Natural products with protein tyrosine phosphatase inhibitory activity

Methods 65 (2014) 229–238

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Natural products with protein tyrosine phosphatase inhibitory activity Gavin Carr, Fabrice Berrue, Saranyoo Klaiklay, Isabelle Pelletier, Melissa Landry, Russell G. Kerr ⇑ Department of Chemistry, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3, Canada

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Article history: Available online 19 September 2013 Keywords: Protein tyrosine phosphatase PTP1B CD45 CDC25 SHP-1 SHP-2 MPTP LAR VHR Natural products

a b s t r a c t Protein tyrosine phosphatases (PTPs) play an essential role in maintaining the proper tyrosine phosphorylation state of proteins. Abnormal tyrosine phosphorylation has been implicated in diseases as diverse as type 2 diabetes, cancer, immune disorders and neurological disorders, and thus inhibitors of PTPs have been investigated as potential treatments of these diseases. Natural products are widely regarded to be privileged structures in drug discovery efforts, and are therefore a good starting point for the development of PTP inhibitors. Here we describe reported natural product PTP inhibitors as well as methods to screen for natural product PTP inhibitors using bioassay-guided fractionation. These methods are illustrated using the example of a family of bromotyrosine-derived PTP inhibitors isolated from two marine sponges. We also identify potential pitfalls and false-positives, in particular compounds that are oxidizing agents that react irreversibly with the PTP. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction The phosphorylation state of proteins is one of the most important mechanisms of protein regulation with profound effects on signaling pathways [1]. Phosphorylation of proteins typically occurs on the side-chain hydroxyl group of serine, threonine or tyrosine residues. Protein phosphorylation at tyrosine residues is controlled by the balancing actions of protein tyrosine kinases (PTKs), which phosphorylate proteins at tyrosine residues, and protein tyrosine phosphatases (PTPs), which dephosphorylate proteins at phosphotyrosine residues. Abnormal tyrosine phosphorylation has been implicated in many diseases, and PTPs have been investigated as possible targets for the treatment of type 2 diabetes [2], cancer [3], and immune disorders [4], among others [5]. The sequencing of the human genome has revealed that there are at least 107 genes encoding PTPs [6]. These PTPs are classified into four families, the largest of which are the class I cysteinebased PTPs. This class is subdivided into the ‘‘classical’’ tyrosinespecific PTPs, which are specific for phosphotyrosine residues, and the dual-specificity PTPs, which can dephosphorylate phosphotyrosine as well as phosphoserine and phosphothreonine residues. The ‘‘classical’’ PTPs are further subdivided into the transmembrane, receptor-like PTPs (RPTPs) and the soluble, nonreceptor PTPs (NRPTPs). The PTP superfamily shares the active-site

⇑ Corresponding author. E-mail address: [email protected] (R.G. Kerr). 1046-2023/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ymeth.2013.09.007

sequence motif (H/V)CX5R(S/T), and the cysteine-based PTPs all share a common catalytic mechanism [7]. In contrast to PTKs, for which commercially successful inhibitors such as imatinib have been developed, there is a lack of potent and specific inhibitors of PTPs. The highly conserved active site of PTPs and the large number of enzymes in this family makes the search for inhibitors that are selective for a particular PTP more challenging. Another challenge is that the active-site cysteine thiol found in cysteine-based PTPs is unusually acidic and is susceptible to oxidation in its deprotonated (thiolate) form [8]. Some of the PTP inhibitors reported in the literature were found to act by irreversibly oxidizing the active-site cysteine residue from a thiolate to a sulfonate moiety through the production of H2O2 [9–12], so it is likely that screening libraries of compounds or natural product extracts for inhibitors of PTPs will produce a large number of ‘‘hits’’ that act by an oxidative mechanism. Additionally, compounds with Michael acceptor systems can react covalently with the nucleophilic thiolate of the active-site cysteine as has been observed for some PTP inhibitors [13,14]. Oxidizers and Michael acceptors are often toxic and non-selective and are therefore not likely to be good drug candidates. Thus, the use of catalase to remove H2O2 is recommended when screening for PTP inhibitors [15], especially in cases where the inhibitors are suspected to contain chemical functionalities capable of acting as oxidizing agents. Alternatively, the active compounds can be tested against other enzymes containing catalytic cysteine residues such as papain. If the compound also inhibits other cysteine-based enzymes it is a good indication that it might be acting as a non-specific oxidant.

