Boronic Acid Inhibitor Research Articles

Subangstrom Crystallography Reveals that Short Ionic Hydrogen Bonds, and Not a His-Asp Low-Barrier Hydrogen Bond, Stabilize the Transition State in Serine Protease Catalysis

To address questions regarding the mechanism of serine protease catalysis, we have solved two X-ray crystal structures of alpha-lytic protease (alphaLP) that mimic aspects of the transition states: alphaLP at pH 5 (0.82 A resolution) and alphaLP bound to the peptidyl boronic acid inhibitor, MeOSuc-Ala-Ala-Pro-boroVal (0.90 A resolution). Based on these structures, there is no evidence of, or requirement for, histidine-flipping during the acylation step of the reaction. Rather, our data suggests that upon protonation of His57, Ser195 undergoes a conformational change that destabilizes the His57-Ser195 hydrogen bond, preventing the back-reaction. In both structures the His57-Asp102 hydrogen bond in the catalytic triad is a normal ionic hydrogen bond, and not a low-barrier hydrogen bond (LBHB) as previously hypothesized. We propose that the enzyme has evolved a network of relatively short hydrogen bonds that collectively stabilize the transition states. In particular, a short ionic hydrogen bond (SIHB) between His57 Nepsilon2 and the substrate's leaving group may promote forward progression of the TI1-to-acylenzyme reaction. We provide experimental evidence that refutes use of either a short donor-acceptor distance or a downfield 1H chemical shift as sole indicators of a LBHB.

Phase II pharmacodynamic study of the fibroblast activation protein inhibitor Val-boro-Pro in patients with metastatic colorectal cancer

3594 Background: Fibroblast activation protein (FAP) is selectively expressed by carcinoma stromal fibroblasts, but not by epithelial carcinoma cells, normal fibroblasts, or other normal tissues. FAP potentiates tumor growth in a murine model of human epithelial cancers. Val-boro-Pro, a boronic acid inhibitor of FAP protease activity, attenuates tumor growth in a mouse colorectal cancer model characterized by FAP expression by tumor-associated fibroblasts. We therefore initiated this first clinical trial of Val-boro-Pro in patients with colorectal cancer. Methods: Patients (pts) with previously treated, measurable metastatic colorectal cancer (CRC) were initially treated with 400 μg of Val-boro-Pro orally twice daily continuously. Due to unacceptable toxicities in the first 3 patients (including 1 grade 5 renal failure), the starting dose was reduced to 200 μg orally twice daily, with intrapatient dose escalation to 300 μg bid permitted after 4 weeks. The primary endpoint is response rate, with secondary endpoints including time to progression, and in vivo laboratory correlates of FAP inhibitory activity. Results: 20 patients (12 women, 8 men) with ECOG performance status 0–1 have been treated at 200 μg orally twice daily. The median age is 58 (range 41–80). At 200 μg bid, grade 1–2 adverse events include fatigue (12 pts), anorexia (7 pts), edema (7 pts), and fever (5 pts). The most common laboratory abnormalities are grade 1–2 anemia (7 pts), elevated alkaline phosphatase (4 pts), and hypoalbuminemia (3 pts). Grade 3–4 events are elevated alkaline phosphatase (1 pt, grade 3) and headache/syncope (1 pt, grade 3). Analyses of the first 9 patient plasma samples demonstrate a 92% (S.D. 2%) inhibition of dipeptidyl peptidase activity, and 22% increase in the FAP substrate α2-antiplasmin. Conclusions: Val-boro-Pro 200 μg orally bid is biologically active and well-tolerated, with toxicities likely related to cytokine upregulation. A planned interim analysis for clinical response will be conducted after treatment of 23 evaluable patients. The selectivity of FAP expression for tumor fibroblasts provides a unique opportunity to investigate and manipulate stromal contributions to tumor growth and invasion. No significant financial relationships to disclose.

