PHK (IC50 = 11 nM); PDK1 (IC50 = 300 nM); PKA (IC50 = 3.3 µM)

In MDR1 cell models, KT5720 (0-8 μM; 72 h) reverses multidrug activation [1]. In recently isolated channel DRG neurons, KT5720 (3 μM) inhibits HCN channel activation kinetics and reduces Ih. DRG neurons' excitability and intracellular Ca2+ levels are both lowered by KT5720 (3 μM) [2].

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Reversal of multidrug-resistance by KT5720 in MDR1 lymphoma cell model [1]
We have investigated the effect of KT-5720 on LM1/MDR mouse lymphoma cell line carrying and expressing a human MDR1 transgene. This cell variant is highly cross-resistant to several cytotoxic agents relative to its parental, drug sensitive, LM1 cell line. LM1/MDR cells are 46, 98, 137 and 178-fold resistant to VBL, ACT D, COL and DOX, respectively, relative to their LM1 parental cells.
Using chemosensitization plate assay, we measured the concentrations of KT-5720 that reversed resistance of LM1/MDR cells to various cytotoxic agents by 50% (IC50 values) at tolerable concentrations of 0.1, 0.3, 0.5 and 1.0 μg/ml to ACT D, VBL, DOX and COL, respectively. The IC50 values of KT-5720 were 1.7, 2.6, 3.0 and 3.3 μM in the presence of ACT D, DOX, VBL and COL, respectively. However, the IC50 values (concentration resulting in 50% inhibition of cell growth) to KT-5720 alone were 37 μM for both LM1 and LM1/MDR cells. Therefore, 50% toxicity value of KT-5720 was in the range of 11–22-fold higher than the 50% chemosensitization values towards the various cytotoxic agents.
As demonstrated in Fig. 1, the sensitization of LM1/MDR cells to COL by KT-5720 is dose dependent. The classical Pgp inhibitor, verapamil (VRP) is a standard chemosensitizer for the comparison of potency and mechanism of action for all subsequently discovered chemosensitizers. Moreover, VRP has demonstrated cooperative activity with other (but not all) chemosensitizers, including those currently under clinical trials. At low concentrations of KT-5720 and VRP, the inhibitory effect of each on the MDR activity is only partial. However, using their combinations indicated that both inhibitors of MDR act in a cooperative manner and their joined effects are accumulative (Fig. 1). Hence, KT-5720 can be included in a subgroup of chemosensitizers that have demonstrated cooperativity in their kinetics of inhibition. [1]

To confirm the biological activity ofKT5720, we measured the cellular accumulation level of the Pgp-fluorescent substrate, Rh123, by LM1/MDR cells in the absence or presence of KT-5720 (Fig. 2A). We also compared the effect of KT-5720 on the accumulation of Rh123 to the effect of other indole carbazole, close derivatives of KT-5720 (K252a and K252b). The structure of these derivatives is depicted in supplement figure (online). As shown in Fig. 2B, the cellular accumulation of Rh123 in LM1/MDR cells is similarly increased by KT-5720 and VRP (positive control chemosensitizer). This increase is approximately three-fold relative to the cellular accumulation of Rh123 in the absence of any Pgp-modulator. The cellular accumulation level of Rh123 in the presence of KT-5720 or VRP by LM1/MDR cells was similar to that measured in the parental, drug sensitive, LM1 cell variant in the absence (or similarly in the presence) of these modulators (Fig. 2C). However, the two other structural close derivatives of KT-5720 (K252a and K252b) had no significant effect on the accumulation of Rh123 by LM1/MDR cells and on the parental, LM1 cells (Fig. 2B and C, respectively).

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Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed in dorsal root ganglion (DRG) neurons, which are involved in diverse mechanisms that regulate DRG functions. Protein kinase A (PKA) is an essential kinase that plays a key role in almost all types of cells; it regulates the ion channel activity, the intracellular Ca(2+) concentration, as well as modulates cellular signals transduction. Nevertheless, the effect of PKA inhibition on the HCN channel activity in DRG neuron remains to be elucidated. Here we investigated the impact of PKA inhibition on the HCN channel activity and DRG neurons excitability. Our patch-clamp experiments both under whole-cell and single-channel conditions demonstrated that PKA inhibition with KT5720, a cell membrane permeable PKA-specific inhibitor, significantly attenuated HCN channel currents. Current clamp recording on freshly isolated DRG neurons showed KT5720 reduced overshoot amplitude and enhanced the threshold of the action potential. Moreover, our live-cell Ca(2+) imaging experiments illustrated KT5720 markedly reduced the intracellular Ca(2+) level. Collectively, this is the first report that addresses KT5720 attenuates the HCN channel activity and intracellular Ca(2+), thus reducing DRG neurons excitability. Therefore, our data strongly suggest that PKA is a potential target for curing HCN and DRG neuron relevant diseases. [2]

