2.1. Assays

Tables 1a through 1d list all of the PubChem AIDs for the KLF5 inhibitor project. Descriptions of the assays follow the tables.

Table 1a

KLF5 PubChem AIDs: SRIMSC Assays (HTS & SAR).

Table 1b

KLF5 PubChem AIDs: Profiling Assays.

Table 1c

KLF5 PubChem AIDs: Assay Provider Cell-Based Assays.

Table 1d

KLF5 PubChem AIDs: Assay Provider Western Blot Assays (SAR).

2.2. Probe Chemical Characterization

The chemical structure of the probe was verified by analysis of its 400 MHz ¹H NMR spectrum (shown in Figure 2). The structure was also corroborated by its LC/MS molecular ion (calc for M+1: 385.1, found 384.9) as well as its expected fragmentation patterns. Purity was measured at >95% (LC/MS analysis, confirmed by analytical HLPC analysis; HPLC purity data is shown in Figure 3).

Figure 2

400 MHz ¹H NMR Spectrum of ML264 in CD₃CN.

Figure 3

HPLC Purity of ML264 >95%, 220, 254, and 280 nM, calculated purity 99.1%.

The solubility of the probe in PBS was determined to be 19 μM (solution used: 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic, pH 7.4, room temperature). This value is the mean of three determinations: 17, 19, and 21 μM. Solubility of the probe in the KLF5 assay buffer was determined to be 35 μM, based also upon three determinations (31, 31, 40 μM). Importantly, its solubility is fully adequate to provide the high potency seem in multiple cell-based assays (30–500 nM) and is also adequate for broad use as a biological probe to be used in a variety of aqueous-based media.

The probe has a half-life of >48 hours in PBS at room temperature, when tested at 10 μM concentration. No erosion of LCMS peak intensity was seen in the 2 day duration of the study.

The probe was found to be unreactive with excess glutathione, indicating that it is not a Michael acceptor under physiologically-relevant conditions. The compound was tested at 10 μM concentration in the presence of 100 μM GSH (10-fold excess) and no erosion of LCMS peak intensity for the probe was seen for duration of the study.

The compounds in Table 2 have been submitted to the SMR collection.

Table 2

Probe and Analogs.

2.3. Probe Preparation

Synthesis Scheme. The probe compound was prepared efficiently in a three-step procedure as shown in Figure 4. An amide coupling reaction of the commercially available cinnamic acid 1 and a suitably glycine methyl ester HCl salt 2 gave the N-acyl glycine ester 3 in 86% yield. Ester hydrolysis to the acid 4 followed by an amide coupling reaction using the commercially available amine 5 then gave the probe molecule, in 49% yield for the final two steps. Detailed experimental procedures are provided in Figure 4. The probe analogs and SAR compounds were prepared by an analogous procedure using available structural variants of reagents 1, 2, and 5.

Figure 4

Four Step Synthesis of ML264.

Alternative synthesis scheme. Because the final step of the route shown in Figure 4 is the coupling of a somewhat hindered secondary amine occurring in modest yield, the (Figure 4) “left-to-right” synthesis can be replaced by a “right-to-left” construction. In this approach the more difficult amide coupling reaction is accomplished first in higher yield using a more active acyl chloride 6. In this manner the probe compound can be efficiently prepared in a three-step procedure that allows for ready late-stage exploration of SAR in the cinnamoyl portion of the molecule. Briefly, as shown in Figure 5, a coupling reaction of acid chloride 6 with the commercially available amine 5 gives the protected glycine amide 7. Deprotection and acylation with cinnamoyl chloride 8 then gives the probe molecule. SAR analogs can be prepared by an analogous procedure using available structural variants of reagents 6, 5, and 8. This construction allows for more exploration in the cinnamide region, including heterocyclic replacements for the cinnamide group. Such structural variants are more challenging to access by the route used in Figure 4 for preparing ML264.

Figure 5

Alternate Synthesis Scheme.

Detailed experimental procedures for the preparation of ML264, (E)-3-(3-chlorophenyl)-N-(2-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)(methyl)amino)-2-oxoethyl)acrylamide. The synthesis scheme from Figure 4 was followed.

Step 1. Preparation of (E)-methyl 2-(3-(3-chlorophenyl)acrylamido)acetate

To (E)-3-(3-chlorophenyl)acrylic acid (1.0 g, 5.48 mmol) in dichloromethane (10 mL) was added anhydrous dimethylformamide (DMF) (0.08 mL, 1.0 mmol). This solution was cooled to 0°C and then oxalyl dichloride (0.57 mL, 6.53 mmol) was added dropwise. The reaction was allowed to warm to room temperature. After two hours at room temperature, the reaction mixture was re-cooled to 0°C and a mixture of the HCl salt of glycine methyl ester (1.38 g, 10.99 mmol) and diisopropyl ethyl amine (DIEA, 3.8 mL, 21.92 mmol) in dichloromethane (10 mL) was added slowly. The reaction was stirred at room temperature overnight. The solvent was removed in vacuo to obtain the crude product, which was purified by flash chromatography (AcOEt/Hex 10~100%) to obtain the title compound, 1.19g (86%). ESI-MS (m/z): 254, [M+1]⁺.