Protection from Anthrax Toxin-Mediated Killing of Macrophages by the Combined Effects of Furin Inhibitors and Chloroquine

Cell surface proteolytic processing of anthrax protective antigen by furin or other furin-related proteases is required for its oligomerization, endocytosis, and function as a translocon for anthrax lethal and edema factors. Countering toxin lethality is essential to developing effective chemotherapies for anthrax infections that have proceeded beyond the stage at which antibiotics are effective. The primary target for toxin is the macrophage, which can be killed by lethal factor via both necrotic and apoptotic pathways. Here we show that three high-affinity inhibitors of furin efficiently blocked killing of murine J774A.1 macrophages by recombinant protective antigen plus lethal factor: RRD-eglin and RRDG-eglin, developed by engineering the protein protease inhibitor eglin c, and the peptide boronic acid inhibitor acetyl-Arg-Glu-Lys-boroArg pinanediol. Inhibition of killing was dose dependent and correlated with prevention of protective antigen processing. Previous studies have shown that weak bases, such as chloroquine, which neutralize acidic compartments, also interfere with toxin-dependent killing. Here we show that combining furin inhibitors and chloroquine strongly augments the inhibition of toxin-dependent killing, suggesting that combined use of antifurin drugs and chloroquine might provide enhanced therapeutic benefits. Reversible furin inhibitors protected against anthrax toxin killing for at least 5 h, but by 8 h, toxin-dependent killing resumed even though furin inhibitors were still active. An irreversible chloromethylketone inhibitor did not exhibit this loss of protection.

Preliminary evidence of efficacy and tolerance for weekly intravenous bortezomib plus mitoxantrone in patients with advanced androgen-independent prostate cancer (AIPCa)

4567 Background: Bortezomib, a dipeptidyl boronic acid inhibitor of the 20S proteasome, has evidence of activity when given on a weekly schedule in patients with AIPCa. Of the cytotoxic agents approved for treatment of AIPCa, experimental models suggest anthracyclines exhibit consistently enhanced activity when given in combination with bortezomib. In an effort to build on these potential additive or synergistic effects, we developed a phase I study of weekly bortezomib (B) with weekly mitoxantrone (M) in patients (pts) with AIPCa. Methods: Twenty-one pts with progressive AIPCa received 63 cycles of B plus M given once weekly 4 weeks out of 5 using dose escalation with a continuous reassessment method. Three pts were treated at dose level 0 (B=1.4 mg/m2, M=2 mg/m2), and 18 at dose level 1 (B=1.4, M=3.0). Results: Median age was 63 yrs (range: 47–78). Nineteen pts received prior systemic chemotherapy (median # regimens: 2, range 0–7); 17 received prior taxane-based therapy. Therapy was well tolerated with only one G4 toxicity (unrelated basal cell cancer). G3 toxicities mainly included fatigue and neutropenia (4 each). Six pts required dose reduction: 4 due to G3 neutropenia (2 in cycle 1), and 2 due to Plt < 75,000. Only 1 pt had G2 neuropathy. Of the 18 pts completing at least 2 cycles and assessable for response, 3 (17%) pts with measurable disease achieved PR (both by PSA and in lymph nodes) and completed 5, 6, and 8 cycles of treatment. Two responders received prior chemotherapy; 1 progressed on non-taxane therapy, the other had 6 prior regimens of which 3 contained a taxane. Two additional heavily pre-treated pts (4 prior chemotherapy regimens each) achieved SD - the first pt had SD throughout all 8 cycles, while the other pt had a minor response in lymph nodes after 2 cycles, and continues on therapy. Conclusions: This schedule of bortezomib with mitoxantrone is well tolerated, and shows evidence of antitumor activity. Accrual to a cohort receiving bortezomib 1.4 mg/m2 and mitoxantrone 3.0 mg/m2 is ongoing. These observations may have broader implications in the development of proteasome inhibitors in combination with cytotoxic chemotherapy. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Millenium Johnson & Johnson Pharmaceutical Research & Development

Arginase: Structure, Mechanism, and Physiological Role in Male and Female Sexual Arousal

AbstractFor Abstract see ChemInform Abstract in Full Text.

