Delanzomib

Myocyte-Damaging Effects and Binding Kinetics of Boronic Acid and Epoxyketone Proteasomal-Targeted Drugs

Brian B. Hasinoff1 · Daywin Patel1

Abstract
The proteasome inhibitors bortezomib, carfilzomib, and ixazomib, which are used in the treatment of multiple myeloma have greatly improved response rates. Several other proteasome inhibitors, including delanzomib and oprozomib, are in clini- cal trials. Carfilzomib and oprozomib are epoxyketones that form an irreversible bond with the 20S proteasome, whereas bortezomib, ixazomib, and delanzomib are boronic acids that form slowly reversible adducts. Several of the proteasome inhibitors have been shown to exhibit specific cardiac toxicities. A primary neonatal rat myocyte model was used to study the relative myocyte-damaging effects of five proteasome inhibitors with a view to identifying potential class differences and the effect of inhibitor binding kinetics. Bortezomib was shown to induce the most myocyte damage followed by delanzomib, ixazomib, oprozomib, and carfilzomib. The sensitivity of myocytes to proteasome inhibitors, which contain high levels of chymotrypsin-like proteasomal activity, may be due to inhibition of proteasomal-dependent ongoing sarcomeric protein turnover. All inhibitors inhibited the chymotrypsin-like proteasomal activity of myocyte lysate in the low nanomolar con- centration range and exhibited time-dependent inhibition kinetics characteristic of slow-binding inhibitors. Progress curve analysis of the inhibitor concentration dependence of the slow-binding kinetics was used to measure second-order “on” rate constants for binding. The second-order rate constants varied by 90-fold, with ixazomib reacting the fastest, and oprozomib the slowest. As a group, the boronic acid drugs were more damaging to myocytes than the epoxyketone drugs. Overall, inhibitor-induced myocyte damage was positively, but not significantly, correlated with their second-order rate constants.

Keywords Bortezomib · Ixazomib · Carfilzomib · Proteasome inhibitors · Cardiac myocytes · Cardiotoxicity

Introduction
The use of the FDA-approved proteasome inhibitors bort- ezomib, carfilzomib, and ixazomib (Fig. 1a) has revolution- ized the treatment of multiple myeloma and has greatly improved response rates. Two other proteasome inhibitors, oprozomib and delanzomib are currently undergoing clinical trials (Fig. 1a).
The citrate com- plex of ixazomib is an orally available boronic acid dipeptide prodrug which is hydrolyzed immediately upon administra- tion [1]. These drugs primarily target the chymotrypsin-like activity of the 20S proteasome [2, 3]. The proteasome is responsible for the degradation of intracellular proteins via the ubiquitin–proteasome system. Inhibition of proteasomal activity disrupts multiple cell signaling and other pathways, leads to cell cycle disruption, and induces apoptosis and active cell death [1]. Bortezomib, which contains a boronic acid moiety, has been shown to form an extremely strong, but slowly reversible, complex with Thr1 in the β5 subunit of the yeast 20S proteasome [1, 4, 5].
Both bortezomib and carfilzomib contain label warnings about possible cardiac toxicity that includes cardiac arrest and cardiac failure with no risk factors for decreased left ventricular ejection fraction (http://www.accessdata.fda. gov). Several recent reviews have compared the relative cardiotoxicity of the proteasome inhibitors [6–12]. A recent large phase III trial that compared carfilzomib head-to-head with bortezomib showed that carfilzomib has a higher risk of cardiovascular toxicity compared to bortezomib [13]. Many patients receiving proteasome inhibitor therapy for multiple myeloma may already have significant age-related cardiovascular disease risk factors and may also have had prior cardiotoxic therapy [11]. In addition, the natural his- tory of multiple myeloma can itself also lead to cardiovascu- lar events [11]. A large retrospective analysis of bortezomib use has also shown that it has no impact on cardiac risk ver- sus non-bortezomib-based treatment [14]. Ixazomib would appear to have a low incidence of adverse cardiac events associated with its use [11, 15–17]. However, because it is the most recently approved proteasome inhibitor there is more limited cardiac safety data available. There is, how- ever, a case report of ixazomib-induced cardiotoxicity [18].
