 4-NPG

+

O CH 2-

O2N 4-nitroacetophenone

O 2N

Figure 4. The proposed chemical mechanism of a reported 4-NPG substrate assay. Enzymatic activity is monitored by the difference of UV absorbance between 4-NPG and 4-nitroacetophenone at 305 nm.

MMA (V) reductase assay. GSTO1-1 can catalyze the reduction of MMA (V) to MMA (III) in the presence of GSH. Notably, GSTO2-2 also possesses MMA (V) reductase activity37. In the proposed chemical mechanism, the thiolate on GSTO1-1 Cys32 acts as an electron donor and forms a conjugated intermediate with MMA (V). Nucleophilic attack by GSH yields a disulfide and an arsenic anion intermediate. After protonation and leaving of a water molecule, MMA (III) is produced. The enzyme can be regenerated when another molecule of GSH serves as a reducing agent (Figure 5). In one assay configuration38, 39, [14C]-MMA (V) is used as the substrate. The resulting product, [14C]-MMA (III) is extracted as complexes with diethylammonium diethyldithiocarbamate (DDDC) into carbon tetrachloride, and quantified by scintillation counting. A non-radioactive, spectrophotometric coupled-enzyme format with non-labelled MMA (V) substrate has also been reported38, 40. In this assay configuration, the GSSG 13 ACS Paragon Plus Environment


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product from the GSTO1-1-catalyzed reduction is used as the substrate for GSH reductase, which catalyzes the reduction of GSSG to GSH and the oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide phosphate (NADP+). Conversion of NADPH to NADP+ can be monitored by optical absorbance at 340 nm, which serves as an indirect measure of GSTO1-1 MMA (V) reductase activity.

Figure 5. The proposed chemical mechanism of a reported MMA(V) reductase assay. Enzymatic activity can be monitored by (A) extraction of radiolabeled MMA (III) or (B) light absorbance of NADPH at 340 nm via an enzyme-coupled reaction.

DHA reductase assay. GSTO1-1 can also catalyze the reduction of DHA to ascorbate in the presence of GSH. Besides GSTO1-1, several other enzymes have DHA reductase activity including GSTO2-241, dehydroascorbate reductase (DHAR)42, chloride intracellular channel protein 1 (CLIC1) and CLIC443 and glutaredoxins44. In the proposed chemical mechanism, the thiolate on GSTO1-1 Cys32 forms a hemiketal intermediate with the DHA 3-carbonyl group. A molecule of GSH then binds to the enzyme active site and forms a disulfide with Cys32, meanwhile a carbanion is generated and stabilized by enolization. Subsequent protonation yields ascorbate


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(Figure 6). GSTO1-1 can be regenerated with another molecule of GSH serving as a reducing agent. Generation of ascorbate is monitored by the increase of optical absorbance at 265 nm45, 46. HO O

O

HO HO O

O

HO

+

H S

O

O

GSTO1-1

O HO S 1-1OTSG HO

S

+

HO O

HO

GSH

GS

GSTO1-1

O

O HO H+ HO O

O HO

HO

HO

HO

O

Figure 6. The proposed chemical mechanism of a reported DHA reductase assay. Enzymatic activity can be monitored by light absorbance of the ascorbate product.

Thioltransferase assay. GSTO1-1 can catalyze the reduction of a disulfide substrate to two molecules of thiol in the presence of GSH, though other enzymes such as glutaredoxins also have this activity47. In this assay, the disulfide substrate 2-hydroxyethyl disulfide is reduced by GSTO1-1 to form BME. The resulting GSSG then serves as the substrate for GSH reductase. Similar to the enzyme-coupled MMA assay, GSTO1-1 thioltransferase activity can be indirectly measured by monitoring the conversion of NADPH to NADP+ by optical absorbance at 340 nm46, 48.

1-Chloro-2,4-dinitrobenzene (CDNB)-GSH conjugation assay. CDNB can also serve as a substrate for most classes of GSTs. In this assay format, CDNB is conjugated to a single molecule of GSH by GSTO1-1. Formation of the resulting CDNB-GSH adduct


O

O OH


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can be monitored by light absorbance at 340 nm49, 50. However, this assay is generally avoided because the rate of the CDNB-GSH conjugation reaction is relatively slow.

Compound-mediated assay interference in GSTO1-1 assays Understanding the underlying biochemical principles of GSTO1-1 assays is essential to interpreting apparent compound responses. Depending on the assay readout, certain types of compound-mediated interferences may be enriched, and others negligible. Variables such as compound incubation time, enzyme and substrate concentrations, buffer composition, and reaction time can profoundly influence observed compound activity. As is the case for any bioassay, each of the GSTO1-1 assay formats is susceptible

to

various

sources

of

technology-related

and

generalized

compound-mediated assay interference (Table 2).

Table 2. Summary of techniques for assaying small-molecule modulation of GSTO1-1. Assay

Advantages

Fluopol-ABPP

Basic instrumentation requirements Homogenous format

Gel-based competitive ABPP

4-NPG

Basic instrumentation requirements Adaptable to cellular analyses Basic instrumentation requirements

Disadvantages Susceptible to compound-probe interactions High enzyme concentrations utilized for sufficient readout Greater complexity/more laborious than fluorpol-ABPP

Technology-related interference susceptibilities

Light-based interferences (compound fluorescence, compound absorbance/inner-filter, fluorescence quenching, light scattering)

Light-based interferences less likely (gel separation)

Ref

33, 35

33, 35, 36

High enzyme concentrations utilized Short wavelength readout

Light-based interferences (compound absorbance, light scattering)

10


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Continuous readout option

End-point readout Analyte separation step required MMA (V) reductase (radiolabel)

High analytical sensitivity

Radiation safety and disposal requirements

Light-based interference (scintillation quenchers)

39

Specialized instrumentation requirements (scintillation counter)

MMA (V) reductase (absorbance, enzyme-coupled)

Basic instrumentation requirements Short wavelength readout

Off-target effect (modulation of coupled enzyme)

Short wavelength readout

Homogenous format


Homogenous format

High enzyme concentrations required

Continuous readout option Basic instrumentation requirements CDNB-GSH conjugation

Light-based interference (compound absorbance, light scattering)

High enzyme concentrations required

Continuous readout option Basic instrumentation requirements Thioltransferase

38

Homogenous format Continuous readout option Basic instrumentation requirements

DHA reductase


45

Reaction with GSH substrate

Light-based interference (compound absorbance, light scattering) 51

Off-target effects (modulation of coupled enzyme) Reaction with GSH substrate


Slow reaction rate

Homogenous format


52

Reaction with GSH substrate

Continuous readout option

Table 3. Summary of reported GSTO1-1 inhibitors. Enzymatic half-maximum Compound chemical No.

Other inhibitory

structure

MW

cLogP

HBD#

HBA#

RTB

tPSA

LogS

LE

LLE

Fsp3

APT

345

2.6

1

5

5

72

-3.1

0.30

2.8

0.29

2

targets concentration (IC50)

1

4.6 µM

NR



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At least 6 2

< 0.4 µM

around 50

264

2.6

0

4

7

41

-2.2

> 0.54

> 3.8

0.80

1

496

5.9

0

5

12

48

-6.7

0.27

1.0

0.34

7

kDaa At least 6 3

120 nM

around 50 kDaa or 0b

4

75 µM

NR

823

3.7

6

16

5

220

-7.1

0.10

0.41

0.47

7

5

28 nM

0c,d

368

2.3

1

9

6

101

-3.5

0.43

5.2

0.38

2

6

54 nM

404

3.3

1

6

6

62

-4.6

0.36

3.9

0.38

5

7

21 nM

0c

404

3.3

1

6

6

62

-4.7

0.38

4.4

0.38

5

8

31 nM

0e

311

1.9

1

5

4

66

-2.9

0.57

5.6

0.30

2

9

2 µM

NR

511

10.2

2

5

14

76

-7.9

0.22

-4.5

0.79

7

10

4 µM

NR

531

11.9

1

5

17

73

-8.3

0.19

-6.5

0.82

7

11

62 µM

NR

332

4.8

3

4

2

78

-5.9

0.24

0.59

0.65

4

12

1.6 µM

NR

286

-0.01

3

6

1

104

-2.4

-0.013

-0.19

0.07

2

13

40 nM

NR

461

5.6

1

5

9

73

-7.5

0.30

1.8

0.38

7

38 kDa, 34 kDac,d


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32 kDa, NR

14

56 kDaf or 0

421

4.8

2

7

6

88

-6.2

NR

NR

0.38

5

416

4.0

2

7

4

88

-5.9

NR

NR

0.38

5

404

3.4

3

6

4

105

-4.6

NR

NR

0.43

5

245

3.2

0

4

3

47

-3.1

NR

NR

0.29

2

g

32 kDa, NR

15

56 kDaf or 0h

22 kDa, 16

NR

17

NR

28 kDai

21 kDa, 55 kDa, 80 kDa

j

18

0.15 µM

NR

720

6.4

4

12

20

158

-6.3

0.18

0.42

0.58

7

19

51.2 nM

0k

465

3.2

0

7

5

88

-6.6

0.30

4.1

0.16

5

Negligiblel

495

0.4

5

11

15

174

-2.9

0.20

4.5

0.45

7

13 µM (in 20 apoptotic cells)