Arginase: Structure, Mechanism, and Physiological Role in Male and Female Sexual Arousal

Mammalian arginases I and II require an intact binuclear manganese cluster for the hydrolysis of L-arginine to generate L-ornithine and urea. Although arginase isozymes differ in terms of their tissue distribution, cellular localization, and metabolic function, each employs a metal-activated hydroxide mechanism for catalysis. To date, the best arginase inhibitors are those bearing N-hydroxyguanidinium or boronic acid "warheads" that can bridge the binuclear manganese cluster. Strikingly, the trigonal planar boronic acids undergo nucleophilic attack by hydroxide ion to form tetrahedral boronate anions that mimic the tetrahedral intermediate and its flanking transition states in the arginase mechanism. Given their affinity and specificity for arginase, boronic acid inhibitors are especially useful for probing the role of arginase in living systems. Arginase can regulate L-arginine bioavailability to nitric oxide synthase by depleting the substrate pool for NO biosynthesis, so arginase inhibition can enhance the substrate pool for NO biosynthesis. Accordingly, arginase inhibition can enhance NO-dependent physiological processes, such as the smooth muscle relaxation required for sexual arousal: administration of arginase inhibitors in vitro and in vivo enhances erectile function and engorgement in the male and female genitalia. Therefore, arginase is a potential therapeutic target for the treatment of sexual arousal disorders in men and women.

Structure–activity relationship for aryl and heteroaryl boronic acid inhibitors of hormone-sensitive lipase

A range of aryl and heteroaryl boronic acids were tested for their in vitro hormone-sensitive lipase inhibitory properties. (2-Benzyloxy-5-fluorophenyl)boronic acid, (2-benzyloxy-5-chlorophenyl)boronic acid and 5-bromothiophene-2-boronic acid were found to be the most potent HSL inhibitors with IC 50 values of 140, 17 and 350 nM, respectively.

Structure and Function of Arginases

The arginases catalyze the divalent cation dependent hydrolysis of L-arginine to produce L-ornithine and urea. Although traditionally considered in terms of its role as the final enzyme of the urea cycle, the enzyme is found in a variety of nonhepatic tissues. These findings suggest that the enzyme may have other functions in addition to its role in nitrogen metabolism. High-resolution crystal structures have been determined for recombinant rat liver (type I) arginase and for recombinant human kidney (type II) arginase, their variants, and complexes with products and inhibitors. Each identical subunit of the trimeric enzyme contains an active site that lies at the bottom of a 15 A deep cleft. The 2 essential Mn(II) ions are located at the bottom of this cleft, separated by approximately 3.3 A and bridged by oxygens derived from 2 aspartic acid residues and a solvent-derived hydroxide. This metal bridging hydroxide is proposed to be the nucleophile that attacks the guanidinium carbon of substrate arginine. On the basis of this proposed mechanism, boronic acid inhibitors of the enzyme have been synthesized and characterized kinetically and structurally. These inhibitors display slow-onset inhibition at the pH optimum of the enzyme, and are found as tetrahedral species at the active site, as determined by X-ray diffraction. The potent inhibition of arginases I and II by these compounds has not only delineated key enzyme-substrate interactions, but has also led to a greater understanding of the role of arginase in nonhepatic tissues.

Antitumor effects of bortezomib (PS-341) on primary effusion lymphomas.

Primary effusion lymphomas (PELs) are a rare type of non-Hodgkin's lymphoma that are resistant to cytotoxic chemotherapy. PELs manifest constitutive activation of nuclear factor kappa B (NF-kappaB), and inhibition of NF-kappaB induces apoptosis of PELs and sensitizes to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced death. Bortezomib (PS-341), a peptidyl boronic acid inhibitor of the proteasome, is a potent agent against a wide range of hematologic malignancies and has been shown to inhibit NF-kappaB. Thus, we examined the cytotoxic effects of bortezomib alone and in combination with various drugs. Bortezomib potently inhibited NF-kappaB in PEL cells in a dose-dependent manner. In addition, bortezomib inhibited growth and induced apoptosis of PEL cell lines (IC(50) values of 3.4-5.0 nM). Results of drug interactions between bortezomib and chemotherapy (doxorubicin and Taxol) were schedule-dependent: synergistic interactions were generally observed when PEL cells were pretreated with bortezomib prior to chemotherapy, whereas additive or even antagonistic interactions occurred with chemotherapy pretreatment or simultaneous treatment with bortezomib and chemotherapy. Most schedules of bortezomib and dexamethasone were synergistic, although pretreatment with dexamethasone resulted in additive interactions. Effects of combinations of bortezomib and TRAIL were generally additive. Thus, bortezomib represents a promising potential therapy for the treatment of PEL.