In a previous study, we utilized a neonatal myocyte model to compare the myocyte-damaging effects of bortezomib and carfilzomib [19]. In this model, nanomolar concentrations of bortezomib and carfilzomib resulted in significant myo- cyte damage as measured by lactate dehydrogenase (LDH) release and induction of apoptosis as indicated by increases in caspase-3/7 activity [19]. Carfilzomib was shown to be less damaging than bortezomib in both these assays. Myo- cytes were determined to have high levels of chymotrypsin- like proteasomal activity. The high sensitivity of myocytes to bortezomib and carfilzomib was posited to be due to inhibition of ongoing proteasomal-dependent sarcomeric protein turnover. I have also compared the slow-binding inhibition of the chymotrypsin-like proteasomal activity of human 20S proteasome under a standard set of conditions in order to compare the kinetic and equilibrium binding proper- ties of bortezomib, carfilzomib, ixazomib, oprozomib, and delanzomib. Progress curve analysis [20, 21] was used to obtain second-order “on” (kon) and first-order “off” (koff ) rate constants, and equilibrium-determined and kinetically- determined inhibitor dissociation constants (KI) (Fig. 1b). In this scheme, the inhibition constant KI is given by the ratio koff/kon. Oprozomib was shown to inhibit the 20S proteasome activity with a second-order binding “on” rate constant that was 60-fold slower than ixazomib, the fastest binding drug [22]. Delanzomib was shown to dissociate from its complex with the 20S proteasome with a half-time that was more than 20-fold slower than ixazomib, the fastest dissociating drug [22]. In this study, we have compared the myocyte-damaging effects of five proteasome inhibitors and have used progress curve analysis to determine the relative second-order bind- ing rate constants (kon) for inhibition of myocyte lysate chy- motrypsin-like proteasomal activity. This was done with a view to relating these kinetic parameters directly to myocyte damage and their relative cardiotoxicity. The study was also carried out with a view to identifying potential class differ- ences in the myocyte damaging and hence the cardiotoxic effects of the boronic acids and the epoxyketones.

Materials and Methods
Myocyte Isolation, Culture, LDH Determination, and K562 Cell Growth Inhibition Assays
Ventricular myocytes were isolated from 2- to 3-day-old Sprague–Dawley mixed sex rats as described [19, 23, 24]. Briefly, minced ventricles were serially digested with colla- genase and trypsin in Dulbecco’s phosphate-buffered saline (pH 7.4) (PBS)/1% (w/v) glucose at 37 °C in the presence of deoxyribonuclease and preplated in large Petri dishes to deplete fibroblasts. The preparation, which was typically greater than 90% viable by trypan blue exclusion, yielded an almost confluent layer of uniformly beating cardiac myo- cytes by day 2. For the LDH release experiments, the myo- cyte-rich supernatant was plated on day 0 in 24-well plastic culture dishes (5 × 105 myocytes/well, 750 µl/well) in DF-15. On days 2 and 3, the medium was replaced with 750 µl of fresh DF-10. In order to lower the background LDH levels, on day 4, 24 h before the drug treatments, the medium was changed to DF-2, and again on day 5 just before the addition of drugs. The animal protocol was approved by the Univer- sity of Manitoba Animal Care Committee.
