Selective HePTP Inhibitors: Probe 1

Sergienko E, Bobkova E, Vasile S, et al.

Publication Details

Tyrosine phosphorylation plays a key role in signal transduction and the regulation of a broad set of physiological processes characteristic of multicellular organisms. Hematopoietic protein tyrosine phosphate (HePTP) is a tyrosine phosphatase expressed in hematopoietic cells with specificity for the dephosphorylation of Erk and p38 MAP kinases (MAPKs). It has been found that HePTP is often dysregulated in the preleukemic disorder myelodysplastic syndrome, as well as in acute myelogeneous leukemia. The identified probe ML119 (CID-1357397) selectively inhibits HePTP activity. Since this probe scaffold was discovered through its activity in the HePTP assays with small molecule substrates, it is proposed that this inhibition occurs through direct targeting of the HePTP active site. The probe inhibits through fast equilibrium as judged by the lack of apparent effect when pre-incubated with phosphatase proteins. Thus, small molecule inhibitors of HePTP would be useful as molecular probes for studying the mechanism of signal transduction and MAP kinase regulation. In addition, these probes may have therapeutic potential for the treatment of hematopoietic malignancies, such as acute myeloid leukemia, where HePTP has been reported to be overexpressed.

Assigned Assay Grant #: 1 X01 MH077603-01

Screening Center Name & PI: Burnham Center for Chemical Genomics & Dr John C Reed

Chemistry Center Name & PI: Burnham Center for Chemical Genomics & Dr John C Reed

Assay Submitter & Institution: Dr Tomas Mustelin w/Dr Lutz Tautz & Burnham Institute for Medical Research

PubChem Summary Bioassay Identifier (AID): AID-2085

Probe Structure & Characteristics

Scaffold 2: Imidazothiazolidinones

Image ml119fu1

Recommendations for the scientific use of this probe

Protein tyrosine phosphatases (PTPs), working with protein tyrosine kinases (PTKs), control the phosphorylation state of many proteins in the signal transduction pathways. HePTP is a tyrosine phosphatase expressed in hematopoietic cells and regulates the MAP kinases Erk and p38. It has been found that HePTP is often dysregualted in the preleukemic disorder myelodysplastic syndrome, as well as in acute myelogeneous leukemia. Small molecule inhibitors of HePTP will be useful as molecular probes for studying the mechanism of signal transduction and MAP kinase regulation, and may have therapeutic potential for the treatment of hematopoietic malignancies for example in acute myeloid leukemia where HePTP has been reported to be overexpressed.

1. Scientific Rationale for Project

Specific aims

The goal of this project was to identify small molecule compounds that were potent and specific inhibitors of HePTP.

Background and Significance

Tyrosine phosphorylation [1] is a key mechanism for signal transduction and the regulation of a broad set of physiological processes characteristic of multicellular organisms, such as integration of signal transduction pathways, decisions to proliferate, differentiate, or die, activation of large gene transcription programs, cell motility and morphology, and the transport of molecules in or out of cells. Tyrosine phosphorylation is also instrumental in the coordination of these processes among neighboring cells, for example within the immune system [2–5]. The importance of tyrosine phosphorylation in normal cell physiology is also well illustrated by the many inherited or acquired human diseases that stem from abnormalities in the protein tyrosine phosphatases (PTPs)[6–8] and protein tyrosine kinases (PTKs) that catalyze the reversible tyrosine phosphorylation of proteins. Severe phenotypes are also observed in many PTP or PTK knock-out mice, and in many cases the immune system is affected.