564 In vitro cytotoxic activity of NBT-272, a novel quassinoid analog

Proteasome inhibitors have a 20 year history in cancer therapy. The first proteasome inhibitor, bortezomib (Velcade, PS-341), a break-through multiple myeloma treatment, moved rapidly through development from bench in 1994 to first approval in 2003. Bortezomib is a reversible boronic acid inhibitor of the chymotrypsin-like activity of the proteasome. Next generation proteasome inhibitors include carfilzomib and oprozomib which are irreversible epoxyketone proteasome inhibitors; and ixazomib and delanzomib which are reversible boronic acid proteasome inhibitors. Two proteasome inhibitors, bortezomib and carfilzomib are FDA approved drugs and ixazomib and oprozomib are in late stage clinical trials. All of the agents are potent cytotoxics. The disease focus for all the proteasome inhibitors is multiple myeloma. This focus arose from clinical observations made in bortezomib early clinical trials. Later preclinical studies confirmed that multiple myeloma cells were indeed more sensitive to proteasome inhibitors than other tumor cell types. The discovery and development of the proteasome inhibitor class of anticancer agents has progressed through a classic route of serendipity and scientific investigation. These agents are continuing to have a major impact in their treatment of hematologic malignancies and are beginning to be explored as potential treatment agent for non-cancer indications.

A phase II trial of PS-341 in metastatic breast cancer (MBC)

3102 Background: The ubiquitin proteasome pathway plays an important role in cell cycle regulation, cancer growth and metastases representing a novel target for therapy. PS-341 (Bortezomib -Velcade) is a depeptidyl boronic acid inhibitor with high specificity for the proteasome and represents the first agent of this class approved for treatment of hematologic neoplasms. We initiated a Phase 2 single Institution trial testing PS-341 in minimally pretreated metastatic breast cancer (MBC). Methods: Twelve patients (pts) with minimally treated (1 previous regimen) measurable disease were enrolled and treated with PS-341 at 1.5mg/m2 intravenously bi-weekly for 2 consecutive weeks in a 21-day cycle. Pts. were evaluated clinically every 3 weeks to assess toxicity. Correlative studies include: level of 20-S proteasome activity in whole blood lysate before and after 1 hour of administration of the first dose (expressed as specific chymotryptic activity, SpA). Additionally we plan for evaluation of angiogenic and i...

A phase II trial of PS-341 in metastatic breast cancer (MBC)

3102 Background: The ubiquitin proteasome pathway plays an important role in cell cycle regulation, cancer growth and metastases representing a novel target for therapy. PS-341 (Bortezomib -Velcade) is a depeptidyl boronic acid inhibitor with high specificity for the proteasome and represents the first agent of this class approved for treatment of hematologic neoplasms. We initiated a Phase 2 single Institution trial testing PS-341 in minimally pretreated metastatic breast cancer (MBC). Methods: Twelve patients (pts) with minimally treated (1 previous regimen) measurable disease were enrolled and treated with PS-341 at 1.5mg/m2 intravenously bi-weekly for 2 consecutive weeks in a 21-day cycle. Pts. were evaluated clinically every 3 weeks to assess toxicity. Correlative studies include: level of 20-S proteasome activity in whole blood lysate before and after 1 hour of administration of the first dose (expressed as specific chymotryptic activity, SpA). Additionally we plan for evaluation of angiogenic and inflammatory cytokines (IL-12p70, TNF-α, IL-6, IL-1B, IL-8). Results: Ten pts are evaluable, 1 died early from disease complications and 1 is still on treatment. The median age was 54 years, (range 30–72 years). Nine pts had predominant visceral disease. 20-S proteasome inhibition compared to the pre-dose administration was documented in all cases. The median number of cycles of PS-341 was 2 cycles, (range 1–5 cycles). There were 1 stable disease (SD) and 9 progressive diseases (PD). The median time-to progression (TTP) was 47 days (range 17–110 days). The most common Grade 3–4 toxicities included: fatigue 70% (n=7) and skin rash 40% (n=4). The median time of onset of the skin rash was 11 days (range 10–27 days). Conclusions: PS-341 inhibits 20S-proteasome in pts with MBC. Administered in this schedule seems to have an acceptable tolerance, but little activity. The role of this agent in the management of breast cancer should be explored using combination with cytotoxics or other biological agents. No significant financial relationships to disclose.