Myocytes were treated with the drugs indicated, for the times indicated. Delanzomib, ixazomib, and oprozomib were from Selleck Chemicals (Houston, TX). Bortezomib was from LKT Laboratories (St. Paul, MN), and carfilzomib was
from LC Laboratories (Woburn, MA). Other materials were from Sigma (Oakville, Canada), unless otherwise specified. By day 6, the myocytes would be essentially non-prolifer- ating [25]. Thus, starting on day 6 after plating, samples (80 µl) of the myocyte supernatant were collected every 24 h for 3 days after treatment. The samples were frozen at
– 80 °C and analyzed within 1 week. Directly after the last supernatant sample was taken the myocytes were lysed with 250 µl of 1% (v/v) Triton X-100/2 mM ethylenediamine- tetraacetic acid (EDTA)/1 mM dithiothreitol (DTT)/0.1 M phosphate buffer (pH 7.6) for 20 min at room temperature as previously described [19, 24]. The total cellular LDH activity was determined from the activity of the lysate plus the activity of the three 80 µl samples previously taken. The LDH activity was determined in quadruplicate in a spec- trophotometric kinetic assay in 96-well plate format in a Molecular Devices (Menlo Park, CA) plate reader as previ- ously described [19, 24]. The spectrophotometric 96-well plate cell (5 × 104 cell/ml, 0.1 ml/well) growth inhibition 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium, MTS CellTiter 96 AQue- ous One Solution Cell Proliferation assay (Promega, Madi- son, WI), which measures the ability of erythroleukemic K562 cells to enzymatically reduce MTS, has been described previously [19, 24] as has the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide, MTT assay [26] for the human colon cancer HCT116 cells. The IC50 values for cell growth inhibition were measured by fitting the absorbance- drug concentration data to a four-parameter logistic equa- tion as we have described [19, 24]. Untreated myocyte cell lysates prepared and stored as described above were also used in determining the concentration dependence of pro- gress curves for the slow-binding kinetics of the inhibition of lysate proteasomal activity by the five proteasome inhibitors.

Proteasomal Enzyme Inhibition Assays
The effect of the proteasome inhibitors on the proteasomal activity of myocyte lysate was determined by measuring the inhibition of the increase in the fluorescence (λEx 340 nm, λEm 460 nm) produced by the cleaved N-succinyl-Leu-Leu- Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC; Enzo, Farmingdale, NY) substrate for chymotrypsin-like activity, basically as we and others have described [2, 19, 22, 27, 28]. The freshly thawed aliquoted lysate, which was kept on ice, was added to a black 96-well plate containing reac- tion buffer [20 mM 4-(2-hydroxyethyl)piperazine-1-ethane- sulfonic acid (HEPES), 0.5 mM EDTA, pH 8.0, 0.035% SDS]. The reaction was started by adding Suc-LLVY-AMC in DMSO (60 µM final) in a final volume of 100 µl. After allowing the reaction to proceed and equilibrate for 8 min,
1µl of proteasome inhibitor in DMSO was added in tripli- cate and the fluorescence increase was followed with time
for 2 h on a BMG Labtech (Cary, NC) Fluostar Galaxy fluo- rescence plate reader at 30 °C.

Analysis of Slow‑Binding Inhibition Progress Curves
As we and others have described, slow-binding inhibitors display progress curves with three phases [19–22]: an ini- tial linear phase that extrapolates to a slope of v0 at t = 0; a final linear phase with a slope vS at long times; and an exponential phase that connects the two linear phases with a pseudo-first-order rate constant kobs. For an inhibitor that forms a covalent bond, ultimately resulting in 100% inhibi- tion, vS approaches 0 at long times, provided the substrate is not significantly depleted over the time course of the assay, the time-dependent product formation P for a substrate that exhibits slow-binding inhibition can be described by Eq. 1 [20–22]. If the concentration of the inhibitor I is similar to the concentration of the enzyme E, Eq. 1 is modified to give Eq. 2 with γ being defined by Eq. 3 [21].
boronic acid inhibitors, bortezomib, ixazomib, and delan- zomib, form slowly reversible covalent bonds in which the enzyme–inhibitor complexes can slowly dissociate with a first-order dissociation rate constant koff . In order to reduce the number of adjustable parameters in the curve fitting the progress curves for the boronic acid inhibitors, the value of v0 in this equation was fixed to the linear least squares calculated value from the DMSO control.