21

NR

Negligiblem

369

4.8

0

3

14

24

-4.8

NR

NR

0.86

7

22

NR

NR

353

3.9

1

4

4

49

-4.6

NR

NR

0.20

2

NR, not reported.

a

MDA-MB-231 soluble proteome, gel-based competitive ABPP, 20 µM compound concentration

b


c




Page 20 of 89

d


e

HCT116p53+/+ soluble proteome, gel-based competitive ABPP, 1 µM compound concentration

f

Human monocyte soluble proteome, autoradiography, 3 µM compound concentration

g

Human monocyte soluble proteome, autoradiography, 0.3 µM compound concentration

h

Human monocyte soluble proteome, autoradiography, 1 µM compound concentration

i

Cytokine release inhibitory drug 3 (CRID 3) elution after addition of 5 mM CRID 3 to elution buffer in the affinity chromatography

experiment, analyzed by autoradiography

j

Cytosolic Madin-Darby canine kidney (MDCK) cell extracts, autoradiography, 1 µM compound concentration

k

NIH/3T3 soluble proteome, in-gel fluorescence, 0.5 µM compound concentration

l

Lysate of staurosporine-induced apoptotic HeLa cells, in-gel fluorescence, 50 µM compound concentration

m

MCF-7 lysate, in-gel fluorescence, 1 µM compound concentration

Hydrogen bond donor number (HBD#) was calculated by adding up the hydrogens bonded to any nitrogen and oxygen without negative

charge in the molecule; Hydrogen bond acceptor number (HBA#) was calculated by adding up any nitrogen, oxygen and fluorine,

excluding nitrogen with positive formal charge or higher oxidation states, pyrrolyl form of nitrogen, and furan oxygen; Rotatable bond

number (RTB) was calculated by following the definition of rotatable bond: any single bond, not in a ring or bound to a nonterminal

heavy atom (Amide C–N bonds are excluded from the count because of their high rotational energy barrier); the other predicted properties

including molecular weight (MW), calculated logarithm of partition coefficient (cLogP), topological polar surface area (tPSA), logarithm

of aqueous solubility (LogS) were generated from ChemDraw Professional 15.0; ligand efficiency (LE) was calculated according to the

equation: LE = 1.37 × pIC50 / heteroatom number; lipophilic ligand efficiency (LLE) was calculated according to the equation: LLE =

pIC50 - cLogP; fraction of sp3 carbon atoms (Fsp3) was calculated according to the equation: Fsp3 = carbon sp3 atoms / total carbon atoms.

Color coding standards: MW: ≤ 460 green, 460-500 yellow, > 500 red. cLogP: -4-4.2 green, 4.2-5.0 yellow, > 5.0 red. HBD#: 0-5 green,

6-8 yellow, > 8 red. HBA#: 0-9 green, 10-12 yellow, > 12 red. RTB: 0-10 green, 10-15 yellow, > 15 red. tPSA: 90-140 green, 75-90


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yellow, ≤ 75 or ≥ 140 red. LogS: ≤ -5, green, -5- -4 yellow, > -4 red. LE: > 0.4 green, 0.3-0.4 yellow, < 0.3 red. LLE: > 4 green, 3-4

yellow, < 3 red. Fsp3: > 0.47 green, 0.30-0.47 yellow, ≤ 0.30 red; Abbott Physicochemical Tiering (APT): 1-3 green, 4-6 yellow, 7-9 red.

Orange: compounds with chemically-reactive moieties.

Unbiased target-based screening to discover inhibitors of GSTO1-1 Target-based screening is a well-established approach to discover biologically-active compounds as starting points for drug and chemical probe discovery53. An industrial screening campaign is typically defined as high-throughput when the screening deck numbers around 100,000 compounds or more, while in an academic setting screening as few as 20,000 compounds may be considered high-throughput. Such approaches have been reported for GSTO1-1 inhibitors.

Bachovchin and co-workers reported the discovery of 38 primary hits with GSTO1-1 inhibitory activity identified from a pilot screening of 2,000 compounds from a validation fraction of the National Institutes of Health Molecular Libraries Small Molecule Repository (NIH MLSMR), using fluopol-ABPP35. Representative hits include the proton pump inhibitor omeprazole (1, IC50 = 4.6 µM), the disulfide compound 2 (IC50 < 0.4 µM), the α-chloroacetamide 3 (IC50 = 120 nM), and the antibiotic rifampicin (4, IC50 = 75 µM), with engagement of purified recombinant GSTO1-1 supported by gel-based competitive ABPP (1, 2, and 4) or 4-NPG assay (3). After assessing their selectivity by performing the gel-based competitive ABPP screening again against the soluble proteome of the human breast cancer cell line 21 ACS Paragon Plus Environment


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MDA-MB-231, the α-chloroacetamide 3 was determined as the most promising compound as it selectively inhibited GSTO1-1 at 1 µM compound concentrations, while at this same concentration the other three compounds were inactive.

The same group then screened the full NIH MLSMR deck of 302,667 small molecules, designating 3,207 compounds as hits from the primary fluopol-ABPP screen33. After activity confirmation by fluopol-ABPP and three rounds of gel-based competitive ABPP secondary screening of cherry-picked active compounds against recombinant GSTO1-1 (1 µM compound concentrations) and the soluble proteome of a human melanoma cell line MDA-MB-435 (1 µM and 100 nM compound concentrations), ten compounds were prioritized, nine of which were α-chloroacetamides. After another gel-based competitive ABPP screening against more than 30 SE-Rh reactive proteins in the MDA-MB-435 soluble proteome (10 µM compound concentrations), three of the compounds were deprioritized as potential leads due to their high off-target rate and reduced binding to GSTO1-1 at 100 nM in MDA-MB-435 proteome. Of these three compounds, two were α-chloroacetamides and one was an α-aryl chloride. The remaining seven α-chloroacetamides can be structurally divided into two scaffolds, respectively represented by 5 (IC50 = 28 nM) and 6 (IC50 = 54 nM). Medicinal chemistry optimization of the latter led to the identification of KT53 (7), a highly selective GSTO1-1 inhibitor with potent in vitro (IC50 = 21 nM) and cellular activity (IC50 = 35 nM). Unfortunately, the chemical stability of this compound is poor due to


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an intramolecular pyridyl N-alkylation that leads to an inactive compound. Note: all the IC50 values reported here were determined by gel-based competitive ABPP.

Based on the potential utility of α-chloroacetamide groups to inhibit GSTO1-1, Ramkumar et al. performed a structural similarity search across a virtual library with approximately 1,000,000 compounds, identifying 141 hits which were grouped into five clusters on the basis of their chemical structures, all featuring an α-chloroacetamide or (cyclized) tertiary-chloroacetamide functional group36. After sourcing and subsequent screening for inhibition of recombinant human GSTO1-1 with an adapted 4-NPG substrate assay in high-throughput format, and then for binding both recombinant GSTO1-1 and endogenous GSTO1-1 in the soluble proteome of a human colorectal cancer cell line HCT116p53+/+ with the in-gel fluorescence binding assay, 43 compounds showed at least 50% activity (inhibition and

binding)

in

all

three

assays,

and

were

progressed

into

further

concentration-response studies. Eventually, C1-27 (8) was identified as the most promising compound, after taking into account its promising inhibitory potency towards purified GSTO1-1 with an IC50 of 31 nM as detected by the 4-NPG substrate assay, its competitive binding to endogenous GSTO1-1 in the HCT116p53+/+ proteome, and its selectivity in binding to endogenous GSTO1-1 in the HCT116p53+/+ proteome. Additionally, for both 5 and 8, strong support for on-target enzymatic inhibition by covalent binding of the active site of GSTO1-1 has been established through x-ray crystallography25. Of all GSTO1-1 inhibitors reported to date, only 5 and 8 have been 23 ACS Paragon Plus Environment


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used in in vivo studies so far. As a potential anti-cancer agent, 8 was shown to restrict the growth of the transplantable tumor HCT116 in nude mice36.

In recent studies, 5 has been used in mice to suppress the severe inflammatory response to bacterial LPS25. Considering that the genetic organization of Gsto1 is similar in mice, rats and humans, and there is no evidence to indicate that the enzyme will respond differently in different species, both these studies could effectively illustrate the clinical potential of 5 and 8 as GSTO1-1 inhibitors.