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order to prevent organ graft rejection and may also be useful in the treatment of autoimmune diseases.

2. Natural product inhibitors of PTPs 2.1. PTP1B 2.1.1. Role of PTP1B in disease When insulin binds to the insulin receptor, the tyrosine kinase activity of the insulin receptor is activated leading to autophosphorylation. In its phosphorylated (active) state the insulin receptor functions as a tyrosine kinase and phosphorylates substrates such as IRS-1, setting off a signal transduction cascade that eventually leads to localization of glucose transporters on the outer membrane of muscle and fat cells where they take up glucose from the blood [16]. The insulin receptor is probably the most important substrate for PTP1B, which dephosphorylates the insulin receptor thereby deactivating it and contributing to insulin resistance. A landmark study showed that PTP1B deficient mice had lower blood glucose levels and were more sensitive to insulin than wild-type mice. When these PTP1B mice were fed a high-fat diet they resisted weight gain and remained insulin sensitive, in contrast to the wildtype mice that gained weight and became insulin resistant [17].

2.1.2. Natural product inhibitors of PTP1B The role that PTP1B plays in insulin signaling has made it an appealing drug target for the treatment of type 2 diabetes, and it has attracted the lion’s share of attention in screening efforts looking for natural product PTP inhibitors. This is reflected in the over 300 reported natural product inhibitors of PTP1B, far more than for any other PTP. Natural product inhibitors of PTP1B were the subject of a recent comprehensive review, so this report will not attempt to describe all natural product PTP1B inhibitors and instead refers readers to that review [18]. Of particular note is the large proportion of flavonoids and other phenolics among natural product PTP1B inhibitors.

2.2. CD45 2.2.1. Role of CD45 in disease CD45, also known as leukocyte common antigen, is a family of RPTPs consisting of various isoforms that are found in high abundance on the surface of hematopoietic cells. In T cells, CD45 dephosphorylates and activates Lck and Fyn, both members of the Src family of PTKs, which are required for T cell activation in response to antigen binding to the T cell antigen receptor [19]. Consistent with its role as a positive regulator of the immune system, treating mice with a monoclonal antibody raised against the RB isoform of CD45 prevented renal allograft rejection [20]. Inhibiting CD45 is therefore one strategy to suppress T cell activation in