Results
Comparison of Proteasome Inhibitor‑Induced Damage to Neonatal Cardiac Myocytes
Using the % LDH release assay [19, 23, 24], we compared the ability of proteasome inhibitors to damage myocytes after 72 h of either continuous (Fig. 2a) drug treatment, or after a 4-h drug treatment (Fig. 2b). The 4-h drug treatment
If a slow-binding inhibitor inhibits the enzyme in a fast equilibrium prior to a subsequent slower inhibition, the v0 term would vary with the inhibitor concentration [20, 21]. But if there is no fast prior inhibition, or if the concentration of the inhibitor is too low to significantly bind, v0 would be independent of the inhibitor concentration. In a previ- ous study using purified human 20S proteasome, we showed that v0 was independent of inhibitor concentration (up to 15 nM, for all inhibitors but oprozomib, which could be studied up to 200 nM) [22]. The lack of v0 dependence on inhibitor concentration was likely due to the low nanomolar concentrations of inhibitors that were used. Thus, a minimal mechanism for slow-binding inhibition under these condi- tions is given in Fig. 1b [20–22]. The pseudo-first-order rate constants kobs for this mechanism were obtained from non-linear least squares curve fitting of progress curves to either Eq. 1 or 2 as appropriate [20–22]. A plot of kobs versus the inhibitor concentration is predicted to vary linearly with the inhibitor concentration [20–22]. The slope of the plot gives the second-order association rate constant kon, and the intercept gives the pseudo-first-order rate constant koff for enzyme binding. For the epoxyketone inhibitors carfilzomib and oprozomib, in which a strong irreversible covalent bond is formed, koff and vS are both 0 [20, 21]. In contrast, the
drug treatment after 72 h is given in Table 1, and shows that myocyte damage increased in the order carfilzomib < opro- zomib < ixazomib ~ delanzomib < bortezomib. The effect of pharmacological concentrations of doxorubicin, a well- known cardiotoxic drug [19, 29, 30], on myocyte damage is shown for comparison. Except for carfilzomib, continuous treatment with 1 µM doxorubicin for 72 h induced about as much myocyte damage as the proteasome inhibitors (Fig. 2a). A 4-h treatment with either 0.3 µM delanzomib or bortezomib induced as much or more myocyte dam- age, respectively, as a 4 h 1.5 µM doxorubicin treatment (Fig. 2b). The relative order for bortezomib and delanzomib to induce myocyte damage was the same for either a 4-h or 72-h treatment. Progress Curve Analysis of the Inhibition of Myocyte Cell Lysate Chymotrypsin‑Like Proteasome Activity by the Boronic Acid Inhibitors Bortezomib, Ixazomib, and Delanzomib The DMSO control progress curves (Figs. 3a, 4a, 5a) for the proteasome-catalyzed hydrolysis of Suc-LLVY-AMC by myocyte cell lysate were highly linear (r2 > 0.99) due to the high concentration (60 µM) of Suc-LLVY-AMC relative to the concentration of product produced during the reac- tion. In the presence of a range of nanomolar concentrations (3–15 nM) of bortezomib, ixazomib, and delanzomib, the progress curves (Figs. 3a, 4a, 5a, respectively) for each of these drugs became progressively and markedly non-linear with time in comparison to the DMSO control, which is characteristic of slow-binding enzyme inhibition [20–22]. The progress curves were similar to what we previously observed for the inhibition of 20S proteasome activity by these drugs [22]. We previously showed that for the inhibi- tion of 20S proteasome activity v0 showed no dependence on proteasome inhibitor concentration [22]. This result implies
that if fast initial complexes are formed prior to covalent bond formation at the active site, their dissociation constants must be much larger than 15 nM [20–22], the maximum concentration at which these three drugs could be studied. Likewise, v0 values, in general, calculated from the first 2min of the myocyte lysate progress curves showed little or no dependence on the inhibitor concentrations. Since under these conditions v0 was the initial velocity both in the absence and in the presence of inhibitors, the DMSO control v0 values were substituted in Eq. 1 or 2 for curve fitting the progress curves. This had the advantage of reducing these equations to two unknown parameters, kobs and vS.