Biased screening GSTO1-1 inhibitor discovery Inspired by the finding that α-tocopherol (vitamin E) was a potent inhibitor of GSTP1-1, a pi-class GST (IC50 = 0.5 µM)54, and that several esterified tocopherols also inhibit GSTP1-1 with IC50 values below 10 µM55, Sampayo-Reyes et al. tested the inhibitory activities of (+)-α-tocopherol phosphate (9) and (+)-α-tocopherol succinate (10), which are both ester derivatives of vitamin E, towards GSTO1-1 by the MMA (V) reductase assay38. The IC50 values of 9 and 10 towards GSTO1-1 were identified as 2 µM and 4 µM respectively. The authors also tested the inhibitory activities of several other reported inhibitors or substrates of general GSTs towards GSTO1-1 via the same MMA reductase assay. Compounds 9 and 10 showed significantly more potent activity than CDNB (GST substrate, GSTO1-1 IC50 = 900 µM), sodium deoxycholate (GST inhibitor, GSTO1-1 IC50 = 1020 µM) and ethacrynic acid (GST inhibitor, GSTO1-1 IC50 = 25 µM). 24 ACS Paragon Plus Environment

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Several other natural products have been tested versus GSTO1-1. Carnosic acid (11) is a bioactive compound found in the herb Rosemary (Rosmarinus officinalis) with reported anti-inflammatory effects by inhibiting signaling through LPS/TLR4 inflammatory pathway, of which GSTO1-1 is a crucial pathway component56. Board et al57 demonstrated 11 inhibits GSTO1-1 with an IC50 value of 30-62 µM in the 4-NPG substrate assay. Protoapigenone (12) is a novel flavonoid firstly isolated from Thelypteris torresiana. Based on reported anticancer properties, Wu et al. showed 12 inhibits GSTO1-1 with an IC50 value of approximately 1.6 µM, as measured by the 4-NPG substrate assay58. After a structure-based modification of this natural product where functional side chains were introduced to mimic the substrate of GSTO1-1, GSH, a novel generation of GSTO1-1 inhibitors was synthesized59. Among them, 13 was the most potent. Its IC50 value towards GSTO1-1 was about 40 nM as determined by the 4-NPG substrate assay.

Serendipitous discovery of GSTO1-1 inhibitors In 2001, Pfizer found that a sulfonylurea-containing drug which had been known as adenosine 5'-triphosphate (ATP)-activated K+ channel inhibitor, glyburide, blocked IL-1β production in a concentration-dependent manner (IC50 = 12 µM). A number of structurally-related analogues were then characterized with an attempt to find agents more potent than glyburide. As a result, a class of compounds designated as diarylsulfonylureas was identified as potential cytokine release inhibitory drugs (CRIDs) which can inhibit the post-translational processing of IL-1β60. In a later study 25 ACS Paragon Plus Environment


Page 26 of 89

designed to seek potential CRID protein targets, GSTO1-1 was identified, and was found to irreversibly bind the epoxide-bearing CRID 1 (14) and CRID 2 (15) via its active-site cysteine (Cys32). Despite the detection of two off-targets (Table 3), 14 and 15 selectively labelled GSTO1-1 within the human monocyte proteome, which suggested that this affinity was relatively specific and not simply driven by a nonspecific electrophilic warhead. In an affinity chromatography experiment where a warhead-conjugated resin of CRID 2 was used as the stationary phase, GSTO1-1 was one of the three proteins recovered after elution with the non-warhead-bearing CRID 3 (16)61. While the biological ramification of the observed affinity of CRIDs for GSTO1-1 remains unclear, it was suggested that GSTO1-1 may be associated with the nucleotide-binding oligomerization domain (NOD)-like receptor family protein nucleotide-binding oligomerization domain-like receptor family protein 3 (NLRP3) inflammasome since it co-immunoprecipitates with the inflammasome component apoptosis-associated speck-like protein containing a carboxy-terminal CARD (ASC)62. Alternatively, it is also possible that GSTO1-1 regulates IL-1β release via the glutathionylation/deglutathionylation of a component of the NLRP3 inflammasome12. Recent studies have identified the functional upstream target of CRID 3 IL-1β modulation as NLRP363. Despite the observations that CRID 1 and 2 covalently bind to GSTO1-1 Cys32 via reaction with their epoxide warheads, there is no definitive evidence that CRID 3 (16), which lacks the epoxide warhead, actually inhibits GSTO1-1 catalytic activity. However CRID 3 (16) may function as a GSTO1-1 inhibitor given it elutes GSTO1-1 from immobilized CRID 2.61 Further studies would 26 ACS Paragon Plus Environment

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help determine if CRID 3 is a competitive inhibitor of GSTO1-1 and the role of GSTO1-1 in the function of the NLRP3 inflammasome.

In 2002, Henry and co-workers found that the oxazolidinone derivative locostatin (17) had the ability to inhibit cellular locomotion in an HTS based on an epithelial wound closure assay system64. While investigating the functional mechanism of 17, GSTO1-1 was identified as one of the four proteins covalently modified by 17 in cytosolic Madin-Darby canine kidney (MDCK) cell extracts, as assessed by DHA reductase assay and thioltransferase assay46. The other three proteins inhibited via covalent modification by locostatin were Rapidly Accelerated Fibrosarcoma (RAF) kinase inhibitor protein (RKIP), aldehyde dehydrogenase 1A1 and prolyl oligopeptidase. Locostatin appeared to inhibit cell motility by disrupting the interaction between RKIP and RAF kinase, while GSTO1-1 was not associated with this function.

With an aim to investigating the possible off-targets of the proteasome inhibitor carfilzomib (CFZ, 18) used clinically for multiple myeloma, Federspiel et al49 utilized a CFZ analogue with an alkyne click chemistry tag to explore off-target binding of CFZ to proteins in HepG2 cells. Targets covalently modified via epoxide reactivity are extracted through click chemistry of the alkyne group and the photocleavable click biotinylation reagent azido-UV-biotin. In this reagent65, the azido and biotin groups are separated by a photocleavable linker. Peptides and proteins modified in this way 27 ACS Paragon Plus Environment


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are affinity purified on streptavidin beads. Photolysis of the beads with a low-intensity ultraviolet (UV) light releases the proteins and/or peptides modified by the CFZ alkynyl analogue. This analogue exhibited a proteasome inhibitory profile as well as chemical stability in hepatocytes similar to CFZ, suggesting it mirrors CFZ biological activity. After a tiered triage process, 12 off-target proteins were identified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) but only two of these were deemed to be of significance based on the MS spectral count results. GSTO1-1 was one of the two remaining off-targets along with CYP27A1. Compound 18 bound GSTO1-1 Cys32 and demonstrated relatively potent enzymatic inhibition (IC50 = 0.15 µM) as determined by a CDNB-GSH conjugation assay. The significance of this GSTO1-1 inhibition is unclear, as CFZ modulates the therapeutically-relevant proteasomal targets with greater potency (i.e. IC50 of the 20S proteasome for CFZ < 10 nM in vitro)66.

In 2010, Son and coworkers50 screened 43 commercially-available reactive fluorescent dyes against NIH/3T3 human fibroblasts, trying to correlate the proteome-staining profiles of these fluorescent compounds with their chemical structures. One CMFDA, the fluorescent protein tag CellTracker Green (19) was found to solely label GSTO1-1 Cys32 via its benzyl chloride moiety. After further investigation, 19 was identified as a potent, selective irreversible inhibitor of GSTO1-1 in vitro with an IC50 value of 51 nM by a CDNB-GSH conjugation assay. A


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molecular modelling analysis suggested that in addition to active site thiol labelling, 19 occupied a hydrophobic sub-site near, but separate from, the GSH-binding site.

With the goal of identifying apoptotic-cell-selective inhibitors of cysteine-mediated protein activities, Pace and coworkers67 designed and synthesized a library of ten cysteine-reactive compounds comprised of a peptide conjugated to an acrylamide or sulfonate ester electrophile, along with an alkyne click chemistry tag. As a result, NJP2 (20) was reported to cause a loss of labelling of GSTO1-1 in apoptotic cells by gel-based competitive ABPP, which suggests inhibition of GSTO1-1 activity (IC50 = 13 µM in apoptotic HeLa cells). Its apoptotic-cell selectivity was attributed to increased cell-permeability during apoptosis, while its selective inhibition of GSTO1-1 was attributed to its specific covalent modification of GSTO1-1 Cys32.

Similarly, Couvertier and coworkers designed and synthesized a library of fifteen cysteine-reactive probes each containing (1) a 4-aminopiperidine scaffold derivatized as a chloroacetamide for covalent modification of cysteines, (2) an alkyne click chemistry tag, and (3) a diversity element intended for targeting specific proteome subsets68. They identified SMC-1 (21), which contains an n-octyl group as the diversity element, as a potent and selective covalent binder of GSTO1-1 Cys32 in GSTO1-1-overexpressing HEK293T cells by click chemistry and subsequent in-gel fluorescence visualization. Complete loss of detectable labelling of a GSTO1-1 C32A



Page 30 of 89

mutant further supported this proposed mechanism. However, the inhibition of GSTO1-1 catalytic activity by 21 is still in need of clarification.

Mechanism-based design of GSTO1-1 inhibitors The known enzymatic activities of GSTO1-1 should allow for mechanism-based design of inhibitors. For example, 4-NPG conjugates could in principle be modified to be non-scissile and hence become inhibitory (Figure 7). However, little has been reported along these lines, presumably because not only would such compounds be unlikely to cell membrane-permeant, but they could be recognized by highly active GSH-conjugate efflux pumps69.

Figure 7. An example of mechanism-based design of GSTO1-1 inhibitor as a non-scissile analogue of 4-NPG. (A) GSTO1-1 catalyzes the reduction of 4-NPG to 30 ACS Paragon Plus Environment

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4-nitroacetophenone by nucleophilic attack of the cysteinyl sulfur of 4-NPG with active-site Cys32. (B) The 4-NPG analogue with cysteinyl sulfur substituted by methylene may still bind to GSTO1-1 at the same site, acting as a competitive inhibitor of the enzyme.