2.2.2. Natural product inhibitors of CD45 The first reported natural product CD45 inhibitor was dephostatin (1), which was isolated from a Streptomyces sp. and found to have an IC50 against CD45 of 7.7 lM [21]. Analogues of dephostatin were later found to have even more potent activity against other PTPs [22]. The alkaloids anonaine (2), roemerin (3) and nornuciferine (4) isolated from Rollinia ulei were found to have CD45 inhibitory activity with IC50 values of 17, 107 and 5.3 lM, respectively [23]. Phosphatoquinones A (5) and B (6) isolated from a Streptomyces sp. were reported to have CD45 inhibitory activity with IC50 values of 28 and 2.9 lM, respectively [24]. Phosphatoquinone B contains a quinone moiety and is approximately 10-fold more potent than phosphatoquinone A, so it is possible that it acts partly through an oxidative mechanism. Dihydrocarolic acid (7) and penitricin D (8) isolated from Aspergillus niger were reported to inhibit CD45 with IC50 values of 1.2 lg/mL (6.7 lM) and 2.3 lg/mL (27.4 lM), respectively, and had only weak activity against PTP1B [25]. However, both compounds contain a,b-unsaturated carbonyl moieties and would be expected to be good Michael acceptors. Finally, the natural product purpurin (9) was recently reported to inhibit CD45 with an IC50 value of 5.97 lM, and inhibited several other PTPs with comparable potency [26]. The structures of 1–9 are shown in Fig. 1. 2.3. CDC25 2.3.1. Role of CDC25 in disease The cell division cycle 25 (CDC25) phosphatases are dual-specificity PTPs with three isoforms in humans: CDC25A, CDC25B and CDC25C. The CDC25 family dephosphorylates and thereby activates cyclin-dependent kinases (Cdks) in a step that is required for cell cycle progression. CDC25A functions primarily at the G1S transition, while CDC25B and CDC25C are important for the G2-M transition [27]. CDC25s also play an important role in blocking cell cycle progression at the G1-S and G2-M checkpoints in response to DNA damage. When DNA damage is detected, the check point kinases Chk1 and Chk2 phosphorylate and thereby inactivate CDC25s leading to cell cycle arrest [28]. Overexpression of CDC25A and CDC25B has been found in a variety of cancer types and their expression is correlated with poor survival [29], making them an attractive target for the discovery/development of inhibitors [30]. 2.3.2. Natural product inhibitors of CDC25s The first reported natural product inhibitor of a CDC25 phosphatase was dysidiolide (10), which was isolated from the sponge

3

Fig. 1. Natural product CD45 inhibitors.

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231

Fig. 2. Natural product CDC25 inhibitors.

Dysidea etheria and inhibits CDC25A with an IC50 value of 9.4 lM [31]. Dysidiolide showed at least some selectivity for CDC25A over other phosphatases as it did not inhibit CD45, LAR or calcineurin at 12.4 lM, and showed weaker activity against CDC25B [32]. Dysidiolide also inhibited the growth of the A-549 human lung carcinoma and P388 murine leukemia cell lines, although it is not clear if that is a direct result of its CDC25A inhibitory activity. Coscinosulfate (11) was isolated from the sponge Coscinoderma mathewsi as a CDC25A inhibitor with an IC50 value of 3 lM [33]. Interestingly, the dimethyl-guanidine salt (12) and the related alcohol (13) were less active against CDC25A with IC50 values of 18 and 30 lM, respectively. A series of polyprenylated hydroquinones and furans isolated from three different sponges were reported to have inhibitory activity against CDC25A, but not against the Ser/Thr phosphatase PP2C-a [34]. Five sesterterpenoids (14–18) and the alkaloid fascaplysin (19) isolated from the marine sponge Thorectandra sp. showed inhibitory activity against CDC25B with IC50 values of 17 lg/mL (42 lM), 15 lg/mL (37 lM), 11 lg/mL (28 lM), 33 lg/mL

(85 lM), 1.6 lg/mL (4.0 lM) and 1.0 lg/mL (3.3 lM), respectively [35]. The same group reported an additional four diterpenoids from a sea anemone with CDC25B inhibitory activity. The most potent analog (20) inhibited CDC25B with an IC50 value of 1.6 lg/mL (4.2 lM), while the other three analogues showed only weak activity [36]. Arenicolsterol A (21) isolated from the marine annelid Arenicola cristata inhibited CDC25A and CDC25B with IC50 values of 0.69 and 0.27 lM, respectively, and also inhibited several other PTPs [37]. Several glycolipid natural products showed selective inhibitory activity against CDC25A over PTP1B, including woodrosin I (22) isolated from the stems of Ipomoea tuberosa, sophorolipd lactone (23) isolated from Candida bombicola and glucolipsin A (24) isolated from Streptomyces purpurogeniscleroticus. All three glycolipids inhibited CDC25A with IC50 values of 7, 45 and 2.2 lM, respectively, but were inactive against PTP1B [38]. More recently, the structurally unusual paracaseolide A (25) from Sonneratia paracaseolaris showed CDC25B inhibitory activity with an IC50 value of 6.44 lM [39], and toonapubesin G (26) from Toona ciliata var. pubescens inhibited CDC25B with an