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The selective PKA inhibitor KT5720 attenuates Ih in freshly isolated rat DRG neurons [2]
The effect of PKA inhibition on DRG neuron Ih was investigated by the conventional whole-cell patch-clamp technique. At the holding potential of −40 mV, whole-cell Ih was elicited with a protocol that hyperpolarized from −60 to −140 mV in −10 mV interval, the hyperpolarize stimulation maintained for 2 s, then returned to the holding potential for 1 s. The cell capacitance was kept unchanged during the recording procedure. In the control condition, Ih was recorded without adding any compound, followed with perfusion with a 5 mL 3 μM KT5720 solution into the recording chamber, which dramatically attenuated the whole-cell Ih. At the end of recording, 10 μM ZD7288 (a selective HCN channel inhibitor) was added into the bath solution, in the presence of ZD7288, the remained inward current was totally inhibited (Fig. 1A). To confirm that the effect of KT5720 on Ih being attributed to PKA inhibition, the same experiments were carried out with H89, it had the same effect as KT5720 (Fig. 1B), which confirmed KT5720 attenuates Ih via PKA inhibition. Furthermore, to test whether KT5720 blocks Ih through HCN channel inhibition, ZD7288 was applied to DRG neurons in the absence of KT5720. The result was consistent with our hypothesis, ZD7288 blocked whole-cell Ih.

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HCN channel activation curve was leftward shifted and its current density was significantly reduced by PKA inhibition [2]
In the above paragraph, we already demonstrated that PKA inhibition with KT5720 induced a dramatic reduction of Ih. This inhibition not only influences the current amplitudes, but also has an impact on the channel active threshold. The aftermath is the HCN channel activation curve shifted to the negative direction. Those points were obtained from the tail current amplitudes following application of hyperpolarizing voltage steps, and were normalized to the largest one at −140 mV. The activation curves have been fitted with a Boltzmann function. As illustrated in Fig. 2B, the HCN channel V1/2 changed from −81.91 mV (control) to −100.85 mV (KT5720) (n=9, N=6). Furthermore, in the presence of 3 μM KT5720, the Ih density was declined as well. To ensure that the HCN channel has been fully activated, we chose −140 mV as the statistical analysis voltage, the current amplitudes averaged from the last 100 ms of the step-stimulation. Statistic results showed that the Ih density decreased from 16.82±1.77 (control) to 3.57±0.52 (KT5720) (n=6, N=5; ***p<0.005) (Fig. 2C).

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KT5720 (3 μM) slows down HCN channel activation kinetics [2]
Since KT5720 (3 μM) attenuated Ih amplitude and increased its activation threshold, thus shifting the V1/2 to more negative potentials. We further postulated that PKA inhibition with KT5720 can slow down Ih activation kinetics. Ih was elicited with a sustained −130 mV stimulation for 2 s. To explore the effect of KT5720 on Ih activation, we first compared the original recording between control and KT5720 (3 μM). The activation time constant was fitted with a monoexponential equation (I=Io+Ae−(x−xo)/τ), within which, τ is the time constant, x represents the time in ms, and I is the current. Our result demonstrated that Ih activation time constant was much longer in the presence of 3 μM KT5720 (Fig. 3A). Moreover, statistic results showed that the Tau value changed from 464.5±35.86 ms (control) to 4164.5±304.33 ms (KT5720) (n=11, N=7; ***p<0.005) (Fig. 3B).