Analysis of the progress curves of Figs. 3a, 4a, and 5a in either Eq. 1 or 2 as appropriate yielded kobs values (Figs. 3b, 4b, 5b) for each concentration of inhibitor. Linear regression of kobs on the inhibitor concentration yielded the second- order rate constants kon from the slopes (Table 1). Because all of the intercepts in these plots were negative, but close to zero, values of koff could not be determined. The problems in determining more accurate koff values were probably due to the small amount of non-linearity of the control progress curves, relative to what we observed previously with puri- fied human 20S proteasome [22]. The reason for the small amount of curvature in the control curves in the myocyte lysate is not known, but could be due to a small loss of activ- ity over the 90-min time course of the reaction. Likewise, vS values could not be accurately determined for the same reason.
Progress Curve Analysis of the Inhibition of Myocyte Cell Lysate Chymotrypsin‑Like Proteasome
Activity by the Epoxyketone Inhibitors Carfilzomib and Oprozomib
Carfilzomib and oprozomib are epoxyketone inhibitors that form a covalent irreversible complex at the proteasome bind- ing site. Thus, the value of koff in the reaction scheme in Fig. 1b for these two drugs is zero. Progress curves obtained
at various concentrations of carfilzomib and oprozomib are shown in Figs. 6a and 7a, respectively. Curve fitting to the progress curves to either Eq. 1 or 2, as appropriate, yielded with vS fixed to zero, kobs values as a function of the inhibi- tor concentrations. Linear regression of kobs on the inhibi- tor concentrations are given in Figs. 6b and 7b and yielded from the slopes values of kon for carfilzomib and oprozomib, respectively (Table 1). The inhibition of the myocyte lysate proteasome-catalyzed hydrolysis of Suc-LLVY-AMC by oprozomib was much slower than any of the other inhibitors studied. This allowed the study of oprozomib at much higher concentrations than the other inhibitors. Both carfilzomib and oprozomib were also examined for the formation of fast initial complexes formed prior to covalent bond formation by measuring v0 as a function of inhibitor concentration. The v0 values were essentially unchanged (results not shown) with varying inhibitor concentrations as we observed before [22]
for inhibition of 20S proteasome activity. This result again indicated that if there was a fast initial complex formed, their dissociation constants were greater than 15 and 200 nM for carfilzomib and oprozomib, respectively, which was the respective maximum concentrations at which these two drugs could be studied.

Correlation of Proteasome Inhibitor‑Induced Damage to Myocytes with kon and the Growth Inhibitory Effects on K562 and HCT116 Cells
The kon values for the reaction of the proteasome inhibitors for inhibition of the proteasome activity of myocyte lysate varied by 85-fold (Table 1). A correlation plot of the rate constant kon for myocyte lysate proteasome inhibition versus the proteasome inhibitor-induced myocyte damage as meas- ured by Δ% LDH values at 300 nM drug is shown in Fig. 8a. Although Δ% LDH was positively correlated with kon, the correlation was neither high (r 0.68) nor significant (p 0.21).
In a previous study on the myocyte-damaging effects of five clinically approved ABL kinase inhibitors, we showed that there was a good correlation of myocyte-induced dam- age with their growth inhibitory effects on K562 cells, a result that suggested that there were common mechanistic determinants for growth inhibition of a proliferating cancer cell and damage to non-proliferating myocytes [31]. IC50 values for growth inhibition after a 72-h drug treatment for the five proteasome inhibitors for erythroleukemic K562 and colon cancer HCT116 cells were all in the low nanomolar range (Table 1). These IC50 values are in the same range of concentrations at which these inhibitors significantly induced myocyte damage (Fig. 2a). However, log IC50 val- ues for either K562 (Fig. 8b) or HCT116 (Fig. 8c) cells were poorly correlated (r2 < 0.01, respectively) with Δ% LDH val- ues for proteasome inhibitor-induced myocyte damage. The straight lines in Fig. 8b, c were linear least squares calculated for the three boronic acid drugs only. While the correlation coefficients were relatively high (r = 0.9), because of the small number of data points significance was not achieved. Discussion We previously used progress curve analysis to measure the inhibition kinetics of these five slow-binding inhibitors of human 20S proteasome [22]. As shown in Table 1, the kon values for inhibition of proteasome activity of rat myocyte and human 20S proteasome were close in value except for bortezomib (myocyte lysate fourfold larger). These results suggest that the rat and the human binding kinetics are very similar and