Structure-based design of GSTO1-1 inhibitors In order to quantify the absolute cellular concentration of GSTO1-1 using inductively coupled mass spectrometry (ICP-MS), Liang and co-workers70 designed and synthesized a GSTO1-1-targeted dibenzylcyclooctyne-modified 2-chloroacetamide (22) as a tool compound based on GSTO1-1 Cys32 and the adjacent H-site. As expected, 22 covalently bound to GSTO1-1 Cys32 via its chloroacetamide group and formed the element/isotope-loadable complex detected by ICP-MS via its alkyne group. In a preliminary study of its selectivity, 22 did not form detectable adducts with the model sulfhydryl-containing protein papain, whereas 22 formed detectable adducts with GSH. However, an expanded selectivity study would be useful to better gauge potential off-targets. This approach is relatively peripheral to structure-based design as the authors did not use crystal structure information as a guidance for rational design. In fact there do not appear to be any such reports, despite the availability of x-ray crystallographic information of relevant host-ligand complexes.

Indeed, the crystal structures of GSTO1-1 in complex with ML175 (5) and C1-27 (8) have been reported25, 36. Both 5 and 8 covalently bind GSTO1-1 Cys32 through 31 ACS Paragon Plus Environment


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alkylation of the cysteine thiol with their α-chloroacetamide warheads, as well as binding in the H-site (Figure 8). Both compounds form a hydrogen bond between their acetamide oxygen atoms and the Phe34 backbone amide nitrogen.

Compound 8 forms an additional hydrogen bond between to the sulfonamide group and the Trp180 indole nitrogen. The residues Gly128, Val127, and Pro124 form hydrophobic interactions with the sulfonamide methyl groups of 8, while the carbonyl oxygen of Pro124 forms a hydrogen bond with the sulfonamide nitrogen atom of 8. The phenyl group of 8 sits in a hydrophobic pocked formed by the side-chains of Pro33, Trp222, Phe225, and Leu226, and forms a partial π-stacking interaction with Tyr229, while its chlorine substituent is oriented toward Ile131. The positioning of the aromatic ring in 8 contrasts with nitrophenyl group of 5, which is directed toward Leu56. The aliphatic trifluoroacetamide group of 5 occupies the aforementioned hydrophobic pocket, and a buried water molecule form bridging hydrogen bonds to Trp222 and the trifluoroacetamide oxygen group of 5.

It is also noteworthy that similar shifts are observed between the inhibitor-bound forms of GSTO1-1 and the GSH-bound form. In both cases, Tyr229 rotates away from the G-site to accommodate the ligands. Similarly, Ile131 rotates so as to prevent steric clashes with the bound inhibitors. Finally, the indole group of Trp222 is rotated approximately 180° in the 8/GSTO1-1 complex relative to the analogous 5- and


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GSH-bound GSTO1-1 structures, allowing the formation of additional hydrophobic interactions with the inhibitor.

With the reported protein-ligand structural information, fragment-based drug design (FBDD) would be a credible avenue for future efforts. As a primary screening strategy, FBDD can often identify hits even when routine HTS campaigns fail to identify tractable chemical matter71. Rational design of fragment libraries and screening formats, followed by careful fragment selection and medicinal chemistry optimization can efficiently lead to the generation of tractable lead compounds. Of relevance to GSTO1-1, reactive fragment libraries are now readily commercially available, though the balance between fragment selectivity and reactivity should be carefully considered throughout any such discovery campaign.



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Figure 8. Stereoscopic images of crystal structures of GSTO1-1 bound to 8 (cyan, PDB ID 4YQM) and 5 (pink, PDB ID 5V3Q). Superimposed on each is the structure of GSTO1-1 in complex with GSH (yellow, PDB ID 1EEM). Hydrogen bonds are indicated as black lines. Images rendered in VMD.

Assay interference: counter-screens, orthogonal assays, and selectivity assays in GSTO1-1 inhibitors Nomenclature. In screening literature, the nomenclature used to distinguish useful screening hits from those that are not is somewhat ad-hoc and the latter will commonly see terms such as anomalous binder, frequent hitter, false-positive, bad actor, and pan assay interference compounds (PAINS) applied to them. However, such terms are often ill-defined and used interchangeably. For the purposes of this manuscript and as we have recently discussed72, we consider an assay-active compound to be a false-positive if it interferes with assay-related or generalized technology, or alternatively non-specifically interferes with the target biomolecule (e.g., aggregation, nonspecific thiol reactivity, redox-activity). We consider that an assay-active compound that has affinity with a specific target site to be a true positive, and while some of these may be true hits with workable tractability, some of these might reveal themselves to be false hits/bad actors, in that they are shown to be inherently and non-specifically protein-reactive.

A fundamental strategy to identify compound-mediated assay interference and to


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support on-target modulation is the use of technology-related and more generalized interference counter-screens, as well as orthogonal assays73. In addition, higher quality bioassays often include specific reagents and experimental protocols to mitigate potential compound-mediated assay interference.

Technology-related

compound-mediated

interferences.

Technology-related

counter-screens are performed to characterize the effects of test compounds on the assay readout independent of on-target effects. Most of the published GSTO1-1 activity assays utilize light-based readouts (Table 2). Compounds that absorb light at excitation or emission channels, interact with fluorophores, or fluoresce at emission channels have the potential to interfere with light-based assay readouts by inner-filter effects, fluorescence quenching, and auto-fluorescence, respectively74-76. In addition, highly insoluble compounds can precipitate and interfere with light-based readouts via light scattering. For light-based assays, such counter-screens often include measuring absorbance in assay buffer, spiking compound post-reaction to investigate fluorescence quenching and light absorbance, or monitoring compound fluorescence under assay-like conditions to assess for compound auto-fluorescence. In certain instances, compounds can interfere with the primary assay readout but still exert on-target activity that can be confirmed with orthogonal assays77.

Generalized compound-mediated interferences. The main sources of generalized compound-mediated

assay

interference

include

compound

aggregation

and 35

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nonspecific reactivity (which includes redox activity)78-81. Other sources of interference can include chelation and membrane disruption (which may reflect aggregation).

Colloidal aggregates represent a significant burden in early drug and chemical probe discovery. Compounds that form aggregates under test concentrations have the potential to modulate GSTO1-1 in any of the aforementioned assay formats. In most cases, aggregators disrupt enzymatic function, though in certain instances aggregators appear to enhance enzymatic activity82, 83.

Generalized interference counter-screens are performed to characterize the effect of test compounds on the assay readout independent of on-target effects. Counter-screens using thiol-based probes (most popularly GSH) are often performed to de-risk nonspecific compound reactivity84. Compound-probe adducts can be detected by several conventional techniques such as LC-MS or fluorescence. However, many thiol-based probes are non-proteinaceous and may not fully model protein microenvironments and reactivity85. In addition to GSH, our group performs a La assay to detect reactive molecules by nuclear magnetic resonance (ALARM NMR), an industry-developed NMR-based counter-screen for nonspecific protein thiol reactivity which utilizes the human Lupus antigen (hLa)86, 87. Such a technique may be especially useful for assessing the specificity of reported GSTO1-1 inhibitors using hLa as a surrogate for nonspecific reactivity. Counter-screens for aggregation are also 36 ACS Paragon Plus Environment

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becoming

commonplace82.

Examples

include

dynamic

light

scattering,

AmpC β-lactamase enzymatic counter-screen, detergent titration, and NMR methods. Redox-active compounds can also be flagged by facile biochemical counter-screens including a facile horseradish peroxidase-phenol red assay, as well as related Amplex® red and rezarufin formats88, 89. In our experience, high-quality studies often perform a combination of the above counter-screens to de-risk bioactive compounds.

Interference mitigation strategies. Higher quality assays usually include several strategies to mitigate both technology-specific and generalized compound-mediated assay interference. Light-based interference can also be mitigated by several strategies. For example, the use of orange/red-shifted fluorophores over blue-shifted fluorophores is beneficial because a greater percentage of compounds in collections tend to fluoresce at relatively short wavelengths in the blue spectral region90, 91. Also, implementation of a pre-read after compound addition but prior to fluorophore addition can help flag auto-fluorescent compounds. Reducing agents such as DTT have the added benefit in biochemical assay buffers to scavenge certain highly electrophilic test compounds86. Compound aggregation can also be mitigated by including low concentrations of nonionic detergents (e.g., Triton X-100, Pleuronics) and/or decoy proteins such as bovine serum albumin (BSA)82, 92. Performing these assay modifications can dramatically attenuate certain interferences. However, they are not completely efficacious and counter-screens are still recommended to de-risk compounds for assay interference. 37 ACS Paragon Plus Environment


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Interference profiling. An additional strategy for understanding compound-mediated assay interference is to investigate the effects of known interference compounds such as aggregators, nonspecific reactive compounds, redox-active compounds, and optically-active compounds (e.g., quenchers) on the assay readout. Depending on the experimental system, assay readouts may be particularly sensitive to certain types of interference. For example, in GSTO1-1, the active-site thiol on Cys32 would raise caution about enriching for nonspecific thiol-reactive compounds.