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PKA inhibition with KT5720 (3 μM) reduced DRG neurons excitability [2]
At the end of AP, it was believed that Ih can depolarize the membrane potential to the threshold thus inducing a new AP (Accili et al., 2002). It also reported that the HCN channel selective inhibitor ZD7288 can attenuate DRG neuron excitability through inhibiting AP (Chaplan et al., 2003). We further hypothesize that KT5720 inhibiting Ih may also have an impact on DRG neuron excitability, therefore, current-clamp recordings were implemented on six rats as shown in Figure 4A. APs were evoked by a 2 s depolarize current, which confirmed our hypothesis. As demonstrated in Figure 4A, after application with 3 μM KT5720, the APs were profoundly inhibited. Furthermore, AP recording was also carried out in the presence of 10 μM ZD7288. As illustrated in Figure 4B, KT5720 did not have a significant effect under this condition, which confirmed that KT5720 attenuates APs via HCN channel inhibition. AP threshold was changed from −25.25±1.21 mV under control condition to −22.42±1.54 mV in the presence of 3 μM KT5720 (n=6, N=6) (Fig. 4C). Moreover, AP overshoot amplitude significantly decreased from 23.77±2.12 mV (control) to 6.93±1.39 mV (KT5720) (n=6, N=6; **p<0.01) (Fig. 4D).

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The effect of KT5720 on HCN single-channel properties [2]
To the best of our knowledge, no published literatures reported single-channel recording on freshly isolated DRG neurons so far. Therefore, to further reveal the mechanism of the PKA pathway on regulating HCN channel activation, cell-attached single-channel recording experiments were implemented as illustrated in Figure 5A. To eliminate irrelevant impacts, only those original gigaseal ≥10 GΩ were considered for further recording. After gigaseal formation, 15 more minutes were waited to ensure the seal was stable. Moreover, no rundown was observed under control condition, with 10 μM ZD7288 added to ensure the recorded currents were Ih (data did not show). The open probability has a voltage-dependent characteristic, which showed it increased from 0.21 at −50 mV to 0.91 at −140 mV, and the V1/2 was −82.18 mV (Fig. 5), which was consistent with our whole-cell recording result and the published report as well (Simeone et al., 2005). In the presence of 3 μM KT5720, the voltage-dependent characteristic of Po was totally dampened. At −90 mV, KT5720 inhibited Po for 71%, it also slightly attenuated the current amplitude from 1.06±0.18 pA (control) to 1.02±0.13 pA (KT5720). Moreover, before and after application of 3 μM KT5720, HCN channel conductance decreased from 12.41±0.69 pS to 11.54±0.76 pS, but except for Po neither the changes of current amplitude nor channel conductance were statistical significant (n=13, N=9) (Table 1). So, our data showed that PKA inhibition with KT5720 reduced HCN channel Po.

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PKA inhibition with KT5720 (3 μM) reduced intracellular Ca2+ concentration [2]
Previous study showed that the HCN channel activity was relevant to intracellular Ca2+ (Hagiwara and Irisawa, 1989), which hints PKA-induced Ih inhibition may be attributed to the change of the intracellular Ca2+ level. To explore the effect of KT5720 on DRG neuron intracellular Ca2+ level, real-time calcium imaging experiments were implemented on freshly isolated DRG neurons. Fura 2-AM (a ratiometric and UV light-excitable dye) was used as the intracellular Ca2+ indicator, the dual-wave excitation UV lights were generated with a TILL's Polychrome V light source. Recording coverslips were coated with poly-l-lysine to fix the cells. At first, the experiment presented in Figure 6A was performed. In the control part, great care was taken to ensure that no intracellular degradation occurred. As showed in Figure 6A, application of KT5720 (3 μM) profoundly reduced the intracellular Ca2+ level, similar results as illustrated in Figure 6A also were obtained from 12 other cells. The fluorescent ratio (340/380 nm) under control conditions was 0.80±0.05 (n=13, N=4). Thereafter, the fluorescent ratio decreased to 0.64±0.06 in the presence of 3 μM KT5720 (n=13, N=4; Fig. 6B). Thus, application of KT5720 reduces the DRG neuron intracellular Ca2+ level, so, PKA inhibition induced Ih attenuation, at least partly, due to reduction of intracellular Ca2+.

In the MDR1 bullet model, KT5720 (5 mg/kg; intraperitoneal; once daily for 8 days) totally reverses the effects of DNR [1].
KT-5720 (at 5 mg/kg) sensitized the bone marrow of MDR1 transgenic mice model towards daunorubicin (at 8 mg/kg) without general toxic effects[1].