Step 2. Preparation of (E)-2-(3-(3-chlorophenyl)acrylamido)acetic acid

To a solution of (E)-methyl 2-(3-(3-chlorophenyl)acrylamido)acetate (1.553 g, 6.14 mmol) in MeOH (10 mL) was added 2.0 N NaOH (6.2 mL, 12.4 mmol). The mixture was stirred at room temperature for 1h, after which time analytical HPLC indicated that the reaction was complete. The mixture was acidified by with a 1N HCl solution. The solvent was removed in vacuo to obtain the crude which was used to the next step with no further purification. ESI-MS (m/z): 240, [M+1]⁺.

Step 3. Preparation of (E)-3-(3-chlorophenyl)-N-(2-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)(methyl)amino)-2-oxoethyl)acrylamide, ML264

To a mixture of (E)-2-(3-(3-chlorophenyl)acrylamido)acetic acid (31.5 mg, 0.13 mmol) in DMF (1 mL) was added DIEA (52 mg, 0.4 mmol) and HATU (50 mg, 0.13 mmol). The mixture was stirred for 5 min, and then 4-(methylamino)tetrahydro-2H-thiopyran 1,1-dioxide hydrochloride (26 mg, 0.13 mmol) was added. The reaction mixture was stirred at room temperature for 30 min. The completion reaction was monitored by analytical HPLC. The solvent was removed in vacuo to obtain the crude which was purified by prep-HPLC (Acetonitrile/MeOH(1:1)/water 40~100%) to obtain the title compound, 24.6 mg (49%). ESI-MS (m/z): 385, [M+1]^{+. 1}H NMR (400MHz, CD₃CN) δ 7.63 (s, 1H), 7.51–7.52 (m, 1H), 7.48 (d, J=16.0 Hz, 1H), 7.39–7.41 (m, 2H), 6.91–7.02 (m, 1H), 6.78 (d, J=16.0 Hz, 1H), 4.65 (m, 1H), 4.08 (d, J=4.8 Hz, 2H, accompanied by smaller d in a 2.2:1 ratio at 4.22 due to amide isomerism), 3.19–3.35 (m, 2H), 2.99–3.04 (m, 2H), 2.87 (s, 3H, accompanied by smaller s at 2.83 in a 2.2:1 ratio due to amide isomerism), 2.04–2.39 (m, 4H).

3.1. Dose Response Curves for Probe

Figure 6Human KLF5 promoter reporter assay reveals that probe ML264 has a potent inhibitory activity against KLF5 expression in DLD-1 4.9 cells

Figure 7DLD-1 cell viability assay shows that probe ML264 is potently cytotoxic to this human colorectal adenocarcinoma cell line

3.2. Cellular Activity

The probe potently halts DLD-1 viability (IC₅₀ = 29 nM) with high maximal effect (>90%). DLD-1 cells are human colorectal adenocarcinoma cells. ML264 has significant effects at submicromolar doses on other cell types as well, including HCT116 (human colorectal carcinoma), HT29 (human colorectal adenocarcinoma), and SW620 (human colorectal adenocarcinoma). The IEC-6 anti-target (a nontransformed rat intestinal epithelial cell line) is largely unaffected, with inhibition below 50% at the highest dose (Figure 8). While the anti-target curve is not flat, 50% inhibition occurs at ~100 μM. The left-shift of the target curves offers promise for useful specificity in the mode of action. Profiling assays (shown later) also further clarify target selectivity.

Figure 8

Cell-based IC50 curves for Probe ML264 in relevant cell types. See AIDs 2749 and 2750 for details.

Western blot analysis shows that the probe ML264 substantially lowers KFL5 expression levels in multiple cell lines, including DLD-1, HCT116, HT29, and SW620 cells (Figure 9). The probe is superior to its structural analogs (top panel) and also superior to the HTS hit CID 5951923 (bottom panel) and also the previous leads from round 1 SAR, CID 46931037 and CID 46931043 (bottom panel). While protein levels do not in all cases linearly track with antiproliferation data, the probe ML264 is clearly improved in all types over probe precursors, and small differences noted may relate to differential uptake or distribution of the probe by the cell lines in question.