Challenging assays with known interference compounds can be especially useful for cell-based assays, where it is considerably more difficult to assess for on-target specificity. In the systematic profiling of reported histone acetyltransferase inhibitors, we have recently shown that many of the reported “specific” inhibitors produced similar readouts to several extensively characterized with PAINS chemotypes, as well as other prototypical interference compounds93.

Analysis of GSTO1-1 assays. Many of the original reports for GSTO1-1 inhibitors do not report such crucial follow-up experiments such as orthogonal assays or assay interference counter-screens (Table 4). This is significant because in principle, the vast majority of the published GSTO1-1 assays are susceptible to light-based interferences (Table 2). This is especially true for those assays monitoring absorbance at shorter wavelengths (e.g., 4-NPG, MMA (V) reductase, and DHA reductase assays), 38 ACS Paragon Plus Environment

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as many conventional screening compounds have high sp2:sp3 ratios and conjugated electron systems which may absorb at 200-400 nm wavelengths94. The lack of detergent in many cases also allows for the possibility that reported GSTO1-1 inhibitors are simply inhibiting by aggregation. The use of orthogonal assays confirming initial bioactivity in some of the reports lends weight to compounds being useful inhibitors. Somewhat ironically, the presence of electrophiles in many of the reported hits is congruent with inhibition of a nucleophilic active site and is therefore supportive of direct inhibition of enzyme function. However, in the case of covalent inhibition, it is possible to have indisputable on-target engagement but also substantial concurrent off-target engagements, which must also be considered. Therefore, it would be highly informative to re-assess many of these compounds for generalized sources of assay interference, notably aggregation and nonspecific thiol reactivity.

Approximately one-half of the reports for GSTO1-1 inhibitors perform selectivity experiments based on proteomics (e.g., 5, 8, 20, 21). Most of these experiments were performed on the soluble proteome, and it is unclear if the reported inhibitors meaningfully engage targets in the remaining proteome or those proteins unamenable to fluorescent or MS analyses (i.e., poorly ionizable, low abundance). Additional biochemical (non-proteome) selectivity testing versus other GSTs, mammalian orthologues, and also a panel of unrelated targets (including those sensitive to electrophiles) would further address questions about target selectivity and also guide appropriate pre-clinical animal models. Additional studies that may be useful in 39 ACS Paragon Plus Environment


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addressing generalized assay interference and selectivity with respect to biological thiols would be counter-screens for GSH and coenzyme A (CoA) adducts, the former being especially relevant given the roles of GSH as a GSTO1-1 substrate.

Further specific details will be included where we subject hits to hit assessment, in the next section.


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Table 4. Summary of individual assay reports for small-molecule modulation of GSTO1-1. GSTO1-1 activity

Fluopol-ABPP

Readout

Fluorescence polarization

Cpd

4-NPG

Absorbance 305 nm, continuous

Reaction time, tempt (min, °C)

Non-GSH scavenging reagents

SE-Rh probe, 75 nM

30, 25

90, 25

NR

GSTO1-1, 1250

SE-Rh probe, 75 nM

30, 25

1200, 37

1 mM DTT

GSTO1-1, 1000

SE-Rh probe, 75 nM

30, 25

90, 25

NR

5, 6, 7

DPBS

GSTO1-1, 250

SE-Rh probe, 10 µM

30, 25

60, 25

8

100 mM Tris HCl, pH 8.0, 1.5 mM EDTA

GSTO1-1, 1000

CMFDA, 500 nM

30, 37

3


GSTO1-1, 10

4-NPG, 0.5

8


GSTO1-1, 90

11 12, 13 MMA (V) reductase (radiolabel) MMA (V) reductase (absorbance, enzyme-coupled)

Compound incubation time, temp (min, °C)

GSTO1-1, 1100

1, 2, 3, 4 In-gel fluorescence

[Substrate] (mM)

50 mM Tris HCl, pH 8.0, 150 mM NaCl 50 mM Tris HCl, pH 8.0, 150 mM NaCl 50 mM Tris HCl, pH 8.0, 150 mM NaCl

1, 2, 3, 4 5, 6

Gel-based competitive ABPPa

Buffer

[Enzyme] (nM)

100 mM Tris HCl, pH 8.0, 1.5 mM EDTA 100 mM Tris HCl, pH 8.0, 1.5 mM EDTA

Detergent, decoy proteins

Counter-screens

Orthogonal assays

0.01% Pluronics, NR 0.01% Pluronics, NR 0.01% Pluronics, NR

NR

NR

NR

35

NR

NR, NR

NR

NR

33

30, 37

1 mM DTT

NR, NR

NR

NR

36

30, 37

60, 25

10 mM BME

NR, NR

NR

4-NPG, 1

30, 37

NR, 37

1 mM DTT

NR, NR

NR

GSTO1-1, 18

4-NPG, 0.5

5, 37

5, 37

10 mM BME

NR, NR

NR

NR

57

GSTO1-1, 2

4-NPG, 0.5

30, 30

NR, 25

10 mM BME

NR, NR

NR

NR

58

NR

Gel-based competitive ABPP Gel-based competitive ABPP

Ref

Fluor, gel-based competitive ABPP Fluor, gel-based competitive ABPP

35

33

35

36

9

100 mM Tris HCl, pH 8.0

GSTO1-1, 290

GSH, 5; [14C]-MMA (V), 0.088

NR, NR

60, 37

NR

NR, NR

NR

NR

38


10


GSTO1-1, 0.3; GSH reductase, 0.8 U

GSH, 5; MMA (V), 18; NADPH, 0.25

NR, NR

NR, 37

NR

NR, NR

NR

NR

38

DHA reductase (absorbance)


17


GSTO1-1, 1000

GSH, 1; DHA, 0.25

NR, NR

10, 25

NR

NR, NR

NR

NR

46

Thioltransferase (absorbance, enzyme-coupled)


17


GSTO1-1, 1000; GSH reductase, 1 U

GSH, 0.5; 2-hydroxyethyl disulfide, 1; NADPH, 0.4

NR, NR

10, 25

NR

NR, NR

NR

NR

46

Intensity of radiation


Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

CDNB-GSH conjugation (absorbance)


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49

18

100 mM KPi, pH 6.5

GSTO1-1, 1

GSH, 1; CDNB, 1

60, 37

6, 25

NR

NR, NR

NR

NR

19

DPBS

GSTO1-1, 9

GSH, 200; CDNB, 100

NR, NR

6, NR

NR

NR, NR

NR

NR

50

NR, none reported. aAssay for recombinant GSTO1-1


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Hit assessment of reported GSTO1-1 inhibitors Physicochemical assessment of screening hits is useful to place any given compound in terms of how advanced and drug-like it is. The most interesting hits are generally those that are relatively more potent while being relatively smaller in size (typically assessed by ligand efficiency, LE) and less lipophilic (typically assessed by lipophilic ligand efficiency, LLE)95. Hits with fewer hydrogen bond donors (HBD) and a smaller polar surface area (PSA) are more likely to be cell permeable96-99 although a PSA that is too small has been linked to a propensity for downstream toxicity100. Relatively greater water solubility is also attractive, while recent evidence suggests preferred compounds for successful development are not excessively planar (measured using fraction of sp3 carbon atoms, Fsp3)94. Additional analyses include Abbott Physicochemical Tiering (APT), an aggregate score (“tier”) based on calculated physicochemical properties and structural features that have been directly linked to suboptimal solubility, cell permeability, as well as aggregation, the latter liability which is especially pertinent to early-phase drug and chemical probe discovery101. In Table 3 we list the compounds discussed along with these hit assessment metrics, color coded according to more acceptable (green), neutral (yellow) or less acceptable (red)102. We stress that these metrics are only a guide for potential optimizability towards orally available therapeutics in a very general sense, but we think they are useful to obtain an overall picture of how advanced or otherwise the field of GSTO1-1 inhibitor discovery and development is. In the discussion below, we allude only to metrics where there is a key point to be made.



Apart from physicochemical calculations, the presence of chemically-reactive moieties may be associated with likely bioassay promiscuity and hence limitations in utility as useful tool compounds. We have colored such compounds orange in Table 3.

Turning attention initially to compounds 1-4, physicochemical assessment reveals 1 and 2 to be favorable in many respects. The LE of 2 is remarkably high (> 0.54) as a result of its small molecular weight and relatively high potency. Such high LE may be useful for medicinal chemistry optimization, as it allows for the common increase of molecular weight throughout the lead generation process. For 3, its larger size and lipophilicity is less desirable as a screening hit while the large molecular size and polarity of 4 is similarly less attractive. Both 3 and 4 are binned in high APT tiers, which may coincide with higher tendencies towards nonspecific target engagement and poor solubility.