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Reversal of multidrug-resistance of hematological malignancies by KT5720 ex-vivo [1]
Since KT5720 reversed resistance of LM1/MDR and other drug resistant cell lines, we have further evaluated its chemosensitizing effects on malignant cells from hematological patients in primary cell cultures (Fig. 3). In this study, we have collected bone marrow samples of two groups of patients with resistant hematological malignancies. The first group was from patients with CML-BC (N = 8; over 95% myeloblasts in the bone marrow) and the second group included MM patients (N = 6; over 85% malignant plasma cells in the bone marrow). The bone marrow samples were tested for RNA expression level of MDR1 and for cell survival in culture in the presence of DOX alone at 50 ng/ml (which is the therapeutically achievable cellular concentration in vivo) or in combination with KT-5720 at 5 μM. This concentration of KT-5720 was chosen since it was previously found most effective on various cell lines expressing similar or higher levels of MDR1 relatively to expression levels seen in primary samples. In these experiments cell counts in the absence of cytotoxic agent (DOX) were considered as control, 100% cell survival. It should be noted that incubation with KT-5720 alone had no effect on cell growth and survival of all samples (with values identical to the control, untreated samples, data not shown) indicating that KT-5720 alone had no cytotoxic activity at the tested dose.

As shown in Fig. 3, primary cells from all patients were resistant to DOX (range of 85–98% cell survival). In the presence of KT5720, however, cellular resistance to DOX was reversed in all multiple myeloma samples (range of 0–20% cell survival). MDR1 was respectively overexpressed in all these samples. Analyses of bone marrow samples from patients with CML-BC revealed that the resistance to DOX was reversed by KT-5720 in the cells of six out of eight patients. Yet, in cells of the two samples that KT5720 had no significant chemosensitizing effects, also MDR1 expression could not be detected. This indicates that an MDR1-unrelated resistance to DOX existed in cells of two out of eight CML-BC patients and that this resistance could not be reversed by KT-5720.

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Effects of KT5720 in vivo [1]
To evaluate whether KT-5720 can reverse MDR in vivo, we tested its activity on MDR1 transgenic mice model. These mice (overexpressing the human MDR1 gene in the bone marrow) are resistant to leukopenia induced by a panel of cytotoxic drugs. The effect of KT-5720 (alone or with DNR) on the bone marrow of normal mice versus MDR1 transgenic mice is shown in Fig. 4. Drug dosage (DNR) was selected in accordance with previous studies to induce a significant reduction (>50%) of leukocytes (WBCs) in normal mice within 5 days after administration and without causing generalized toxicity. At a dose of 5 mg/kg of KT-5720, the leukopenia induced by DNR in the MDR mice was equivalent to that measured in non-MDR mice treated with DNR alone (Fig. 4). This indicates that KT-5720 at this dose reversed completely DNR resistance in this animal model. Moreover, KT-5720 alone had no effect on the WBC counts of both normal mice and MDR-mice, indicating that KT-5720 alone has no toxic effects on the bone marrow at the dose needed to reverse DNR-resistance in MDR-mice.

Because first and second-generation chemosensitizers have pharmacological side effects in vivo, we tested whether KT5720 administration into mice demonstrate general toxicities in terms of survival of mice after double administration of DNR or KT-5720 alone or in combination as described in Section 2. This study indicated that KT-5720 was apparently not toxic at the concentration (5 mg/kg) needed to reverse drug resistance in MDR1 transgenic mice. Combination of KT-5720 and DNR had no additional toxicity to that of DNR administration alone (95% mice survival). Therefore, KT-5720 does not increase the toxicity to the cytotoxic agent (DNR) as reported for first-and second-generation Pgp modulators

PKA inhibition with KT5720 (3 μM) reduced intracellular Ca2+ concentration[2]
Previous study showed that the HCN channel activity was relevant to intracellular Ca2+ (Hagiwara and Irisawa, 1989), which hints PKA-induced Ih inhibition may be attributed to the change of the intracellular Ca2+ level. To explore the effect of KT5720 on DRG neuron intracellular Ca2+ level, real-time calcium imaging experiments were implemented on freshly isolated DRG neurons. Fura 2-AM (a ratiometric and UV light-excitable dye) was used as the intracellular Ca2+ indicator, the dual-wave excitation UV lights were generated with a TILL's Polychrome V light source. Recording coverslips were coated with poly-l-lysine to fix the cells. At first, the experiment presented in Figure 6A was performed. In the control part, great care was taken to ensure that no intracellular degradation occurred. As showed in Figure 6A, application of KT5720 (3 μM) profoundly reduced the intracellular Ca2+ level, similar results as illustrated in Figure 6A also were obtained from 12 other cells. The fluorescent ratio (340/380 nm) under control conditions was 0.80±0.05 (n=13, N=4). Thereafter, the fluorescent ratio decreased to 0.64±0.06 in the presence of 3 μM KT5720 (n=13, N=4; Fig. 6B). Thus, application of KT5720 reduces the DRG neuron intracellular Ca2+ level, so, PKA inhibition induced Ih attenuation, at least partly, due to reduction of intracellular Ca2+.