The proteasome inhibitor bortezomib synergizes with gemcitabine to block the growth of human 253JB-V bladder tumors in vivo

Abstract Bortezomib (PS-341, Velcade) is a dipeptidyl boronic acid inhibitor of the 20S proteasome that was developed as a therapeutic agent for cancer. Here, we investigated the effects of bortezomib on the growth of human 253JB-V bladder cancer cells. Although the drug did not stimulate significant increases in levels of apoptosis, it inhibited cell growth in a concentration-dependent fashion and augmented the growth inhibitory effects of gemcitabine in vitro. These effects were associated with accumulation of p53 and p21 and suppression of cyclin-dependent kinase 2 activity. Bortezomib also inhibited secretion of the proangiogenic factors matrix metalloproteinase-9, interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF). In vivo studies with 253JB-V tumors growing in nude mice demonstrated that bortezomib (1 mg/kg) did not inhibit tumor growth when it was delivered as a single agent, although it reduced tumor microvessel density and inhibited expression of VEGF and IL-8. However, combination therapy with bortezomib plus gemcitabine produced synergistic tumor growth inhibition associated with strong suppression of tumor cell proliferation. Together, our results demonstrate that bortezomib has significant antiproliferative activity in aggressive bladder cancer cells, which is best exploited within the context of combination chemotherapy.

P1 Phenethyl peptide boronic acid inhibitors of HCV NS3 protease

A series of peptide boronic acids containing extended, hydrophobic P1 residues was prepared to probe the shallow, hydrophobic S1 region of HCV NS3 protease. The p-trifluoromethylphenethyl P1 substituent was identified as optimal with respect to inhibitor potency for NS3 and selectivity against elastase and chymotrypsin.

Structure-based approach for binding site identification on AmpC beta-lactamase.

Beta-lactamases are the most widespread resistance mechanism to beta-lactam antibiotics and are an increasing menace to public health. Several beta-lactamase structures have been determined, making this enzyme an attractive target for structure-based drug design. To facilitate inhibitor design for the class C beta-lactamase AmpC, binding site "hot spots" on the enzyme were identified using experimental and computational approaches. Experimentally, X-ray crystal structures of AmpC in complexes with four boronic acid inhibitors and a higher resolution (1.72 A) native apo structure were determined. Along with previously determined structures of AmpC in complexes with five other boronic acid inhibitors and four beta-lactams, consensus binding sites were identified. Computationally, the programs GRID, MCSS, and X-SITE were used to predict potential binding site hot spots on AmpC. Several consensus binding sites were identified from the crystal structures. An amide recognition site was identified by the interaction between the carbonyl oxygen in the R1 side chain of beta-lactams and the atom Ndelta2 of the conserved Asn152. Surprisingly, this site also recognizes the aryl rings of arylboronic acids, appearing to form quadrupole-dipole interactions with Asn152. The highly conserved "oxyanion" hole defines a site that recognizes both carbonyl and hydroxyl groups. A hydroxyl binding site was identified by the O2 hydroxyl in the boronic acids, which hydrogen bonds with Tyr150 and a conserved water. A hydrophobic site is formed by Leu119 and Leu293. A carboxylate binding site was identified by the ubiquitous C3(4) carboxylate of the beta-lactams, which interacts with Asn346 and Arg349. Four water sites were identified by ordered waters observed in most of the structures; these waters form extensive hydrogen-bonding networks with AmpC and occasionally the ligand. Predictions by the computational programs showed some correlation with the experimentally observed binding sites. Several sites were not predicted, but novel binding sites were suggested. Taken together, a map of binding site hot spots found on AmpC, along with information on the functionality recognized at each site, was constructed. This map may be useful for structure-based inhibitor design against AmpC.