The hematopoietic phosphatase HePTP

HePTP [9,10] is a 38-kDa enzyme, which consists mainly of a PTP domain with only a short (~50 residues) N-terminal extension (Fig. 1). HePTP is expressed in bone marrow, thymus, spleen, lymph nodes, and all myeloid and lymphoid lineages and cell lines [9–11]. Studies from our laboratory have shown that transient expression of HePTP in T cells caused a clear reduction in TCR-induced transcriptional activation of a reporter gene driven by an NFAT/AP-1 element taken from the IL-2 gene promoter [12]. In contrast, a catalytically inactive C270S mutant of HePTP had no effect, suggesting that the PTP activity of HePTP was required for inhibition. HePTP also reduced the TCR-induced activation of the MAP kinases Erk1, Erk2, and p38α, but not of the N-terminal c-Jun kinase (Jnk). HePTP also did not affect the activity or phosphorylation of Mek, the upstream activator of Erk, and all examined tyrosine phosphorylation events upstream of Mek appeared intact. Furthermore, we found that HePTP bound specifically to Erk1, Erk2 and p38α in intact T cells and dephosphorylated the critical PTyr in their activation loop [13]. This reaction caused rapid inactivation of the bound Erk or p38. We mapped the region of HePTP responsible for the specific binding of Erk and p38 to amino acids 10 – 30 in the non-catalytic N-terminus of HePTP [13]. Moreover, it was reported [14,15] that the two related PTPs, STEP and PCPTP, also bind Erk and p38 through a short motif termed the kinase interacting motif (KIM), which is well conserved also in HePTP within residues 15 – 30. Thus, the binding and regulation of MAP kinases is common property of this subgroup of PTPs.

Figure 1. Crystal structure and aa sequence of HePTP.

Figure 1

Crystal structure and aa sequence of HePTP. In the sequence, the KIM is in blue, the PTP domain in green with the PTP signature motif underlined, and the phosphorylation sites in red. In the structure, colors indicate α-helices, β-sheets (more...)

It is now well established that HePTP associates through its KIM with the inactive unphosphorylated forms of Erk and p38 in resting leukocytes, at least in T cells and in K652 erythroleukemia cells [13,16–20]. This complex can be disrupted by phosphorylation at S23 in the KIM by cAMP-dependent kinase [16] or perhaps by phosphorylation at T45 and S72 by the bound MAP kinases [13]. Interestingly, TCR signaling also induces some S23 phosphorylation and dissociation of Erk and p38 from the inhibitory HePTP [19]. HePTP remains in the cytosol and apparently cannot reach activated Erk and p38 in the nucleus, which therefore are allowed to remain active. Some 30 – 60 min later, the transcription of the genes for several MKPs is induced and these proteins begin to accumulate in the nucleus, where they dephosphorylate both phosphoamino acids in the activation loop of Erk. This results in complete inactivation of Erk, which shuttles back to the cytosol and reassociates with HePTP. This is referred to as the “sequential phosphatase model” [21] and it is as an example of how different PTPs are used in a spatially and temporally ordered manner to control the extent, location, and duration of MAP kinase activation. However, reality is clearly even more complex in live cells, which also express many other MAP kinase phosphatases.

HePTP in hematopoietic malignancies

The HePTP gene is located on chromosome 1q32 [22], which is often found in extra copies (usually partial trisomy) in bone marrow cells from patients with myelodysplastic syndrome [23,24], which is characterized by disturbed hematopoiesis and an increased risk of acute leukemia. Amplification and overexpression of HePTP was reported in a case of acute myelogenous leukemia [22]. Conversely, deletions of 1q32 have been reported in non-Hodgkin lymphomas and chronic lymphoproliferative disorders [25]. These findings suggest that excess HePTP may correlate with reduced proliferation (in myelodysplastic syndrome) and loss of HePTP with increased cell proliferation and/or survival. The finding that the HePTP gene is transcriptionally activated in T cells treated with IL-2 [9,26] supports a connection with proliferation. Although mRNA levels also increased several fold upon stimulation of normal mouse lymphocytes with phytohemagglutinin, lipopolysaccharide, Concanavalin A or anti-CD3 [26], the HePTP protein was present in resting cells and its amount increased only moderately.

2. Project Description

a. Original goal for probe characteristics

No CPDP was written (or required) for this MLSCN phase of the project (cycle 2). However, the ideal probe would have a potency of 1 µM or better. The main goal was to identify compounds that inhibit HePTP and are selective against other phosphatases.

b. Information for each Assay Implemented and Screening Run

i. PubChem Bioassay Name(s), AID(s), Assay-Type (Primary, DR, Counterscreen, Secondary)

Table 1. PubChem Assay Summaries for Probe project.