The fact that this grouping of hits contains some known pharmacology and even Food and Drug Administration (FDA)-approved drugs might appear, on the surface of it, to be an attractive feature and lead one to question the relevance of hit assessment exercises. For example, the H+/K+-adenosine triphosphatase (ATPase) inhibition of 1 (omeprazole) is used clinically for the treatment of gastroesophageal reflux disease and peptic ulcers, while 4 is an antibiotic that acts through inhibition of bacterial DNA-dependent RNA polymerase103, 104. However, their inhibition of GSTO1-1 is much less potent than the therapeutically-relevant target, and we argue such 44 ACS Paragon Plus Environment

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bioactivity should be re-assessed as if they were screening hits. The fact that they are already drugs should have less relevance especially if they are discovered using experimental conditions (i.e. compound concentrations) that markedly deviate from clinical use105. Indeed, 1 contains a redox-active sulfoxide and is chemically unstable, readily forming the bioactive thiol-reactive species in mildly acidic conditions in accordance with its clinical use106. Further, it is known to be a thiol-reactive screening hit80, 107 and has been reported to inhibit other unrelated targets108, 109. Likewise, 4 contains potentially reactive PAINS motifs such as hydroquinone105, 110 and has also turned up in unrelated screens111, 112.

Similarly, the reported biological activities for 2 is strongly suggestive of nonspecific chemical reactivity113-116, which is not surprising since it contains a reactive and potentially redox-active disulfide group117. It is very closely related to the FDA-approved drug disulfiram, which is itself a well-known thiol-reactive compound107,

118

. Unsurprisingly, 2 modulates a number of

off-targets when screened against the whole proteome instead of the purified GSTO1-1. Redox activity or electrophilicity in 1, 2, and 4 represent potential interference mechanisms for a reactive thiol-containing enzyme such as GSTO1-1. Oxidation of the GSTO1-1 active site thiol would abolish enzymatic function, as would covalent labelling by electrophilic compounds. It is uncertain how GSTO1-1 is being inhibited by these particular compounds and in the case of 1 and 2 it could simply be labelling via a disulfide bond. In the case of 4, the mechanism is more obscure but its activity is presumably insufficiently potent to warrant further investigation. Of 45 ACS Paragon Plus Environment


note, while the potency of 2 is superficially impressive, this alone does not support the notion that it is of any interest as a starting point for optimization.

In contrast, chloroacetamides 3 and 5-8 are likely to inhibit GSTO1-1 through covalent bonding of the active site thiol. Many physicochemical metrics for 5-8 such as cLogP, HBD#, HBA#, RTB, LE, and LLE show promising lead-like profiles. Additionally, all are relatively potent inhibitors of GSTO1-1, and appear selective. A drawback for 7 is the aforementioned chemical instability while 5 contains a nitro group, a toxicophore with mutagenic potential though it needs to be realized that nitro group toxicity is context-dependent and marketed nitro group-containing drugs are known119. Furthermore, the presence of a potential toxicophore is less relevant in early-stage drug and chemical probe discovery, provided there are appropriate risk-mitigation plans including replacement with less liable but acceptably efficacious bioisosteres120.

The chloroacetamide group in 3 and 5-8 merits special discussion. It has been shown that amongst arrays of electrophilic functional groups, chloroacetamides can be relatively less reactive yet selective for thiols and may be considered reasonable starting points for optimization in certain contexts121, 122. From this perspective, the chloroacetamide functionality in 5-8 does not pose an obvious selectivity problem. By contrast, the relative promiscuity of 3 could be driven by its great hydrophobicity and so this would be an example of how physicochemical properties could convert a more acceptable compound to a less acceptable compound even though inherent 46 ACS Paragon Plus Environment

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reactivity is the same123. Although there are no chloroacetamide-containing drugs currently approved for clinical use or undergoing investigation in clinical trials, this observation should not preclude such compounds from becoming drugs or exclude consideration of such compounds as useful biochemical tools if selectivity and other relevant properties are sufficient124. Notably, another enzyme using GSH as a cofactor, glutathione peroxidase 4 (GPX4), was also reported to be selectively inhibited by some small-molecules containing chloroacetamide groups125. Indeed, we have performed ALARM NMR on 5 as a counter-screen for nonspecific protein thiol reactivity. The compound did not perturb the La antigen conformation under standard testing conditions, suggesting it is not a grossly nonspecific reactive compound.25 Undoubtedly, such orthogonal assays will be useful in periodically assessing selectivity especially for compounds with reactive warheads.

Indeed, the potential utility of electrophilic compounds versus GSTO1-1 would represent an important vindication of one principle on which the NIH Molecular Libraries Program was founded, which was to allow reactive compounds into the HTS library as potentially useful molecular probes126. For example, Tsuboi et al. sought to deliberately identify an irreversible mechanism preferred GSTO1-1 inhibitor as a probe to investigate the role of GSTO1-1 in several biological pathways, rather than aim solely for a therapeutic candidate33. As a result, compounds with reactive functionalities which can be prone to be irreversible covalent binders were not eliminated from the screening deck. On the contrary, such compounds were enriched and 47 ACS Paragon Plus Environment


prioritized by the specific design of assay conditions and the cherry-pick among the primary hits in full-deck screening33.

Out of all the α-chloroacetamides, we would consider 8 as perhaps the most attractive hit in terms of its lead-likeness, notably its relatively low molecular weight, promising values of LE and LLE, as well as its favorable synthetic accessibility. As a final selection from the HTS campaign, 8 covalently binds to GSTO1-1 Cys32 with promising selectivity. Its inhibition of GSTO1-1 activity is confirmed by orthogonal assays including fluopol and gel-based competitive ABPP. Based on its potency, apparent selectivity, physicochemical profile, and reported mechanism of action, 8 may nevertheless be suited for use as a tool compound or even a starting point towards therapeutic candidature even though it possesses an α-chloroacetamide. Tellingly, α-chloroacetamides 5-8 have not been reported as hits for other targets based on search via SciFinder®, even though inaccessibility and hence lack of wider testing by others would not appear to be an obvious reason for this lack of observed bioactivity.

By contrast, compounds 9 and 10 most likely represent poorly tractable starting points with respect to GSTO1-1 inhibitors. For 9 and 10, considering their large molecular weights, high lipophilicities, and the presence of long flexible alkyl chains in their structures, they are highly suboptimal leads, particularly as activity is only in the low micromolar compound concentration range and such structures are likely to have surfactant and/or aggregation properties and could be 48 ACS Paragon Plus Environment

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nonselective and membrane-active as low micromolar compound concentrations. The high APT tiers of 9 and 10 would be consistent with a tendency to form colloidal aggregates. As further support of this point, the tocopherol chemotype has been identified by others to be a frequent hitter based on statistical analysis127, and the closely related tocopherol succinate (10) is reported active at low micromolar compound concentrations in a variety of different bioassays128-130.

Carnosic acid (11) represents another compound with poor odds of progressing to a viable chemical probe or drug targeting GSTO1-1, based on weak potency and behaviors consistent with aggregation. Like the tocopherols 9 and 10, it was tested in the absence of detergent, decoy proteins, and not subjected to common counter-screens for aggregation. It has been reported as a promiscuous inhibitor in an analysis of multiple (n = 11) mammalian DNA polymerases131, and also as an inhibitor of the β-catenin/ B-cell lymphoma 9 (BCL9) complex132. Both it and closely related carnosol possess the catechol motif, a prototypical PAINS and frequent hitter motif105, 110. Indeed, carnosol has also been reported to be a transient receptor potential ankyrin 1 (TRPA1) agonist with low micromolar activity133. Both carnosic acid and carnosol were also identified as potent C-C chemokine receptor type 5 (CCR5) receptor antagonists134. Although the physicochemistry of 11 is largely acceptable as a screening hit, its lipophilicity is relatively high (cLogP = 4.8). Moreover, its relatively weak inhibition of GSTO1-1 (62 µM) coupled with concerns about promiscuity would make us weary to further investigate this compound and derivatives. 49 ACS Paragon Plus Environment


Protoapigenones 12 and 13 physicochemically are largely acceptable as hits, with the exception of the lipophilicity of 13 (cLogP = 5.6) that is the penalty associated with its increased potency of 40 nM relative to 12 (1.6 µM). However, the major demerit points are incurred by the enone Michael acceptor, a known promiscuity moiety106. Although the potency of 13 is excellent, a thorough scan for promiscuity would need to be undertaken before further optimization is attempted. Indeed, 12 has been reported to be active in unrelated assays at low micromolar concentrations135,

136

. Interestingly, both 12 and 13 show steep Hill slopes in the available

concentration-response data, which may be suggestive of aggregation contributing to reported GSTO1-1 inhibition and worthy of a more thorough work-up for such interference (Table 5)137. The high APT tier of 13 is also consistent with these observations.

Sulfonylureas 14-16 are intriguing. As screening hits, their physicochemistry is generally acceptable by conventional metrics, and while the epoxide group in 14 and 15 is a classic covalent modifier, there is no evidence that these compounds are highly promiscuous by a conventional literature search via SciFinder®. As such, these sulfonylureas may be considered more optimizable, the aforementioned ambiguity about their mechanism of anti-inflammatory activity notwithstanding.

On the other hand, chemists would recognize locostatin (17) to be a highly reactive acyl transfer reagent and Michael acceptor which has rather obvious potential for off-target binding in the 50 ACS Paragon Plus Environment

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proteome. Indeed, it is known to be a promiscuous covalent modifier, reacting with greater than 30% of the 6,150 quantified cysteines from 2,900 proteins in human cancer cell lysate138. Hence, although its physicochemistry is generally acceptable by conventional metrics, it holds little promise as a starting point for optimization.