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IC50 value of 2.1 lM [40]. The structures of 10–26 are shown in Fig. 2. Caulibugulones A–E (27–31) were found to inhibit all three CDC25s, and evidence suggests that they act at least in part through irreversible oxidation of the active-site cysteine to the corresponding sulfonate [41]. Several other quinone and quinone-like natural products are known to inhibit CDC25s including nocardiones A (32) and B (33) [42], halenaquinone (34), xestoquinone (35), adociaquionones A (36) and B (37), 3-ketoadociaquinones A (38) and B (39), tetrahydrohalenaquinones A (40) and B (41), 13O-methyl xestoquinol sulfate (42) [43], cryptotanshinone (43) [44], physcion (44), emodin (45), questin (46), chrysophanol (47) and rhein (48) [45]. While the mechanism of inhibition for compounds 32–48 has not been studied, it seems likely that they act through a similar oxidative mechanism. The structures of 27–48 are shown in Fig. 3.

sensitivity, so inhibitors of SHP-1 may be useful against type 2 diabetes [48]. Finally, SHP-1 is the likely target of the anti-Leishmania drug sodium stibogluconate, so other SHP-1 inhibitors may be useful against leishmaniasis [49]. In response to growth factors, SHP-2 activates the Ras/ERK pathway leading to cell proliferation and protection from apoptosis [50]. Consistent with its role as a positive regulator of cell cycle progression, overexpression of SHP-2 has been implicated in the progression of leukemia [51], neuroblastoma [52] and breast cancer [53], and gain of function mutations in the gene encoding SHP-2 have been observed in childhood leukemia [54,55]. Gain of function mutations in the gene encoding SHP-2 are also the cause of over 50% of the cases of Noonan syndrome, a disease whose symptoms include congenital heart disease, short stature, learning difficulties and abnormal facial features [56]. Inhibiting SHP-2 may be a viable strategy to treat Noonan syndrome, leukemia and other cancers where overactive or overexpressed SHP-2 is involved.

2.4. SHP-1 and SHP-2 2.4.1. Role of SHP-1 and SHP-2 in disease The SH2 (Src homology 2) domain-containing PTPs, SHP-1 and SHP-2, each contain two SH2 domains in addition to a phosphatase domain, which allows them to interact with receptor tyrosine kinases (RTKs). Despite their similarity in structure, SHP-1 is primarily a negative regulator of RTK signaling and cell growth and survival, while SHP-2 is primarily a positive regulator [46]. Since SHP-1 functions as a tumour suppressor and loss of function mutations in the gene encoding SHP-1 are associated with hematopoietic cancers, inhibiting SHP-1 would appear to be a poor strategy in cancer therapy. However, since SHP-1 is a negative regulator of the immune system, inhibitors of SHP-1 could activate the immune system to fight cancer [47]. Deficiencies in SHP-1 have also been associated with improved glucose tolerance and insulin

2.4.2. Natural product inhibitors of SHP-1 and SHP-2 To the best of our knowledge the first reported natural product inhibitor of SHP-1/SHP-2 is corosolic acid (49), which inhibits SHP1, SHP-2 and PTP1B with IC50 values of 24.56, 10.50 and 5.49 lM, respectively [57]. Somewhat surprisingly, the structurally similar compound oleanolic acid (50) was later found to enhance SHP-2 activity [58]. Tautomycetin (51) was originally isolated as an antifungal from Streptomyces griseochromogenes [59] and was later found to be a potent inhibitor of the Ser/Thr phosphatases PP1 and PP2A [60] and an immunosuppressant [61]. More recently, tautomycetin was found to inhibit SHP-1 and SHP-2 with IC50 values of 14.6and 2.9 lM, respectively, and it was proposed that the immunosuppressant activity of tautomycetin was due at least in part to its inhibition of SHP-2 [62]. Caffeic acid (52) and various fatty acids isolated from Angelica dahurica were also found to have

Fig. 3. Quinone and quinone-like natural product CDC25 inhibitors.