Figure 9

Western blotting assays (representative blots) show Probe ML264 reduces KL5 protein levels. See AID 485336 (DLD1 Round0), AID 588617 (DLD1, Round 1), AID 588652 (HCT116 Round 1), AID 588660 (HT29 Round 1), AID 588661 (SW620 Round 1) for methods.

3.3. Profiling Assays

The cell-based assays used in screening support a clear mode of action for ML264: reduction of KLF5 expression. It should be noted that the precise molecular mechanism of action by which this is achieved is not established, though future studies will aim to ascertain the mechanism. To support the mode of action hypothesis, we studied the properties of the probe and its precursors.

Analysis of PubChem assay results for the initial HTS hit suggested that the hit (and perhaps the scaffold itself) is not assay-promiscuous, with a hit rate of 3.3% (less when KLF5-related assays are excluded). The probe’s lack of activity in validated counterscreens provided evidence of target-specific KLF5 activity as well. A number of kinases, however, are associated with the KLF5 pathway and could theoretically give cell-based assay results that would be falsely consistent with the intended mechanism of direct KLF5 inhibition. For this reason, the SRIMSC submitted the probe for targeted kinase profiling. It was found to be inactive against a selected panel of 47 kinases (Figure 10). Each individual kinase activity was measured at a ATP concentration equivalent to its Km value for each kinase, as duplicate in presence of 10 μM of the probe ML264, and in the presence of an additional 10-point, 3:1 serial dilution of Staurosporine as a positive control to verify IC₅₀ value. The 47 kinases screened were selected by the assay provider based upon their connection, in literature studies, to the KLF5 pathway. The lack of kinase activity, in combination with KLF5 western blotting data, demonstrates that probe ML264 is not a general kinase modulator.

Figure 10

Kinase Panel Screen for ML264 (no significant kinase activity was seen). See PubChem AID 588725 for data and methods.

To gauge selectivity in a broader sense, ML264 was submitted for analysis in a lead profiling screen with Ricerca. ML264 showed no significant inhibition (<<50%) at 10 μM vs. 67 protein targets of therapeutic and/or toxicological interest. These targets, all of which are human unless indicated, are listed in Figure 11.

Figure 11

ML264 Has No Known Off-target Activity, based upon a Lead Profiling Screen^® at Ricerca. Data/methods are available on request.

4.1. Comparison to Existing Art and How the New Probe is an Improvement

No prior art KLF5 inhibitors are highly effective in reducing KLF5 expression with suitably high mechanistic specificity. A few active compounds were identified by Dr. Yang and co-workers in a screen of the LOPAC collection using assay conditions that were subsequently optimized for our uHTS effort (12). As shown in Figure 12, each prior art structure has in all cases severe concerns with respect to mechanistic specificity, and in the case of all compounds besides ouabain, potency issues as well.

Figure 12

Prior art. None has potency, chemical tractability, stability, and mechanistic specificity.

Ouabain is a cardiac glycoside with notable KLF5 activity and emerged in our uHTS campaign as a KLF5 inhibitor. The ouabain family of molecules, while having in some cases impressive antitumor effects, is notorious for broad-spectrum cytotoxicity, including promiscuity in the NCI60 assay. Ouabain’s toxicity and polypharmacology makes it unsuitable for KLF5-specific probe development.

AG17, or 3,5-Di-tert-butyl-4-hydroxybenzylidene)-malononitrile, is a promiscuous KLF5 inhibitor, as is the related compound AG879. AG17 has been studied mostly for its ability to inhibit a number of tyrosine kinases. Its structure contributes to its high reactivity, since it is a very strong Michael acceptor due to two conjugated nitrile groups. In addition, it bears a free phenol and is thought to undergo redox chemistry. It also shows promiscuity in the NCI60 assay. For all of these reasons, AG17 is also unsuitable for KLF5-specific probe development.

Tamibarotene, also called retinobenzoic acid or Am80, is a synthetic retinoid that affects KLF5 levels by targeting an active transcriptional complex containing retinoic acid receptor RARα and KLF5. Its modest potency renders tamibarotene ill-suited for KLF5-specific probe development. ML264 is structurally dissimilar to Am80 and likely unrelated in mode of action, though RARα activity of ML264 has not yet been determined (discussed in future work).

Finally PI3K and MAPK inhibitors (e.g., Wortmannin, LY294002) show weak effects on KLF5, perhaps due to indirect effects upon the pathway. Their low potency renders PI3K and MAPK inhibitors ill-suited for KLF5-specific probe development. ML264 is structurally dissimilar to these families of inhibitors and likely unrelated in mode of action. Several MAP kinases were included in probe profiling (Figure 10) and no activity was seen. PI3K activity of ML264 has not been determined (discussed in future work), but is unlikely due to lack of structural elements thought necessary for PI3K activity.