Carfilzomib (18) is an FDA-approved proteasome inhibitor for the treatment of relapsed/refractory multiple myeloma. Like 14 and 15, it too contains an epoxide warhead and labels its therapeutic target covalently, as it does for GSTO1-1 based on its Cys32 (and quite potently at 150 nM), which in this sense is an off-target. It has been shown to label a few other off-targets including Estrogen Receptor α, Organic Anion Transporting Polypeptides 1B1 and 1B3, albeit at low micromolar compound concentrations not otherwise expected in therapeutic contexts139, 140. For this reason, even though its physicochemistry and pharmacological profile makes it challenging as a hit for optimization towards GSTO1-1, as an intravenous anticancer injectable, these hit assessment metrics are less relevant.

CellTracker Green (19) is physicochemically favorable and although its molecular weight is relatively large for a screening hit (MW = 465), its potency (IC50 = 51 nM) leads to a respectable LE (0.30). It covalently labels GSTO1-1 Cys32 based on two-dimensional high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) analysis, and its ester groups which are labile to the esterase in vivo would likely need to be modified during 51 ACS Paragon Plus Environment


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optimization to render the bioactive portion more metabolically stable, if they are required for most potent activity. It is an unusual structure for a hit and at first glance it is difficult to know if the benzyl chloride group would be unworkably reactive. However, the literature suggests that this is indeed the case as 19 is prone to react non-specifically via its benzyl chloride with biological thiols including GSH141-143. Considering the high cytosolic concentration of GSH (approximately 8 mM)144, in most medicinal chemistry circles 19 would not be considered a sensible starting point for optimization as its GSTO1-1 inhibition would presumably be first deactivated by endogenous GSH.

Benzenesulfonyl ester 20 was designed as an electrophile, is physicochemically relatively unattractive (e.g., less promising tPSA, LogS, LE, and APT), and the evidence linking its weak cell activity to GSTO1-1 inhibition is tenuous. Although it should in principle inhibit GSTO1-1 activity, its inhibitory activity against the sole enzyme is not reported.

Covalent labellers 21 and 22 are physicochemically more favorable and belong to the α-chloroacetamide cohort of reported inhibitors. However, to-date there is insufficient evidence to suggest they are potent and selective GSTO1-1 inhibitors worthy of optimization. Additional assays with the sole enzyme and proteome are needed to more convincingly demonstrate their inhibitory activity and selectivity.


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Of note, compounds 20-22 are not associated with notable literature evidence of promiscuity but the presumed reason for this is that they are proprietary compounds and not more widely accessible for further testing. Therefore, an absence of indicting literature does not necessarily mean a compound is benign, but rather a reflection that few others have ever tested it in biological assays. Table 5. Selected concentration-response analyses of reported GSTO1-1 inhibitors. Compound 3 7 8 9 10 12 13 19 a

Assay 4-NPG Gel-based competitive ABPP 4-NPG MMA (V) reductase MMA (V) reductase 4-NPG 4-NPG CDNB-GSH conjugation

IC50 120 nM

# points 6

Estimated Hill slopea 2

Ref

21 nM

9

2

33

31 nM

7

1-1.5

36

35

2 µM

4

Indeterminate

38

4 µM

6

Indeterminate

40

1.6 µM 40 nM

5 5

>2 2

58

51 nM

5

1-1.5

50

58

Hill slopes were estimated by plotting reported concentration-responses in GraphPad Prism 7.0 and fitting curves to four-parameter variable

slope equation.

Future perspectives Advances in all aspects of biomedical research continues to allow for increasingly easier access to compound libraries, proteins for assay development, and the technology to rapidly, cheaply, and efficiently screen for small-molecule modulators. However, while compounds that appear to modulate target activity are relatively easy to discover, it is much harder to prove that such compounds are genuine on-target modulators that are suitable starting points for medicinal chemistry optimization towards useful tool compounds or therapeutic candidates. Ultimately, 53 ACS Paragon Plus Environment


comprehensive and logical structure-activity relationship (SAR) is a key arbiter that determines this. However, such an effort is resource-intensive and since it may not be necessary for publication of screening results, a natural consequence is a large volume of screening hit literature disclosing compounds of uncertain and at times problematic utility. Along with others, we have tried to help raise awareness of this issue110, 145-147, but the problem continues in a relatively unfettered manner.

It is particularly the case that for relatively unusual, new, or otherwise difficult targets, much of the literature reporting small molecule modulators lies within this category. For this reason, in this Perspective, we have chosen to review a protein target that might be considered to be relatively difficult to drug due to its open binding pocket and the high concentration of its endogenous substrate in cell, and in our review of the small-molecule modulator literature, we have deliberately focused on the integrity of hit discovery, triage, and assessment. We believe this approach might usefully guide the format of reviews for similarly difficult targets.

The protein we review is GSTO1-1, an enzyme with a reactive active site cysteine residue. What is clear from our literature analysis is that many of the reported modulators are electrophiles. Physicochemical properties, assay design, and concentration-responses analyses also suggest a handful are likely aggregators (i.e, 9, 10). In most cases, the evidence that compounds containing reactive groups behave as promiscuous modulators suggests reactivity will be excessive and 54 ACS Paragon Plus Environment

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unworkable, while in others the door remains ajar, in particular for some of the more polar chloroacetamides such as 8 and epoxides 14 and 15. The acceptable track record of epoxides in drug discovery suggests this group may be a warhead that is particularly worthy of consideration for inhibition of enzymes with active site thiols. However, in all cases, medicinal chemistry optimization is absent and such elaboration is required to ensure that SAR can be discerned amongst structure-interference relationships (SIR)86.

Carrying an inherited warhead through optimization is fraught and demands constant attention to off-target liabilities148, 149. Such continued testing for selectivity can be resource-intensive and often disheartening, and is usually not reported in most manuscripts. Screening a mutant GSTO1-1 enzyme where thiol reactivity has been abrogated might usefully remove the noise that otherwise muddies primary screening data, though the resulting loss of enzymatic activity would necessitate biophysical or target-binding assays to monitor target engagement. It would also be interesting to assess the overall susceptibility of recombinant GSTO1-1 to various nonselective electrophiles. This could estimate the overall likelihood of identifying relatively specific covalent inhibitors in any given screen versus GSTO1-1 and also other electrophile-sensitive targets. In other words, this could help address whether the existing screens were extremely lucky in the identification of their original hits, or whether nonspecific hits can be optimized into more useful, specific agents.



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Alternatively, there is precedent for warhead-containing compounds to be useful. However, many of the more viable targeted covalent modulators are developed and optimized with rigorous characterization of the covalent and non-covalent contributions of target engagement150, 151. Why is this important? Characterizing the covalent and non-covalent contributions allows for more informative SAR interpretation, thereby facilitating the weakest-allowable reactive warhead in efforts to mitigate off-target reactivity.

A potentially useful component for systemically studying covalent modulators of GSTO1-1 is determining the covalent and non-covalent contributions to target engagement. One potentially useful approach would be to more systematically study the effects of various warheads versus GSTO1-1 to inform warhead optimization121. Another approach involves a more rigorous evaluation of enzyme kinetics. Covalent inhibition can be modelled by Equation (1), where a compound (I) initially binds to a target (E) by non-covalent interactions (measured by inhibition constant, Ki), thereby forming an initial complex (E • I), which then forms a covalent bond with the target (measured by rate of enzyme inactivation, Kinact, or k2) to form a covalently-linked compound-target complex (E-I). These parameters are often assessed by measuring enzymatic activity as a function of time and compound concentration to determine Ki and Kinact.

(1) 56 ACS Paragon Plus Environment

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In general, most of the reports on GSTO1-1 inhibitors report IC50 values. Our literature review could not identify any reports of Ki and Kinact for reported GSTO1-1 inhibitors. This can be problematic when IC50 and Ki/Kinact values diverge because of time-dependent and other mechanistic effects, as well as experimental factors such as enzyme and substrate concentrations. One barrier to determining these parameters is the relatively higher concentrations of enzyme and substrate utilized for some of the GSTO1-1 biochemical assays (Table 4). This suggests new and more sensitive assays might be required in order to develop ways to determine Ki/Kinact values. Another potential barrier would be the potential for electrophilic compounds to react with GSH substrates. Potential strategies to resolve these technical barriers include reconfiguring existing assays to explicitly address studying time-dependent bioactivity, as well as the use of high-throughput MS assays, whose extraordinarily analytical sensitivities can facilitate decreases enzyme and substrate concentrations, though we note such instrumentation is still not standard in most HTS settings152-154.

More rigorous determination of Ki and Kinact may facilitate structure-based drug design where people can design-in favorable non-covalent interactions. Simultaneously, one could utilize published rate constants for model electrophiles and their reaction with biological thiols to combine improved non-covalent interactions with decreased electrophilicity to maintain or even increase overall potency, which should concomitantly increase selectivity155. However, 57 ACS Paragon Plus Environment


determination of Ki and Kinact in the specific context of GSTO1-1 would be a necessary step to rationally guide this process.