Table 1

PubChem Assay Summaries for Probe project.

ii. Assay Rationale & Description

The primary HTS and dose-response confirmation assays (AID-521) were based on utilization of a small molecule substrate of HePTP, p-nitrophenol phosphate (pNPP), which is dephosphorylated by the enzyme and a number of other phosphatases to generate p-nitrophenol and phosphate. Each of the products could be detected independently. In our reaction, the phosphate product was measured with Biomol Green™ reagent. The resulting phosphomolybdylate complex with this malachite green-based indicator yields a deep green color. Any compound that inhibits the p-NPP-based reaction is expected to inhibit any other catalytic event of catalyzed HePTP.

In this assay 112,438 compounds (comprising the MLSCN library at the time of screening and DIVERset of Chembridge available at Burnham HTS core facility) were tested. The average Z′ for this assay was 0.8, the signal to background ratio was 6.4, the signal to noise ratio was 300 and the signal window was 12.3.

Assay materials
Table 2. Reagents used for the uHTS experiments.

Table 2

Reagents used for the uHTS experiments.

HePTP assay materials

  1. HePTP protein was provided by Dr. Mustelin (Burnham Institute for Medical Research, La Jolla, CA). The pNPP pellets were obtained from Sigma-Aldrich (S0942). Biomol Green reagent was purchased from BIOMOL Research Laboratories, Inc (AK-111)
  2. Assay Buffer: 50 mM Bis-Tris, pH 6.0, 2.5 mM DTT, 0.0125% Tween 20.
  3. HePTP working solution contained 6.875 nM HePTP in assay buffer. Solution was prepared fresh prior to use.
  4. pNPP working solution contained 1 mM pNPP in MQ water. Solution was prepared fresh prior to use.
  5. Vanadate working solution - 45 mM Na3VO4 in 10% DMSO

HePTP HTS protocol

  1. 4 uL of 100 µM compounds in 10% DMSO were dispensed in columns 3–24 of Greiner 384-well clear microtiter plates (781101).
  2. 4 uL of the following solutions were using the Multidrop bulk dispenser (Thermo):
    1. 10% DMSO column 1 (negative control)
    2. 45 mM Na3VO4 in 10% DMSO - column 2 (positive control).
  3. 8 uL of HePTP working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
  4. 8 uL of pNPP working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
  5. Final concentrations of the components in the assay were as follows:
    1. 20 mM Bis-Tris, pH 6.0, 1.0 mM DTT, 0.005% Tween 20.
    2. 2.75 nM HePTP (columns 1–24)
    3. 0.4 mM pNPP (columns 1–24)
    4. 9 mM Na3VO4 (column 2)
    5. 2 % DMSO (columns 1–24)
    6. 20 µM compounds (columns 3–24)
  6. Plates were incubated for 1h at room temperature.
  7. 40 uL of Biomol Green reagent was added to the entire plate using the WellMate bulk dispenser (Matrix).
  8. Plates were incubated for 30 mins at room temperature
  9. Absorbance at 620 nm was measured on the Envision plate reader (PerkinElmer).
  10. Data analysis was performed using CBIS software (ChemInnovations, Inc).
Rationale for confirmatory, counter and selectivity assays

The confirmatory assay for HePTP is based on O-methyl fluorescein phosphate substrate (Sigma-Aldrich, cat # M2629) that is hydrolyzed into highly fluorescent O-methyl fluorescein and phosphate products. This assay was preferred to the colorimetric assay since the progress curves of the reaction could be easily acquired and utilized for weeding out time-dependent inhibitors.

HePTP is a protein tyrosine phosphatase with specificity for dephosphorylation of Erk and p38 MAP kinases (MAPKs). Thus for future utility of the chemical probes, it is important to establish their selectivity against other phosphatases involved in regulation of MAPKs, particularly the ones with overlapping substrate specificity. We utilized two dual-specificity phosphatases, MKP-3 and VHR1 dephosphorylating Erk and Erk/Jnk kinases, respectively, to establish the selectivity of the probes. The assays for MKP-3 and VHR were based on the O-methyl fluorescein phosphate substrate, similar to an alternative assay developed for HePTP.