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KT5720 (3 μM) slows down HCN channel activation kinetics[2]
Since KT5720 (3 μM) attenuated Ih amplitude and increased its activation threshold, thus shifting the V1/2 to more negative potentials. We further postulated that PKA inhibition with KT5720 can slow down Ih activation kinetics. Ih was elicited with a sustained −130 mV stimulation for 2 s. To explore the effect of KT5720 on Ih activation, we first compared the original recording between control and KT5720 (3 μM). The activation time constant was fitted with a monoexponential equation (I=Io+Ae−(x−xo)/τ), within which, τ is the time constant, x represents the time in ms, and I is the current. Our result demonstrated that Ih activation time constant was much longer in the presence of 3 μM KT5720 (Fig. 3A). Moreover, statistic results showed that the Tau value changed from 464.5±35.86 ms (control) to 4164.5±304.33 ms (KT5720) (n=11, N=7; ***p<0.005) (Fig. 3B).

Cell Viability Assay[1]
Cell Types: LM1/MDR cells (carrying and expressing human MDR1 transgene)
Tested Concentrations: 0-8 µM
Incubation Duration: 72 hrs (hours)
Experimental Results: Sensitivity of LM1/MDR cells to colchicine in a dose-dependent manner way increased.

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Cell lines and cell culture [1]
The drug sensitive, LM1 cell line and its drug resistant, LM1/MDR cell variant were used for in vitro studies. LM1 is a mouse lymphoma cell line of BALB/c origin. LM1/MDR is a subline of LM1 cells that were transduced with retroviral vector containing the human MDR1 cDNA. These cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. COL at 700 ng/ml or as otherwise indicated was additionally supplemented to the incubation media of LM1/MDR cells. Cells were incubated at 37 °C in 5% CO2.

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Primary cell culture [1]
Bone marrow samples from patients with refractory CML-BC and MM were collected. Patients gave informed written consent prior to the sampling procedure. Mononuclear cells were isolated from bone marrow samples by Ficoll–Hypaque gradient centrifugation, washed with phosphate-buffered saline, and resuspended at 1 × 106 cells/ml in RPMI 1640 medium containing 10% fetal-calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells incubated in suspension at 37 °C in 5% CO2.

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RNA preparation and analyses [1]
Total RNA was isolated from bone marrow mononuclear cells by RNeasy Mini kit, according to the manufacturer instructions. RNA samples (10 μg each) were mixed with RNA sample loading buffer, electrophoresed on denatured 1% agarose gel and transferred to Hybond-N nylon filter for Northern blotting as described previously. Briefly, a 645 bp 32P-radiolabeled, PCR product was used as a probe for Northern analyses. For PCR, the plasmid pHaMDR was used as template and the primers were: 5′-AAGCTTAGTACCAAAGAGGCTCTG-3′ (sense); 5′-CTTCCCAGCACCTTCTAGT-3′ (anti-sense). Thirty-five cycles (1 min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C) were performed in Paltier Thermal Cycler, PTC-200 (MJ Research, MA, USA). Northern hybridization was performed using the MDR1 probe or β-actin nick-translated probe as previously described.

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Flow cytometric analysis [1]
The cellular uptake of the Pgp substrate, Rh123, by cells was measured as described previously. Briefly, 1 × 106 cells were pre-incubated in tissue culture medium in the absence or presence of chemosensitizer (20 μM) for 30 min at 37 °C. After additional incubation in the presence of Rh123 (0.5 μg/ml) for 1 h at 37 °C, cells were washed with phosphate buffer solution at 4 °C and the fluorescence of accumulated Rh123 was measured by FACS scan flow cytometer equipped with 488-nm argon laser. The green fluorescence of Rh123 was collected after a 530-nm band pass filter. Samples were gated on forward scatter versus side scatter to exclude cell debris and clumps and the intrinsic-fluorescence of the cells (blank, cells without exposure to Rh123) was subtracted.