There is wider relevance of compounds reported as inhibitors of GSTO1-1. Those examples that are FDA-approved drugs such as 1, 4, and 18 or present in naturally-ingested materials such as 9 and 10 reflect a common hope that clinical progression of discovered actives could be rapid. The possibility that an existing FDA-approved drug could be repurposed for a new therapeutic use is a beguiling one that has garnered intense interest in recent times. A clear attraction is that such a compound is already highly advanced, having been shown to be efficacious to treat a given human affliction at doses deemed to be tolerable, and that is approved for clinical use in humans. Further, such an FDA-approved drug is usually associated with a large amount of formulation, pharmacological, safety, and toxicity data that can streamline repurposed clinical development.

However, drug repurposing is not without limitations, as concisely discussed by Nosengo156. If any structural modification or significant reformulation is required for improved performance, then the drug development process necessarily starts anew, from preclinical progression to clinical trials. Even if no modification is required, phase II and phase III trials may still need to be conducted for the new indication. If the dosing regimen required for efficacy in the repurposed indication is to be significantly altered from an approved regimen, additional trials may be necessary. In the absence of a patent position, traditional licensing partners may also be 58 ACS Paragon Plus Environment

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difficult to engage. That being said, clinical progression of a compound closely related to a known drug would likely be advantaged relative to a novel chemotype. In addition to this, intellectual property can be generated and protected through novel formulation strategies applied to the new indication.

Drugs, just like any screening compound, can influence biological pathways in a variety of non-specific and non-progressable ways at the micromolar compound concentrations typically used in screening105. Hence, potency is key, and a compound active at low micromolar concentrations should not be subject to any favoritism simply because it is a known drug or natural product.

Conclusion In summary, GSTO1-1 is an atypical glutathione transferase which features a catalytic cysteine residue in the active site, and the enzyme is involved in different pathological pathways. Approximately 20 compounds have been reported in the literature as inhibiting GSTO1-1, many of which feature reactive warheads that appear to target GSTO1-1 Cys32. Several of these compounds may represent viable starting points for medicinal chemistry operations, including compound 5-8, 14-16, and 18. Analysis of the reports shows many compounds were not subjected to common counter-screens for technology-related and generalized assay interference. Many were not tested by orthogonal assays and selectivity assays, though several were subjected 59 ACS Paragon Plus Environment


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to proteome-level chemoproteomic analyses that support promising selectivity. Several of the reported compounds have suboptimal physicochemical properties, which should be considered when interpreting reported bioactivity, potential tractability, and medicinal chemistry optimization. A major challenge to developing useful small-molecule inhibitors of GSTO1-1 will be optimizing the warheads for target selectivity. Future work may benefit from developing new assays more suitable to systematically profiling the covalent and non-covalent contributions to target engagement, as well as improving and performing additional counter-screens and orthogonal assays to better de-risk assay interference and assessing target specificity.

While our review is focused on GSTO1-1, we believe it serves as a useful template for literature reviews on those many biological targets that are relatively new or difficult to drug, where an increased emphasis of screening hit critical analysis, understanding of the assay technology and the potential for assay interference would be of general benefit.

Corresponding author information Philip G. Board: [email protected] Jonathan B. Baell: [email protected] Author contributions Wrote the manuscript: YX, JLD, AJO, PGB and JBB. Contributed with revisions: JLD, MGC, PGB, and JBB. Analyses of crystallography: AJO. 60 ACS Paragon Plus Environment

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Biographies Yiyue Xie graduated from China Pharmaceutical University in 2016 with a Bachelor of Science in Pharmaceutical Sciences. She is now a Ph.D. candidate under the supervision of Professor Jonathan B. Baell at the Monash Institute of Pharmaceutical Sciences. Her research mainly focuses on the development of GSTO1-1 inhibitors for the treatment of inflammatory conditions. Jayme L. Dahlin graduated with an M.D. from Mayo Medical School and a Ph.D. in Molecular Pharmacology and Experimental Therapeutics from Mayo Graduate School. He is currently Chief Resident in Clinical Pathology at Brigham and Women’s Hospital (Boston, MA) and a postdoctoral research fellow in the laboratory of Dr. Stuart L. Schreiber at the Broad Institute of Harvard/Massachusetts Institute of Technology (Cambridge, MA). His graduate and postdoctoral work have focused on chemical mechanisms of biological assay interference and post-HTS triage. Aaron J. Oakley graduated with a Ph.D. from the University of Melbourne. He has published on the structure and function of glutathione transferases for over 20 years. He is currently Associate Professor at the School of Chemistry, Faculty of Science, Medicine and Health, University of Wollongong. Marco G. Casarotto graduated with a Ph.D. from the Department of Pharmaceutical Sciences at the University of Melbourne. He later took up a Wellcome Trust postdoctoral position at the Biological NMR Centre, University of Leicester (UK) before being recruited to the John Curtin School of Medical Research (JCSMR) at The Australian National University (Canberra, 61 ACS Paragon Plus Environment


ACT). His current position is Associate Professor and group leader of the Biomolecular Interactions Laboratory at JCSMR. His long-term interests are in the structure/function relationship of biological systems in health and disease. Philip G. Board graduated with a Ph.D. from the Physiology Department at the University of New England. He has been Head of the Department of Molecular Medicine and Deputy Director of the John Curtin School of Medical Research at The Australian National University (Canberra ACT). His long-term interests have been in the structure, function, and genetics of the glutathione transferases and other enzymes involved in glutathione metabolism. He is currently Emeritus Professor in the ACRF Department of Cancer Biology and Therapeutics at the John Curtin School of Medical Research. Jonathan B. Baell graduated with a Ph.D. from the Pharmaceutical Science Department at the University of Melbourne. Since 2012, he has been a Professor in Medicinal Chemistry and Larkins Fellow at Monash Institute of Pharmaceutical Sciences, and Co-Director of the Australian Translational Medicinal Chemistry Facility. His research interests include design and synthesis of peptidomimetics, HTS and design of high quality HTS libraries, medicinal chemistry optimization in neglected, tropical and parasitic diseases, epigenetics and cancer, and targeted drug delivery. Acknowledgements Jayme L. Dahlin gratefully acknowledges Dr. Michael A. Walters for performing APT calculations and helpful discussions. This work was supported by Project Grant APP1124673 62 ACS Paragon Plus Environment

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from the National Health and Medical Research Council of Australia (NHMRC) to Philip G. Board, Marco G. Casarotto and Aaron J. Oakley. The NHMRC is thanked for Fellowship support for Jonathan B. Baell (2012-2016 Senior Research Fellowship #1020411, 2017- Principal Research Fellowship #1117602). Acknowledged is Australian Federal Government Education Investment Fund Super Science Initiative and the Victorian State Government, Victoria Science Agenda Investment Fund for infrastructure support, and the facilities, and the scientific and technical assistance of the Australian Translational Medicinal Chemistry Facility (ATMCF), Monash Institute of Pharmaceutical Sciences (MIPS). ATMCF is supported by Therapeutic Innovation Australia (TIA). TIA is supported by the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS) program.

Abbreviations used ABPP, activity-based protein profiling; AD, Alzheimer’s disease; ALARM NMR, A La assay to detect reactive molecules by nuclear magnetic resonance; APT, Abbott Physicochemical Tiering; ASC, apoptosis-associated speck-like protein containing a carboxy-terminal CARD; BCL9, B-cell lymphoma 9; BME, 2-mercaptoethanol; CCR5, C-C chemokine receptor type 5; CDNB, 1-chloro-2,4-dinitrobenzene; CFZ, carfilzomib; CLIC, chloride intracellular channel; CMFDA, 5-chloromethylfluorescein diacetate; CRID, cytokine release inhibitory drug; CYP27A1, Cytochrome P450 Family 27 Subfamily A Member 1; DDDC, diethylammonium diethyldithiocarbamate; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase; fluopol, 63 ACS Paragon Plus Environment


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fluorescence polarization; Fsp3, fraction of sp3 carbon atoms; GPX4, glutathione peroxidase 4; GSH, glutathione; G-site, glutathione-binding site; GSSG, oxidized glutathione; GSTP1-1, glutathione transferase pi-1; GSTO1-1, glutathione transferase omega-1; GSTO2-2, glutathione transferase omega-2; hLa, human Lupus antigen; H-site, hydrophobic binding site; ICP-MS, inductively-coupled mass spectrometry; IL-1β, interleukin 1β; JNK1, c-Jun N-terminal kinase 1; Kinact, rate of enzyme inactivation; LogS, logarithm of solubility; LPS, lipopolysaccharide; MDCK, Madin-Darby canine kidney; MLSMR, Molecular Libraries Small Molecule Repository; MMA, monomethylarsonate; NLRP3, nucleotide-binding oligomerization domain-like receptor family

protein

3;

NOD,

nucleotide-binding

oligomerization

domain;

4-NPG,

S-4-(nitrophenacyl)glutathione; PAINS, pan assay interference compounds; RAF, Rapidly Accelerated Fibrosarcoma; RKIP, Rapidly Accelerated Fibrosarcoma kinase inhibitor protein; RTB, rotatable bonds; SE-Rh, phenyl sulfonate-rhodamine; shRNA, short hairpin RNA; SIR, structure-interference relationships; TLR4, Toll-like receptor 4; tPSA, topological polar surface area; TRPA1, transient receptor potential ankyrin 1.

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