Palmitoyl lactic acid induces adipogenesis and a brown fat-like phenotype in 3T3-L1 preadipocytes.

Brown adipose tissue is specialized to generate heat by dissipating chemical energy and may provide novel strategies for obesity treatment in humans. Recently, advances in understanding the pharmacological and dietary agents that contribute to the browning of white adipose tissue have been made to alleviate obesity by promoting energy expenditure. Krill oil is widely used as a health supplement in humans. In this study, the components from krill oil that promote adipogenesis of 3T3-L1 cells were screened to reveal palmitoyl lactic acid (PLA) as a promoter of adipogenesis. The PLA-induced adipocytes contained large number of small lipid droplets. Moreover, similar to the peroxisome proliferator-activated receptor (PPAR)γ agonists, pioglitazone and rosiglitazone, PLA significantly enhances adipogenesis in the presence of dexamethasone compared with PLA alone. Treatment with PLA causes a brown fat-like phenotype in 3T3-L1 cells by enhanced expression of various brown/beige cell-specific genes, such as PR domain containing 16 (Prdm16) and peroxisome proliferative activated receptor, gamma, coactivator 1 alpha (Pgc1a), as well as adiponectin gene. The expression profile of the brown/beige cell-specific genes induced by PLA was similar to that of the PPARγ agonist in 3T3-L1 cells. Our findings suggest that PLA induces a brown fat-like phenotype and, thus, likely has therapeutic potential in treating obesity.

Acinetobacter baumannii Lipopolysaccharide Influences Adipokine Expression in 3T3-L1 Adipocytes.

Acinetobacter baumannii is one of the most important nosocomial opportunistic pathogen worldwide. In addition, obesity has been associated with an increased risk of nosocomial infection, suggesting that there may be an association between A. baumannii and white adipose tissue. However, the effects of A. baumannii on adipocytes have not been well studied at the molecular level. Here, we investigated the potential role of A. baumannii-derived lipopolysaccharides (LPS) as signaling molecules that affect adipocyte functionality. We tested the effect of increasing concentrations of A. baumannii-derived LPS (10, 100, or 1000 ng/mL) on the 3T3-L1 adipocyte cell line. Exposure to LPS was found to increase the expression of several adipokines (e.g., MIP-2, MCP-1, TNF-α, IL-6, lipocalin-2, and FABP4) in 3T3-L1 adipocytes and significantly reduced the expression of leptin and adiponectin. The effects of A. baumannii-derived LPS on MIP-2 expression were similar in comparison with that of LPS prepared from Pseudomonas aeruginosa and Escherichia coli in our cell culture-based system. This study suggests that A. baumannii-derived LPS functions as a signaling molecule that impacts the inflammatory function of white adipose tissue on the level of gene expression.

Development of a new class of proteasome inhibitors with an epoxyketone warhead: Rational hybridization of non-peptidic belactosin derivatives and peptide epoxyketones.

Proteasome inhibitors are currently a focus of increased attention as anticancer drug candidates. We recently performed systematic structure-activity relationship studies of the peptidic natural product belactosin A and identified non-peptidic derivative 2 as a highly potent proteasome inhibitor. However, the cell growth inhibitory effect of 2 is only moderate, probably due to the biologically unstable ß-lactone warhead. Peptide epoxyketones are an important class of proteasome inhibitors exhibit high potency in cellular systems based on the efficient α,ß-epoxyketone warhead. Importantly, belactosin derivatives bind primarily to the primed binding site, while peptide epoxyketones bind only to the non-primed binding site of proteasome, suggesting that hybridization of them might lead to the development of a new class of proteasome inhibitors. Thus, we successfully identified a novel chemotype of proteasome inhibitors 3 and 4 by rational structure-based design, which are expected to bind to both the primed and non-primed binding sites of proteasome.

Longevity control by supersulfide-mediated mitochondrial respiration and regulation of protein quality.

Supersulfides, which are defined as sulfur species with catenated sulfur atoms, are increasingly being investigated in biology. We recently identified pyridoxal phosphate (PLP)-dependent biosynthesis of cysteine persulfide (CysSSH) and related supersulfides by cysteinyl-tRNA synthetase (CARS). Here, we investigated the physiological role of CysSSH in budding yeast (Saccharomyces cerevisiae) by generating a PLP-binding site mutation K109A in CRS1 (the yeast ortholog of CARS), which decreased the synthesis of CysSSH and related supersulfides and also led to reduced chronological aging, effects that were associated with an increased endoplasmic reticulum stress response and impaired mitochondrial bioenergetics. Reduced chronological aging in the K109A mutant could be rescued by using exogenous supersulfide donors. Our findings indicate important roles for CARS in the production and metabolism of supersulfides-to mediate mitochondrial function and to regulate longevity.

Design and synthesis of the stabilized analogs of belactosin A with the unnatural cis-cyclopropane structure.