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Cytotoxicity and chemosensitization plate assays [1]
The sensitivity of LM1/MDR cell line to cytotoxic agents and/or Pgp-modulators was determined in a monolayer growth assay as described previously. Briefly, 4 × 104 cells in drug-containing medium were plated in each well of a 96-well tissue culture plate in the absence or presence of various concentrations of cytotoxic drug and/or Pgp-modulator, as indicated. Seventy-two hours later, plates were washed with phosphate-buffered saline, stained with 0.5% (w/v) methylene blue in 50% methanol for 1 h, washed with H2O and air-dried. In this procedure, only surviving stained cells remain adherent to the plastic dish. The density of stained cells was measured by EDAS 290 Documentation and Analysis System. For each modulator, the IC50 (50% inhibitory concentration on cell growth) values were extracted from curves of cell survival measurements in the presence of various concentrations of modulator and a given (fixed) concentration of cytotoxic agent. For each concentration of chemosensitizer, the EC50 (50% effective concentration) values were calculated. EC50 is the concentration of cytotoxic agent that is needed for killing of 50% of the cells in the presence of a given concentration of chemosensitizer.

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Electrophysiological whole-cell patch-clamp recording [2]
Whole-cell currents were recorded with Axopatch 200B, Digidata 1440A, and pClamp 10.2 software (Molecular Devices, Union city, CA), signals have been filtered with an eight-pole Bessel filter 900CT/9L8L (Frequency Devices, Ottawa, IL).. Ten millimeter length borosilicate glasses were used to prepare for the patch pipettes. The pipettes were pulled with a Narishige PP-830 Two-step puller, and then polished with a Micro Forge MF-830 fire polisher to obtain a final resistance of 3–5 MΩ.

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Electrophysiological single-channel patch-clamp recording [2]
Single-channel recording was implemented in a cell-attached mode. Fire-polished borosilicate glass pipettes with a final resistance of 8–10 MΩ were chosen for recording, those seal resistance ≥10 GΩ were considered for data analyses, 10 μM ZD7288 was added to ensure that the recorded single-channel currents were Ih (data did not show). Only those rounds, shinny looking DRG neurons were chosen for experiments. All single-channel experiments were implemented at room temperature 25°C–26°C.

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Live-cell calcium imaging [2]
For intracellular Ca2+ imaging experiments, 250 μL poly-l-lysine was added to each 35-mm-diameter glass bottom dish, incubated at 37°C, poly-l-lysine was put on the coverslip and incubated for 1 h, then dishes were aspirated and washed with 2 mL sterile water three times. Cell suspension was added into the dishes, kept at room temperature for 30 min to let the cells adhere to the bottom of the dishes. The Fura 2-AM solution was diluted with a bath solution to a final concentration of 2 μM. The supernatant was then removed from the dishes, 250 μL of the Fura 2-AM working solution was added into the dishes, and stored in darkness at room temperature for 30 min. The Fura 2-AM working solution was then removed and cells were washed with a bath solution 3 times. Ca2+ imaging at ×40 oil objective was acquired with an OLYMPUS IX81 motorized inverted research microscope, the corresponding software was MetaFluor 7.7.2.0.

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Solutions and drugs [2]
The DRG tissue-containing solution has the following compositions (in mg/mL): DMEM 10 pH 7.4 with NaOH. The DRG digestion solution contains (in mg/mL) DMEM 10, type I collagenase 1, trypsin 0.5 pH 7.4 with NaOH. The whole-cell patch-clamp recording bath solution contains (in mM) NaCl 80, KCl 5, BaCl2 2, NaH2CO3 26, CoCl2 2, TEAC 40, CaCl2 1, 4-AP 4, CdCl2 0.2, D-Glucose 10 pH 7.4 with NaOH, 1 μM TTX was added to block irrelevant sodium channels. The whole-cell patch-clamp recording pipette solution contains (in mM) NaCl 5, K+-gluconate 135, ATP-Mg2+ 2, GTP-Na+ 0.4, EGTA 0.5, HEPES 10 pH 7.3 with KOH. The single-channel patch-clamp recording bath solution contains (in mM) potassium gluconate 100, KCl 50, NaCl 4, CaCl2 1, EGTA 5, HEPES 10, and glucose 10 pH 7.25 with KOH. The single-channel patch-clamp recording pipette solution contains (in mM) NaCl 140, KCl 5, CaCl2 2, MgCl2 1, HEPES 10 pH 7.4 with NaOH, plus 1 μM TTX, 10 mM TEA, 0.2 mM CdCl2, 10 μM picrotoxin was supplemented to block other voltage- and ligand-gated channels.