thus the rat is a good model. In the previous study, koff values were obtained for two of the three boronic acid inhibitors and KI values for all three boronic acid ana- logs [22]. However, the DMSO-treated control curves for rat myocyte lysate were not as linear as the progress curves for human 20S proteasome, and thus KI values could not be accurately determined. We previously compared the myocyte-damaging effects of bortezomib and carfilzomib [19]. In that study, an initial velocity method was used to measure kobs and kon. Rather than fitting the whole progress curve to Eq. 1 or 2, the myocyte lysate was incubated with inhibitor for a series of fixed times, and the concentration dependence of the initial velocities was measured to give kobs. The previously deter- mined [19] kon values of 2.6 × 105 and 0.7 × 105 M- 1 s- 1, for bortezomib and carfilzomib, respectively, compared well to values of 2.8 × 105 and 0.33 × 105 M- 1 s- 1 from the progress curve analysis results (Table 1), and thus validates the pro- gress curve analysis. We previously speculated that the greater myocyte-dam- aging effects of bortezomib compared to carfilzomib might be due its much faster inhibition of proteasome activity [19]. While the results of Fig. 8a showed that inhibitor-induced myocyte damage increased with kon, statistical significance was not achieved. However, as a group the boronic acid inhibitors, bortezomib, ixazomib, and delanzomib, reacted faster and induced greater myocyte damage than the epox- yketone inhibitors carfilzomib and oprozomib. In principle, at equimolar concentrations, the slower epoxyketone reac- tions would result in comparatively less inhibition at longer times, and hence could produce reduced myocyte-damaging effects. The fact that the boronic acid drugs form reversible complexes with the proteasome, compared to the irreversible covalent binding epoxyketone drugs, did not translate to less myocyte damage, at least in the myocyte model. We previ- ously showed that chymotrypsin-like activity in myocytes was largely recovered 24 h after a pulse (6.5 h) bortezomib treatment [19]. Recovery of proteasome activity in tissues due to new proteasome synthesis has also been shown to occur within 24 h in mice treated with either carfilzomib or bortezomib [2]. Thus, partial recovery of proteasome activ- ity could more readily occur with a slower reacting inhibitor resulting in less myocyte damage. In support of this there was much less myocyte damage when the myocytes were treated with inhibitors for 4 h compared to 72 h (Fig. 2a, b). This result suggests that myocyte-damaging effects may require continual and high inhibition of proteasome activity. Even though the concentrations at which the protea- some inhibitors inhibited cell growth of proliferating K562 and HCT116 cells, and the concentrations that induced myocyte damage were both in the submicromolar concentration range, there was no significant overall cor- relation between cancer cell growth inhibition log IC50 values and myocyte damage Δ% LDH values (Fig. 8b, c). It is worth noting, however, that within the boronic acid group of inhibitors (bortezomib, ixazomib, and delan- zomib) myocyte damage progressively decreased with a decrease in inhibitor potency in either K562 or HCT116 cells. This lack of overall correlation suggests that there may not be common mechanistic determinants for protea- some inhibitor-induced myocyte damage and cell growth inhibition. However, it should be noted that the range of IC50 values for the inhibitors (4- and eightfold for K562 and HCT116 cells, respectively) may not have been large enough to accurately show a correlation. This lack of cor- relation cannot be due to any large differences in protea- somal activity present in myocytes and K562 cells as we previously showed that the chymotrypsin-like specific activity of myocyte lysate on a total cellular protein basis is almost equal to that of K562 cell lysate [19]. This is inspite of the fact that neonatal myocytes are essentially non-proliferating by day 5 after isolation [25] when drug treatments were commenced. Cardiac myofibrillar proteins are in a dynamic state of continual degradation and resyn- thesis [32–35], likely due to the constant mechanical work myocytes perform. Also the ubiquitin–proteasome system is known to play a critical role in protein turnover in the heart [32, 34, 35]. While we were not able to measure koff for myocyte lysates for the boronic acid inhibitors, we did previously determine koff rates for human 20S proteasome [19]. These koff values varied nearly 20-fold with ixazomib dissociat- ing the fastest, followed by bortezomib and delanzomib with k off values of 23 × 10 - 4 , 3.8 × 10 - 4 , and < 1.2 × 10- 4 s- 1, respectively. These koff values convert to half-time t1∕off2 values of 5, 30 and > 98 min, respectively.