The belactosin A analog 2a, having the unnatural cis-cyclopropane structure instead of the trans-cyclopropane structure in belactosin A, is a much more potent proteasome inhibitor than belactosin A. However, its cell growth inhibitory effect is rather lower than that expected from its remarkable proteasome inhibitory effect, probably due to its instability under cellular conditions. We hypothesized that the instability of 2a was due to chemical and enzymatic hydrolysis of the strained ß-lactone moiety. Thus, to increase the stability of 2a by chemical modification, its analogs with a sterically more hindered ß-lactone moiety and/or cyclopropylic strain-based conformational restriction were designed and synthesized, resulting in the identification of a stabilized analog 6a as a proteasome inhibitor with cell growth inhibitory effects. Our findings suggest that the chemical and biological stability of 2a is significantly affected by the steric hindrance around its ß-lactone carbonyl moiety and the conformational flexibility of the molecule.

Identification of novel kynurenine production-inhibiting benzenesulfonamide derivatives in cancer cells.

Kynurenine (Kyn), a metabolite of tryptophan (Trp), is known to be a key regulator of human immune responses including cancer immune tolerance. Therefore, abrogation of Kyn production from cancer cells by small molecules may be a promising approach to anticancer therapy. Indeed, several small molecule inhibitors of indoleamine 2,3-dioxygenase (IDO), a rate-limiting enzyme in the catabolism of Trp to Kyn, exert antitumor effects in animal models. We screened our chemical libraries using a cell-based Kyn production assay to identify a new type of small molecules that regulate Kyn production, and for the first time identified a benzenesulfonamide derivative (compound 1) as a hit with the ability to inhibit Kyn production in interferon-γ (IFN-γ)-stimulated A431 and HeLa cells. Unlike the previously identified S-benzylisothiourea derivative, compound 2, compound 1 had little effect on the enzymatic activity of recombinant human IDO in vitro but suppressed the expression of IDO at the mRNA level in cells. Furthermore, compound 1 suppressed STAT1-dependent transcriptional activity and DNA binding, whereas no decrement in either the expression or phosphorylation level of STAT1 was observed. The inhibition of IDO expression by several benzenesulfonamide derivatives is associated with the suppression of STAT1. Thus, compound 1 and its analogs might be useful for analyzing the regulation of IDO activation, and STAT1-targeting could be an alternative to the IDO-directed approach for the regulation of Kyn levels by small molecules in the tumor microenvironment.

Immunomodulatory gene expression analysis in LPS-stimulated human polymorphonuclear leukocytes treated with antibiotics commonly used for multidrug-resistant strains.

Conventional antibiotics used for the treatment of severe infections such as sepsis and septic shock confer immunomodulatory benefits. However, the growing problem of multidrug resistant infections has led to an increase in the administration of non-conventional last-resort antibiotics, including quinolones, aminoglycosides, and polypeptides, and the effects of these drugs on immunomodulatory gene expression in activated human polymorphonuclear leukocytes (PMNs) have not been reported. In this study, lipopolysaccharide-stimulated PMNs were incubated with piperacillin, rifampicin, fosfomycin (FOM), levofloxacin (LVFX), minocycline (MINO), colistin, tigecycline, or amikacin, and the mRNA expression levels of pattern recognition receptors (TLR2, TLR4, and CD14), inflammatory cytokines (TNFα and IL6), and chemokine receptors (IL8Rs and ITGAM) in these cells were quantitated using real-time qPCR. Many of the tested antibiotics altered the expression of the investigated cytokines. Notably, FOM, LVFX, and MINO significantly downregulated the expression of IL6, which is associated with pro- and anti-inflammatory defense mechanisms. Treatment of FOM and LVFX reduced IL-6 production as well as observed for IL6 gene expression. These findings indicated transcription and translation cooperation under the used experimental conditions. Therefore, our findings suggest that administration of these antibiotics suppresses the host anti-inflammatory response.

Identification of a small-molecule inhibitor of the interaction between Survivin and Smac/DIABLO.

The protein Survivin is selectively overexpressed in a variety of cancers, but not in normal tissues. It has been reported to be involved in cell survival and cell division. However, the molecular mechanisms involved in its function are not clear, although several binding partner proteins have been proposed to date. Here, we report the identification of a novel small molecule Survivin antagonist, which disrupts the Survivin-Smac/DIABLO interaction in cells. In order to identify Survivin-directed antagonists, we developed a high-throughput screening system based on AlphaScreen technology, which allows the identification of small molecules with the ability to inhibit the interaction of Survivin with Smac/DIABLO or INCENP in vitro. We screened chemical libraries, generated in-house, using this system and identified a 5-deazaflavin analog (compound 1) as a hit compound that selectively inhibited the interaction of Survivin with Smac/DIABLO but not INCENP. In cultured cells, compound 1 abrogated the formation of the complex between Survivin and Smac/DIABLO. In addition, this compound was able to sensitize cultured cells to doxorubicin-mediated DNA damage stress and synergistically enhance apoptotic cell death. Thus, the small-molecule inhibitor described here may serve as a proof-of-principle agent for discriminating between the multiple functions of Survivin.