Animal/Disease Models: MDR1 transgenic mouse model [1].
Doses: 5 mg/kg
Route of Administration: intraperitoneal (ip) injection; one time/day for 8 days
Experimental Results: Daunorubicin-induced leukopenia in MDR mice was comparable to non-MDR mice treated with daunorubicin alone The measured results are the same.

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Evaluation of general toxicity of KT5720 in combination chemotherapy in mice [2]
Female BALB/c mice (8-week-old, 20–22 g body weight) were were divided into groups, 20 mice each and treated at day 0 and 10 with DNR, KT5720 or their combination. All treatments were via i.p. injection. Mice survival was monitored within 2 months post the second drug administration.

For in vivo use, stock solutions of KT5720 was diluted in 5% (v/v) dextrose in phosphate-buffered saline solution, at appropriate concentrations for intraperitoneal (i.p.) administration.

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Cytotoxicity and chemosensitization of primary malignant cells ex vivo [2]
Mononuclear cells were separated from bone marrow samples of patients with CML-BC or MM and incubated in 24-well plates at 37 °C. For each patient, cells at initial density of 2 × 105 cells/ml were incubated in the absence or presence of DOX, KT5720 or their combination. After incubation for 72 h, a sample of cells in suspension was stained with 0.4% trypan blue solution and viable unstained cells were counted by ultraplane Neubauer's hemocytometer. Cell counts of control, untreated samples were considered as 100% cell survival. The percentage of cell survival was calculated dividing cell counts of treated samples with cell counts of control, untreated samples.

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Test conditions [2]
Eight-week-old, female littermates were investigated. DNR was administered by i.p. injection in the lower right quadrant of the abdominal cavity. In chemotherapy/chemosensitization combination experiments, KT5720 was injected i.p. in the lower left quadrant of the abdominal cavity, 2 h before DNR administration. Drug and chemosensitizers such as KT5720 concentrations were adjusted in 5% dextrose-phosphate-buffered saline so that a maximum volume of 0.2 ml was injected per mouse.

[1]. In vitro and in vivo reversal of MDR1-mediated multidrug resistance by KT-5720: implications on hematological malignancies. Leuk Res. 2006 Sep;30(9):1151-8.

[2]. Novel role of KT5720 on regulating hyperpolarization-activated cyclic nucleotide-gated channel activity and dorsal root ganglion neuron excitability. DNA Cell Biol. 2013 Jun;32(6):320-8.

[3]. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J. 2000 Oct 1;351(Pt 1):95-105.

KT5720 is an organic heterooctacyclic compound that is 1H,1'H-2,2'-biindole in which the nitrogens have undergone formal oxidative coupling to positions 2 and 5 of hexyl (3S)-3-hydroxy-2-methyltetrahydrofuran-3-carboxylate (the 2R,3S,5S product), and in which the 3 and 3' positions of the biindole moiety have also undergone formal oxidative coupling to positions 3 and 4 of 1,5-dihydro-2H-pyrrol-2-one. It has a role as an EC 2.7.11.11 (cAMP-dependent protein kinase) inhibitor. It is an organic heterooctacyclic compound, a gamma-lactam, a tertiary alcohol, a carboxylic ester, a hemiaminal, a semisynthetic derivative and an indolocarbazole. It is functionally related to a K-252a.

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Multidrug resistance (MDR) due to over-expression of the MDR1 (ABCB1) gene and its P-glycoprotein (Pgp) product is an obstacle in the treatment of hematological malignancies. In this study, we have evaluated the potency of KT-5720 to reverse Pgp-dependent MDR in vitro and in vivo. KT5720 (but not its close derivatives, K252a and K252b) reversed multidrug resistance of LM1/MDR cell line at non-toxic concentrations and increased accumulation of rhodamine 123 (Rh123). KT-5720 significantly reversed MDR1-dependent resistance of primary malignant cells from patients with chronic myelogenous leukemia in blast crisis (CML-BC) and advanced multiple myeloma (MM). Moreover, KT-5720 (at 5 mg/kg) sensitized the bone marrow of MDR1 transgenic mice model towards daunorubicin (at 8 mg/kg) without general toxic effects. Therefore, KT-5720 can be considered as candidate for combination therapy in various hematological malignancies where Pgp activity is a major impediment for cure.[1]