Boron-11 pure quadrupole resonance investigation of peptide boronic acid inhibitors bound to alpha-lytic protease.

Pure quadrupole resonance is a potentially useful spectroscopic approach to study the coordination of quadrupolar nuclei in biological systems. We used a field-cycling NMR method to observe boron pure quadrupole resonance of two peptide boronic acid inhibitors bound to alpha-lytic protease. The method is similar to our earlier field-cycling experiment [Ivanov, D., and Redfield, A. R. (1998) Z. Naturforsch. A 53, 269-272] but uses a simple Hartmann-Hahn transfer from proton to (11)B before field cycle and direct (11)B observe after it. Pure quadrupole resonance is sensitive to the boron coordination geometry. For example, trigonal boron in neutral phenylboronic acid, which was used as a model compound, resonates at 1450 kHz, while the resonance of the tetrahedral phenylboronic acid anion appears at approximately 600 kHz. In the complex of the MeOSuc-Ala-Ala-Pro-boroVal inhibitor with the enzyme the quadrupole resonance signal was observed at 600-650 kHz, which indicates tetrahedral boron coordination in the active site. The quadrupole frequency of the MeOSuc-Ala-Ala-Pro-boroPhe enzyme-inhibitor complex, in which a boron-histidine bond is known to be formed, was found to be the same within experimental error as in the MeOSuc-Ala-Ala-Pro-boroVal enzyme-inhibitor adduct, suggesting that the boron coordination geometry in the enzyme-MeOSuc-Ala-Ala-Pro-boroPhe adduct is also close to tetrahedral.

Hepatitis C Virus NS3 Protease Requires Its NS4A Cofactor Peptide for Optimal Binding of a Boronic Acid Inhibitor as Shown by NMR

NMR spectroscopy was used to characterize the hepatitis C virus (HCV) NS3 protease in a complex with the 24 residue peptide cofactor from NS4A and a boronic acid inhibitor, Ac-Asp-Glu-Val-Val-Pro-boroAlg-OH. Secondary-structure information, NOE constraints between protease and cofactor, and hydrogen-deuterium exchange rates revealed that the cofactor was an integral strand in the N-terminal β-sheet of the complex as observed in X-ray crystal structures. Based upon chemical-shift perturbations, inhibitor-protein NOEs, and the protonation state of the catalytic histidine, the boronic acid inhibitor was bound in the substrate binding site as a transition state mimic. In the absence of cofactor, the inhibitor had a lower affinity for the protease. Although the inhibitor binds in the same location, differences were observed at the catalytic site of the protease.

Structure-based design guides the improved efficacy of deacylation transition state analogue inhibitors of TEM-1 beta-Lactamase(,).

Transition state analogue boronic acid inhibitors mimicking the structures and interactions of good penicillin substrates for the TEM-1 beta-lactamase of Escherchia coli were designed using graphic analyses based on the enzyme's 1.7 A crystallographic structure. The synthesis of two of these transition state analogues, (1R)-1-phenylacetamido-2-(3-carboxyphenyl)ethylboronic acid (1) and (1R)-1-acetamido-2-(3-carboxy-2-hydroxyphenyl)ethylboronic acid (2), is reported. Kinetic measurements show that, as designed, compounds 1 and 2 are highly effective deacylation transition state analogue inhibitors of TEM-1 beta-lactamase, with inhibition constants of 5.9 and 13 nM, respectively. These values identify them as among the most potent competitive inhibitors yet reported for a beta-lactamase. The best inhibitor of the current series was (1R)-1-phenylacetamido-2-(3-carboxyphenyl)ethylboronic acid (1, K(I) = 5.9 nM), which resembles most closely the best known substrate of TEM-1, benzylpenicillin (penicillin G). The high-resolution crystallographic structures of these two inhibitors covalently bound to TEM-1 are also described. In addition to verifying the design features, these two structures show interesting and unanticipated changes in the active site area, including strong hydrogen bond formation, water displacement, and rearrangement of side chains. The structures provide new insights into the further design of this potent class of beta-lactamase inhibitors.