Both MKP-3 and VHR are targets of specific MLSCN/MLPCN projects assigned to our center. We opportunistically utilized them for counter screening the hits of the HePTP inhibitor discovery project.

iii. Summary of Results

The current probe originated from the historical HePTP project supported by the MLSCN grant X01 MH077603-01. Compound CID2861757 (MLS-0046352, SID-7977434,) was identified as a primary hit of the HePTP screen. Dry powder compound, CID1357397 (MLS-009081), was purchased from a commercial source for confirmation. After performing the counter screen of this HePTP hit in MKP-3 and later VHR assays, we realized that this molecule was very selective towards HePTP. The compound was active in a cell-based assay assessing the phosphorylation state of Erk MAPK in Jurkat cells. Commercial compounds were purchased for the SAR study. From the purchased compounds we were able to nominate CID1357397 (MLS-0090801) as a probe (see SAR section 2bi below). This selective HePTP inhibitor could provide an invaluable tool to study HePTP pharmacology and potentially lead to a discovery and development of HePTP-specific therapeutic agents.

Image ml119fu2

c. Probe Optimization

i. Description of SAR & chemistry strategy (including structure and data) that led to the probe

SAR was developed by the analog-by-catalog approach for this scaffold. Compounds were purchased to explore R1 and R2 (Figure 1). Ortho –COOH as R1 is preferred. Replacing the ortho –COOH at R1 with –NO2 (CID5341934,(MLS-0091942)) or –Cl (CID2300608 (MLS-0091956)) reduces the inhibition by more than 20 folds (Table 3). For R2, Bromide as a substituent contributes more to the inhibition than methyl substituent (CID5341943, (MLS-0091943)) and H (CID2243732 (MLS-0091939)).

Figure 1. Probe analogs R1 & R2 variations.

Figure 1

Probe analogs R1 & R2 variations.

Table 3. SAR studies on probe CID1357397 [ML119].

Table 3

SAR studies on probe CID1357397 [ML119].

3. Probe

a. Chemical name of probe compound

2-{5-[(7-bromo-3-oxopyridino[3′,2′-4,5]imidazo[2,1-b]1,3-thiazolidin-2-ylidene)methyl]-2-furyl}benzoic acid [ML119]

b. Probe chemical structure including stereochemistry

Image ml119fu9

c. Structural verification information of probe SID

Probe SID number is 26514203

Purity: >95% (HPLC-MS)

Image ml119fu10

ES-MS m/z 470 [M+H]

Image ml119fu11

d. PubChem CID (corresponding to the SID)

PubChem CID is CID1357397

e. Availability from a vendor

This probe is commercially available from vendor, Labotest, cat. No. LT00237558.

f. MLS#'s of probe molecule and five related samples that were submitted to the SMR collection

Table 4. Submission of Probe and 5 analogs to the MLSMR.

Table 4

Submission of Probe and 5 analogs to the MLSMR.

g. Mode of action for biological activity of probe

The reported probe compound CID1357397 (MLS-0090801) selectively inhibits HePTP activity. Since the probe scaffold was discovered through its activity in the HePTP assays with small molecule substrates, it is most likely that it inhibits via a direct targeting of the HePTP active site.. The probe compound inhibits through fast equilibrium as judged by no apparent effect of their pre-incubation with phosphatase proteins. No time-dependent inhibition is observed as demonstrated by the linear progress curves of the HePTP phosphatase reaction in the presence of various concentrations of the probe compound CID1357397 (MLS-0090801).

Figure 2. The progress curves are strictly linear, showing slowing down with incubation time.