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This study shows that KT5720 effectively reverse Pgp-dependent drug resistance in various hematological models. Therefore, this study provides support for developing KT-5720 as a potential candidate for future application in combination therapy against various hematological malignancies including CML-BC (not responding to imatinib), lymphoma and advanced MM. [1]

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Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed in dorsal root ganglion (DRG) neurons, which are involved in diverse mechanisms that regulate DRG functions. Protein kinase A (PKA) is an essential kinase that plays a key role in almost all types of cells; it regulates the ion channel activity, the intracellular Ca(2+) concentration, as well as modulates cellular signals transduction. Nevertheless, the effect of PKA inhibition on the HCN channel activity in DRG neuron remains to be elucidated. Here we investigated the impact of PKA inhibition on the HCN channel activity and DRG neurons excitability. Our patch-clamp experiments both under whole-cell and single-channel conditions demonstrated that PKA inhibition with KT5720, a cell membrane permeable PKA-specific inhibitor, significantly attenuated HCN channel currents. Current clamp recording on freshly isolated DRG neurons showed KT5720 reduced overshoot amplitude and enhanced the threshold of the action potential. Moreover, our live-cell Ca(2+) imaging experiments illustrated KT5720 markedly reduced the intracellular Ca(2+) level. Collectively, this is the first report that addresses KT5720 attenuates the HCN channel activity and intracellular Ca(2+), thus reducing DRG neurons excitability. Therefore, our data strongly suggest that PKA is a potential target for curing HCN and DRG neuron relevant diseases.[2]

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In summary, this is the first study that addressed the effect of PKA inhibition on the HCN channel activity and further elucidated the effect on DRG neuron excitability. Our whole-cell and single-channel electrophysiological data indicated that PKA inhibition robustly attenuated the HCN channel activity. We provided further evidence that KT5720 selectively inhibits PKA reduced the intracellular Ca2+ level and further reduced DRG neuron excitability. Thus, our data strongly suggests PKA is a pivotal mediator of the HCN channel activity and DRG excitability. Therefore, it may represent a potential pharmacological target for the treatment of HCN channel and DRG neuron-relevant diseases. [2]

C32H31N3O5

537.62

537.226

C, 71.49; H, 5.81; N, 7.82; O, 14.88

108068-98-0

454202

White to off-white solid powder

1.49g/cm3

715ºC at 760mmHg

386.2ºC

1.88E-21mmHg at 25°C

1.752

5.934

2

5

7

40

1060

3

CCCCCCOC([C@]1(O)C[C@H]2N3C4=CC=CC=C4C5=C3C6=C(C7=C5C(NC7)=O)C8=CC=CC=C8N6[C@@]1(O2)C)=O

ZHEHVZXPFVXKEY-RUAOOFDTSA-N

InChI=1S/C32H31N3O5/c1-3-4-5-10-15-39-30(37)32(38)16-23-34-21-13-8-6-11-18(21)25-26-20(17-33-29(26)36)24-19-12-7-9-14-22(19)35(28(24)27(25)34)31(32,2)40-23/h6-9,11-14,23,38H,3-5,10,15-17H2,1-2H3,(H,33,36)/t23-,31+,32+/m0/s1

hexyl (15R,16S,18S)-16-hydroxy-15-methyl-3-oxo-28-oxa-4,14,19-triazaoctacyclo[12.11.2.115,18.02,6.07,27.08,13.019,26.020,25]octacosa-1,6,8,10,12,20,22,24,26-nonaene-16-carboxylate

KT-5720; KT5720; KT 5720; KT-5720; 58HV29I28S; Hexyl (15R,16R,18S)-16-hydroxy-15-methyl-3-oxo-28-oxa-4,14,19-triazaoctacyclo[12.11.2.115,18.02,6.07,27.08,13.019,26.020,25]octacosa-1,6,8,10,12,20,22,24,26-nonaene-16-carboxylate; hexyl (15R,16S,18S)-16-hydroxy-15-methyl-3-oxo-28-oxa-4,14,19-triazaoctacyclo[12.11.2.115,18.02,6.07,27.08,13.019,26.020,25]octacosa-1,6,8,10,12,20,22,24,26-nonaene-16-carboxylate; (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester; KT 5720

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

MEthanol : ~5 mg/mL (~9.30 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)