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moderate inhibitory activity against SHP-2 [63]. The structures of 49–52 are shown in Fig. 4. 2.5. PTPs from pathogenic bacteria 2.5.1. Role of bacterial PTPs in pathogenesis The bacterium Mycobacterium tuberculosis, the causative agent of tuberculosis, utilizes two PTPs, MptpA and MptpB, as virulence factors to suppress the host immune system [64]. MptpA and MptpB are required for the survival of M. tuberculosis in human hosts, so inhibiting MptpA and/or MptpB is one strategy to treat tuberculosis. Likewise, pathogenic Yersinia spp. evade the host immune system with the help of YopH, a PTP that is required for virulence [65]. Other pathogenic bacteria that utilize PTPs to inhibit the host immune system include Salmonella enterica, Listeria monocytogenes, Shigella flexneri, Legionella spp., Coxiella burnetti and Pseudomonas syringae [66]. Therefore, PTP inhibitors may be useful in treating a variety of bacterial infections. 2.5.2. Natural product inhibitors of bacterial PTPs The cyanobacterial hexapeptides brunsvicamides A-C (53–55) isolated from Tychonema sp. inhibit MptpB with IC50 values of 64.2, 7.3 and 8.0 lM, respectively [67]. Brunsvicamides A–C showed some selectivity towards MptpB, as they did not inhibit CDC25A, VHR, PTP1B, SHP-2 or MptpA at concentrations as high as 100 lM. Ascochitine (56), a polyketide isolated from the marine-derived fungus Ascochyta salicorniae inhibited MptpB with an IC50 value of 11.5 lM and showed weaker activity against PTP1B (IC50 = 38.5 lM) and CDC25A (IC50 = 69 lM) [68]. The new bromotyrosine alkaloids pseudoceramines A-D (57–60) were isolated along with the known bromotyrosine alkaloid spermatinamine (61) from the marine sponge Pseudoceratina sp. [69]. Pseudoceramine B and spermatinamine were found to inhibit YopH with IC50 values of 33 and 6 lM, respectively, and had antibiotic activity against Yersinia pseudotuberculosis with IC50 values of 40 and 4 lM, respectively. Recently, the novel sesterterpenoid asperterpenoid A (62) was reported as an inhibitor of MptpB with an IC50 value of 2.2 lM [70]. The structures of 53–62 are shown in Fig. 5. 2.6. Other PTPs 2.6.1. Role of other PTPs in disease The leukocyte antigen-related protein (LAR) is a receptor-like PTP that acts as a negative regulator of insulin signaling [71]. Overexpression of human LAR in mice causes insulin resistance and since LAR is often overexpressed in type 2 diabetics, inhibitors of LAR may be useful in the treatment of this disorder [72]. LAR overexpression has also been associated with breast [73] and thyroid [74] cancers, so inhibitors of LAR may find use in the treatment of cancer.