26S Proteosome inhibition induces apoptosis and activation of p21 Cip1-Waf-1 in human pancreatic cancer

Intact 26S proteosome activity promotes the transcription of anti-apoptotic genes. New highly selective 26S proteosome inhibitors are becoming available for testing against human malignancies. We examined whether selective inhibition of the 26S proteosome alone alters proliferation and apoptosis in human pancreatic cancer. Methods:BxPC-3 human pancreatic cancer cells were serum starved for 60 hours. Cells were repleted with media containing 10% serum (FCS) alone or combined with the boronic acid inhibitor PS-341 (loonM; Leukosite, Inc., Cambridge, MA). Proliferation was measured by MTT assay. Apoptosis was determined by FACS analysis (sub-Go peak) and nuclear morphology (Pap stain). Data were comgared by two-tailed t-test (*p<0.05). Activation of caspase-3 and p21 ,pl-Waf-I by lOOnM PS-341 were determined by Western blot. Results: Proteosome inhibition with PS-341 significantly blocked mitogen (FCS) induced BxPC-3 cell proliferation. PS-341 alone caused apoptosis to occur as indicated by FACS analysis and nuclear morphology. This corresponds with activation of p21 within 3 hours (Fig), and subsequent activation of caspase-3 within 12 hours (Western not shown). Conclusions:In BxPC-3 pancreatic cancer cells, targeted inhibition (PS-341) of the 26S proteosome alone blocks proliferation and induces apoptosis as pro-apoptotic kinases become activated. These novel findings support further study of 26S proteosome inhibition in the therapy of human pancreatic cancer.

Characterization of peptidyl boronic acid inhibitors of mammalian 20 S and 26 S proteasomes and their inhibition of proteasomes in cultured cells

Proteasomes are large multisubunit proteinases which have several distinct catalytic sites. In this study a series of di- and tri-peptidyl boronic acids have been tested on the chymotrypsin-like activity of purified mammalian 20 S and 26 S proteasomes assayed with succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (suc-Leu-Leu-Val-Tyr-AMC) as substrate. The inhibition of 20 S proteasomes is competitive but only slowly reversible. The Ki values for the best inhibitors were in the range 10-100 nM with suc-Leu-Leu-Val-Tyr-AMC as substrate, but the compounds tested were much less effective on other proteasome activities measured with other substrates. Free boronic acid inhibitors exhibited equivalent potency to their pinacol esters. Both benzoyl (Bz)-Phe-boroLeu and benzyloxycarbonyl (Cbz)-Leu-Leu-boroLeu pinacol ester inhibited 20 S and 26 S proteasomes with non-ideal behaviour, differences in inhibition of the two forms of proteasomes becoming apparent at high inhibitor concentrations (above 3×Ki). Both of these compounds were also potent inhibitors of 20 S and 26 S proteasomes in cultured cells. However, gel filtration of cell extracts prepared from cells treated with radiolabelled phenacetyl-Leu-Leu-boroLeu showed that only 20 S proteasomes were strongly labelled, demonstrating differences in the characteristics of inhibition of 20 S and 26 S proteasomes. The usefulness of peptidyl boronic acid inhibitors for investigations of proteasome-mediated protein degradation was confirmed by the observation that Bz-Phe-boroLeu and Cbz-Leu-Leu-boroLeu pinacol ester inhibited NFĸB activation with IC50 values comparable to their Ki values for purified proteasomes. The latter result supports the view that the chymotrypsin-like activity of proteasomes assayed with suc-Leu-Leu-Val-Tyr-AMC is a critical one for protein degradation in cells.