Figure 2The progress curves are strictly linear, showing slowing down with incubation time

This is consistent with the absence of time-dependent inhibition of HePTP phosphatase reaction in the presence of the probe compound CID1357397 (MLS-0090801)

Cell-based activity and specificity evaluation of HePTP inhibitors

For the development of HePTP lead structures, membrane permeability and inhibition of HePTP activity inside intact cells was crucial for compound classes to being further considered. In T cells, HePTP is known to dephosphorylate the activation loops of the Erk and p38 kinases to terminate signaling via the T cell receptor (TCR). Therefore, bona fide HePTP inhibitors should affect TCR-induced activation levels of Erk and p38. To probe these phosphorylation sites with specific antibodies, we incubated Jurkat T cells with selected compounds at 40 µM concentration or vehicle (DMSO) at 37°C for 45 min (Figure 3). Cells were then TCR-stimulated (with OKT3) for 5 min, and reactions were stopped by adding lysis buffer. Immunoblotting was done with pErk and pp38 phospho-specific antibodies. An anti-phospho-MEK blot was included as a specificity control within the Ras-Raf-MEK-MAPK signaling cascade. MEK directly activates Erk and p38. Therefore, by verifying unchanged MEK activation, augmented pErk and pp38 levels should not be the result of a more active kinase upstream of Erk and p38. Antibodies towards Erk, p38, and MEK were used on stripped membranes in order to verify equal loading between lanes. Results (Figure 3) show that probe CID1357397 (MLS-0090801) and analogs CID5341934 (MLS-0091942), CID2258411 (MLS-0091944) and CID2300608 (MLS-0091956) augment both pErk and pp38 levels, while pMEK was not affected. In addition, the probe compound CID1357397, which had the lowest IC50 value (0.24 µM) in the in vitro assay with recombinant HePTP, was also subjected to a dose-response immunoblot experiment using the same assay format (Figure 4). Taking the slight differences in loading between the DMSO control and the 4 µM assay point into account, the effect of CID1357397 (MLS-0090801) on Erk and p38 activation peaks between 1 and 4 µM, indicating that a concentration that is 10 fold higher than the in vitro IC50 value yields the highest effect in cells.

Figure 3. Immunoblot analysis of HePTP inhibitors in Jurkat T cells.

Figure 3

Immunoblot analysis of HePTP inhibitors in Jurkat T cells. Cells were incubated with vehicle (DMSO, 0.2%) or inhibitors at 40 µM concentration for 45 min at 37°C prior to T cell receptor stimulation with OKT3 for 5 min. Cell lysates were (more...)

Figure 4. Dose-response immunoblot analysis of HePTP Probe compound CID1357397 (MLS-0090801) in Jurkat T cells.

Figure 4

Dose-response immunoblot analysis of HePTP Probe compound CID1357397 (MLS-0090801) in Jurkat T cells. Cells were incubated with vehicle (DMSO, 0.2%) or inhibitor at the given concentrations for 45 min at 37°C prior to T cell receptor stimulation (more...)

h. Detailed synthetic pathway for making probe

This probe is commercially available from vendor, Labotest cat. No. LT00237558

i. Summary of probe properties (solubility, absorbance/fluorescence, reactivity, toxicity, etc.)

The probe compound appear soluble in the assay conditions up to 100 µM concentration. Although specifically not tested, we have not observed any adverse effect of the compound on either fluorescent or colorimetric assay.

As the probe compound CID1357397 [ML119] (MLS-0090801) contains a carboxylic acid, it exhibited low solubility at pH 5.0 pH but good solubility at pH 6.2 and 7.4. It had high permeability at the lower pHs tested to low permeability at pH 7.4, as more of the carboxylate anion forms. It exhibits fairly high plasma protein binding (both human and mouse). It has low stability in both human and mouse plasma. But, it shows high stability in the presence of both human and mouse microsomes (determined by the exploratory pharmacology group). The probe compound has a LD50 >50µM towards Fa2N-4 immortalized human hepatocytes.

Table 5. Summary of in vitro ADMET/PK Properties of HePTP Inhibitor Probe.

Table 5

Summary of in vitro ADMET/PK Properties of HePTP Inhibitor Probe.

j. Probe properties

Table 6. Properties computed from Structure.

Table 6

Properties computed from Structure.

4. Appendices

a. Comparative data on (1) probe, (2) similar compound structures (establishing SAR) and (3) prior probes

See Table 3 above

b. Comparative data showing probe specificity for target

See Table 3 above

5. Bibliography

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