The koff values for human 20S proteasome would appear not to be correlated with the myocyte-damaging effects of these inhibitors which are in the order from most damag- ing to least damaging: bortezomib > delanzomib ~ ixa- zomib (Table 1). The KI values for human 20S proteasome for delanzomib, bortezomib, and ixazomib varied only 3.5-fold with values of 1.4, 1.6, and 4.9 nM, respectively [19]. Thus, while delanzomib is the strongest binding inhibitor of 20S proteasome activity, it was not the most myocyte damaging, and thus binding strength would also appear not to be correlated with myocyte-damaging effects either.
The plasma Cmax values for bortezomib, ixazomib, delanzomib, carfilzomib, and oprozomib have been deter- mined to be 146 nM [36], 305 nM [37], 801 nM [38], 3800 nM [39], and 1640 nM [40], respectively. Thus, the results of Fig. 2a show that all proteasome inhibitors potently damaged myocytes at submicromolar concentra- tions within clinically relevant concentrations.
A head-to-head clinical trial that compared carfilzomib with bortezomib showed that carfilzomib has a higher risk of cardiovascular toxicity compared to bortezomib [13]. This result does not accord with the results of this study that showed that bortezomib had greater myocyte-damag- ing effects than carfilzomib (Table 1). Ixazomib, being the most recent proteasome inhibitor to gain approval has not been as well studied as either bortezomib or carfilzomib. Ixazomib is thought to have a low incidence of cardiotox- icity [15] and may be less cardiotoxic than either bort- ezomib or carfilzomib. Little or no safety or cardiotoxicity data are available for delanzomib and oprozomib, the other two proteasome inhibitors of this study, and thus no com- parison can made with their clinical cardiotoxicity if any.
Cardiovascular complications of proteasome inhibitor therapy range from accelerated hypertension to congestive heart failure, cardiomyopathy, and arrhythmias. In addition, vascular side effects that include venous and arterial throm- boembolic events have also been reported and may be due to off-target effects [7]. Recent reviews have examined the pos- sible mechanisms of proteasome inhibitor-induced cardio- toxicity. Apart from activating apoptotic pathways in cardiac myocytes as we showed earlier [19], proteasome inhibitors may affect nitric oxide synthase activity and have effects on smooth muscle that can lead to plaque instability as well as inhibit p53 which regulates cell growth [6, 7].
In conclusion, this study compared the myocyte-damag- ing effects of five proteasome inhibitors with their binding kinetics. Three of these drugs were boronic acid reversible inhibitors and two were epoxyketone irreversible inhibitors. All inhibitors, except for carfilzomib, were shown to result in myocyte damage at submicromolar pharmacological drug concentrations at levels that were comparable to those induced by the cardiotoxic drug doxorubicin. As a group, the two irreversible inhibitors were less myocyte damaging than the boronic acid inhibitors. Neither the human 20S protea- some dissociation rate constants, koff , nor the dissociation constants, KI, for the boronic acid inhibitors were correlated with their myocyte-damaging effects. As a group, the bind- ing rate constants kon for the epoxyketone inhibitors were smaller than for the boronic acid inhibitors, which may be a factor in their reduced myocyte-damaging effects, possibly due to partial recovery of proteasome activity.
Acknowledgements This Research was supported by Grants from the Canadian Institutes of Health Research (Grant MOP13748), the Canada Research Chairs Program, and a Canada Research Chair in Drug Devel- opment to Brian Hasinoff. The authors declare no competing financial interests. The funding sources had no involvement in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

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