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The Vaccinia H1-related (VHR) protein is a dual-specificity PTP and functions as a positive regulator of cell cycle progression. Blocking expression of VHR using RNA interference leads to cell cycle arrest at the G1-S and G2-M transitions [75]. Overexpression of VHR has been associated with cervical [76] and prostate [77] cancers, making VHR an attractive target for the development of anticancer agents [78]. To the best of our knowledge, the only reported natural product inhibitor of LAR is illudalic acid (63), which was originally isolated from the fungus Clitocybe illudens [79] and later found to inhibit LAR with a Ki of 330 nM [80]. Illudalic acid was found to have good selectivity for LAR and the related enzyme PTPr over other PTPs. The natural product RK-682 (64) was isolated from Streptomyces sp. 88–682 and found to inhibit VHR with an IC50 value of 2.0 lM [81]. RK-682 was selective for VHR over CD45, against which it showed only weak activity (IC50 = 54 lM). Sulfiricin (65) was the first reported inhibitor of PTP1B (IC50 = 29.8 lM), but showed even more potent activity against VHR with an IC50 value of 4.7 lM [82]. Sulfiricin also inhibited CDC25A with an IC50 value of 7.8 lM. Stevastelins A (66), A3 (67), B (68), B3 (69), C3 (70), D3 (71) and E3 (72) were originally isolated from Penicillium sp. as immunosuppressants [83,84], and later found to inhibit VHR with IC50 values of 2.7, 3.6, 10.8, 13.7, 16.0, 1.7 and 13.6 lM, respectively [85]. Finally, the anthraquinone 73 inhibited VHR with an IC50 value of 3.0 lM, and showed weaker activity against PTP1B with an IC50 value of 38.0 lM [86]. The structures of 63–73 are shown in Fig. 6. 3. PTP assay 3.1. Buffers Enzyme reactions are performed in 50 mM HEPES buffer (pH 7.0) containing 3 mM dithiothreitol (DTT) and 1 mg/mL bovine serum albumin (BSA). The use of double-distilled water in order to minimize the presence of metal ions in the assay buffer is highly recommended. Since H2O2 can be generated in the presence of metal ions and DTT, thereby inhibiting the phosphatase activity of PTPs by oxidizing the catalytic cysteine [87], EDTA is added to the buffer at a concentration of approximately 1–2 mM in order to chelate any metal ions. 3.2. Determination of kinetics constants using DiFMUP or pNPP as substrate Phosphatase assays are performed using artificial substrates such as DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) or pNPP (p-nitrophenyl phosphate). The hydrolysis of DiFMUP is conducted in black 96-well plates (Corning) in a final volume of 100 lL at 25 °C and the reaction is monitored by measuring the fluorescence

Fig. 4. Natural product modulators of SHP-1 and SHP-2.

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3

3

Fig. 5. Natural product inhibitors of PTPs from pathogenic bacteria.

Fig. 6. Natural product inhibitors of LAR and VHR.

(excitation 358 nm, emission 455 nm). For the pNPP assay, a clear bottom 96-well plate (Corning) is used and the absorbance is monitored at 405 nm. All reactions are monitored over 10 min in 30-s intervals with a Varioskan plate reader (Thermo Electron) and rates are calculated using a non-linear least-square fitting procedure. The enzyme concentration used in the DiFMUP assay is chosen such that the reaction rate causes a change in fluorescence in the range of

5–20 FU/min. For the pNPP assay, the enzyme concentration is chosen to give a change in absorbance of 0.05 AU/min. The same selected concentration for each enzyme is used for all kinetic assays and also to determine the specific Km at various substrate concentrations using the Michaelis–Menten equation. In order to determine the specificity of an inhibitor against PTPs, the IC50 value is calculated. Inhibitors are dissolved in DMSO and a

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235

Fig. 7. Bromotyrosine-derived natural products from Aplysina spp.

serial dilution starting at 100 lM is made in assay buffer (1–2% DMSO final) containing the substrate at Km concentration. The reaction starts when the enzyme is added and the reaction is monitored as described above. IC50 values are derived by a sigmoidal dose–response (variable slope) curve using GraphPad Prism software. 3.3. Identification of non-selective inhibitors 3.3.1. Eliminating oxidizing effects using catalase In the presence of a reducing agent such as DTT, compounds that can act as oxidizing agents can produce H2O2 leading to inhibition of PTPs through oxidation of the active-site cysteine from a thiolate to a sulfonate moiety. One strategy to deal with oxidizing compounds is to add catalase directly to the assay buffer. Catalase breaks down H2O2 into water and oxygen and thus should prevent H2O2-mediated oxidation of the active-site cysteine residue. Addition of catalase to the assay at 500 U/mL abolished the inhibitory activity of a series of ortho-quinones that were acting through an oxidative mechanism [9]. If a compound loses inhibitory activity against PTPs in the presence of catalase, it is probably acting through an oxidative mechanism and is therefore not a good drug lead since it will likely be toxic. 3.3.2. Identification of non-selective inhibitors using a secondary assay with papain An alternative strategy to disregard non-selective PTP inhibitors that act through an oxidative mechanism is to employ a secondary

assay against papain. The advantage of this secondary assay is that it can also identify non-selective PTP inhibitors that act as electrophilic Michael acceptors that react irreversibly with the thiol of the active-site cysteine residue. Papain is a cysteine protease enzyme that can be oxidized and inactivated in the same manner as PTPs, and like PTPs it is also prone to inhibition by reacting with electrophilic Michael acceptors. This assay is performed in the same manner as the phosphatase assay, except that the substrate is the synthetic peptide Z-F-R-pNA (supplied by BACHEM) and the absorbance is monitored at 405 nm. If a compound inhibits papain activity in addition to possessing PTP inhibitory activity, it is likely that it does so either by oxidation of the active-site cysteine or by acting as a Michael acceptor and thus is not a good drug lead. A caveat here is that some natural product inhibitors with particularly large structures may be too large to access the catalytic cysteine of papain and lead to a false negative result against this enzyme. 4. Purification of natural product PTP inhibitors – a case study One of the challenges of natural product drug discovery is to differentiate between genuine biological activity and artifacts that interfere with elements of the assay [88]. For example, assay interference causing false-positive results were observed for fluorescent molecules or compounds that form aggregate structures sequestering the target enzyme. PTP enzymatic assays are also affected by oxidative molecules and are generally too sensitive for the screening of complex mixtures of natural products present in crude

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extracts. Therefore, fractionation steps using normal-phase or reversed-phase chromatography are highly recommended prior to the biological screening of extracts. Using the recent chemical investigation of two marine sponges (Key238 and Key284) as a case study, the following section describes an overview of the protocols used in our laboratory for the isolation, purification and structure elucidation of selective PTP1B inhibitors from marine invertebrates. More comprehensive protocols on the various strategies used for the fractionation and purification of natural products have been reviewed elsewhere [89,90]. 4.1. Purification methodology 4.1.1. Preliminary screening Extracts of a total of 300 marine invertebrates were subjected to the following processing and screening protocol. The preliminary screening involved extracting a small quantity of the bulk organism (5 g) in a DCM:MeOH (1:1) solvent mixture, followed by fractionating the extract on a C18 Sep-PakÒ using the following elution system: Water:MeOH (9:1), Water:MeOH (5:5), Water: MeOH (2:8), Ethanol, Acetone, DCM:MeOH (1:1), and MeOH. This fractionation separates the metabolites according to their polarity and allows for the discarding of very lipophilic or hydrophilic metabolites in which could interfere with the assay. The seven fractions were dried, labeled (Sample_fr 1–7) and evaluated in the preliminary assay. Samples Key238_fr3 and Key238_fr4 obtained from the marine sponge Aplysina fisturalis exhibited moderate activity (inhibition > 50% at 100 lg/ml), whereas fractions 3 and 4 from the marine sponge Aplysina sp. (Key284) exhibited PTP1B inhibition above 80% at the same concentration. From the LC-HRMS analyses of the active fractions using a Thermo Orbitrap Exactive mass spectrometer hyphenated to PDA and LT-ELSD Sedex 80, it appeared that a class of bromotyrosine-derived compounds was responsible for the observed PTP1B inhibitory activity of Key238 and Key284. 4.1.2. Scale-up extraction and C18 flash chromatography In order to build-up a small library of brominated metabolites, scale-up extraction of the whole organisms (Key238 and Key284) was performed using DCM:MeOH (1:1). The extracts were then fractionated over C18 as described in Section 4.1.1. The resulting samples were submitted for biological testing to ensure that the activity is located in the fractions previously identified during the small-scale screening. 4.1.3. Low pressure automated chromatography This second purification step aims to further fractionate samples often comprising hundreds of milligrams of material in order to obtain enriched fractions with amounts more suitable for highperformance liquid chromatography (HPLC) purification. The samples containing brominated secondary metabolites (as identified by LC-HRMS) were loaded onto a C18 RediSepÒ column and

separation was performed on an automated Teledyne Combiflash Rf200 low-pressure chromatographic system using a gradient from 5% MeOH in water to 100% MeOH over 30 min. The resulting fractions were monitored and collected into separate tubes based on their UV profiles.

4.1.4. HPLC purification HPLC purifications were carried out on a Thermo Surveyor HPLC system coupled with a Sedex 55 evaporative light-scattering detector (ELSD) or a Waters HPLC system equipped with a fraction collector and both UV/visible and ELSD detectors. The HPLC column and gradient conditions were adjusted for each fraction as necessary to give optimal separation. Three different HPLC columns were used in this study: a Gemini C18 column (10  250 mm, 5 lM), a Luna perfluorophenyl (PFP) column (10  250 mm, 5 lM) and a Luna Phenyl-Hexyl column (10  250 mm, 5 lM). Both isocratic and gradient elution systems were investigated using either MeOH or ACN as the organic solvent with or without the addition of 0.1% formic acid. The addition of 0.1% formic acid can help to sharpen peaks, especially when acidic functional groups are known to be present on the molecule of interest; however it should be avoided if the compound of interest is unstable in acidic solutions.

4.1.5. Structural elucidation Structural elucidation was undertaken using a variety of analytical techniques, such as LC-HRMS, as well as 1D and 2D NMR experiments recorded on a 600 MHz Bruker Avance III NMR spectrometer.

4.2. Results The chemical investigation of two taxonomically related marine sponges Aplysina spp. (Key 238 and Key 284) led to the purification of eleven known bromotyrosine-derived natural products (74–84, Fig. 7). Their structures were elucidated by analysis of spectroscopic data and by comparison with spectroscopic data previously reported [91]. Several other bromotyrosine-derived natural products are known to inhibit PTPs [69] suggesting that other members of this class of natural products may have PTP inhibitory activity as well. The pure compounds (74–84) were tested at a concentration of 50 lM against a panel of seven different PTPs: PTP1B, Tc-PTP, Sigma D1D2, SHP-1, MKPX, LAR D1D2 and PRL2 A/S (Table 1). Compounds 74 and 84 both exhibited selective inhibitory activity against the two non-receptor like PTPs: PTP1B and Tc-PTP, compound 82 showed moderate PTP1B inhibitory activity, while compound 83 exhibited broader activity against PTP1B, Tc-PTP, Sigma D1D2 and MKPX.

Table 1 Activity of PTP enzymes in the presence of 50 lM of compounds 74–84 (% of control). Compound

PTP1B

Tc-PTP

Sigma D1D2

SHP-1

MKPX

LAR D1D2

PRL2 A/S

74 75 76 77 78 79 80 81 82 83 84

65 99 98 98 73 77 77 83 68 41 44

53 88 111 85 88 88 80 80 74 34 53

89 98 87 101 84 94 85 92 79 66 78

107 134 127 102 74 106 85 112 121 72 89

92 105 88 89 87 98 91 93 95 69 94

125 120 121 115 117 116 106 115 113 95 117

102 100 101 112 84 93 92 106 96 105 103

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5. Concluding remarks Despite playing a crucial role in many biological processes that are implicated in human diseases such as diabetes and cancer, there are still no therapeutically available PTP inhibitor drugs. This is in stark contrast to the hugely successful PTK inhibitor imatinib and related drugs. While finding potent and selective inhibitors is proving to be more challenging for PTPs than for PTKs, all hope is not lost. The rich stereochemical properties and evolutionary history of natural products makes them ideal candidates in the search for inhibitory compounds that can distinguish between closely related biological targets such as the PTP family. It is important to consider whether active compounds exert their effects by classical reversible inhibition of the enzyme or by reacting non-selectively and irreversibly with the enzyme, as the later has implications for the potential toxicity of the compounds. Employing secondary screens using catalase to mitigate the oxidizing effects of compounds or assaying the compounds against other cysteine-based enzymes can help to overcome these challenges.

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