S-benzylisothiourea derivatives as small-molecule inhibitors of indoleamine-2,3-dioxygenase.

S-benzylisothiourea 3a was discovered by its ability to inhibit indoleamine-2,3-dioxygenase (IDO) in our screening program. Subsequent optimization of the initial hit 3a lead to the identification of sub-muM inhibitors 3r and 10h, both of which suppressed kynurenine production in A431 cells. Synthesis and structure-activity relationship of S-benzylisothiourea analogues as small-molecule inhibitors of IDO are described.

The dynamic structure of Rab35 is stabilized in the presence of GTP under physiological conditions.

Rab proteins, a family of small guanosine triphosphatases, play key roles in intracellular membrane trafficking and the regulation of various cellular processes. As a Rab isoform, Rab35 is crucial for recycling endosome trafficking, cytokinesis and neurite outgrowth. In this report, we analyzed dynamic structural changes and physicochemical features of Rab35 in response to different external conditions, including temperature, pH, salt concentration and guanosine triphosphate (GTP), by circular dichroism (CD) spectroscopy. CD spectra revealed that the α-helix content of Rab35 varies under different conditions considerably. The addition of GTP increases the α-helix content of Rab35 when the temperature, pH and salt concentration match physiological conditions. The results suggest that the external environment affects the secondary structure of Rab35. In particular, the presence of GTP stabilized the α-helices of Rab35 under physiological conditions. These structural changes may translate to changes in Rab35 function and relate to its role in membrane trafficking.

Lipopolysaccharide-Deficient Acinetobacter baumannii Due to Colistin Resistance Is Killed by Neutrophil-Produced Lysozyme.

Acinetobacter baumannii causes nosocomial infections due to its multidrug resistance and high environmental adaptability. Colistin is a polypeptide antibacterial agent that targets lipopolysaccharide (LPS) and is currently used to control serious multidrug-resistant Gram-negative bacterial infections, including those caused by A. baumannii. However, A. baumannii may acquire colistin resistance by losing their LPS. In mouse models, LPS-deficient A. baumannii have attenuated virulence. Nevertheless, the mechanism through which the pathogen is cleared by host immune cells is unknown. Here, we established colistin-resistant A. baumannii strains and analyzed possible mechanisms through which they are cleared by neutrophils. Colistin-resistant, LPS-deficient strains harbor mutations or insertion sequence (IS) in lpx genes, and introduction of intact lpx genes restored LPS deficiency. Analysis of interactions between these strains and neutrophils revealed that compared with wild type, LPS-deficient A. baumannii only weakly stimulated neutrophils, with consequent reduced levels of reactive oxygen species (ROS) and inflammatory cytokine production. Nonetheless, neutrophils preferentially killed LPS-deficient A. baumannii compared to wild-type strains. Moreover, LPS-deficient A. baumannii strains presented with increased sensitivities to antibacterial lysozyme and lactoferrin. We revealed that neutrophil-secreted lysozyme was the antimicrobial factor during clearance of LPS-deficient A. baumannii strains. These findings may inform the development of targeted therapeutics aimed to treat multidrug-resistant infections in immunocompromised patients who are unable to mount an appropriate cell-mediated immune response.

Three-dimensional structure-activity relationship study of belactosin A and its stereo- and regioisomers: development of potent proteasome inhibitors by a stereochemical diversity-oriented strategy.

The development of potent proteasome inhibitors based on the stereochemical diversity-oriented strategy using a conformationally rigid cyclopropane structure was investigated. Thus, a series of stereo- and regioisomeric analogs of belactosin A (2), a cyclopropane amino acid (methanoamino acid)-containing tripeptidic proteasome inhibitor, were designed, in which the central cyclopropane amino acid part was replaced with the corresponding stereo- or regioisomer. Using a series of stereoisomeric cyclopropane amino acid equivalents with the cis/trans, D/L, and syn/anti stereochemical diversity, which were previously developed by us, as key units, the target compounds were successfully synthesized. Biological evaluation showed that, as expected, compound activity changed depending on the stereochemistry of the central cyclopropane amino acid part: the trans/L-syn-isomer 7 and the cis/L-anti-isomer 9 were more than twice as potent as natural belactosin A (trans/L-anti-isomer). Furthermore, the tripeptidic compound 13, the synthetic precursor for the unnatural cis/L-anti-isomer 9, was identified as a highly potent proteasome inhibitor. This compound, which is 20 times as potent as belactosin A and is even more potent than the well-known inhibitor lactacystin (4), may be an effective lead for developing clinically useful anticancer drugs. These results show that the stereochemical diversity-oriented approach can be a powerful strategy for the development of highly active compounds in medicinal chemical studies.

Multidrug-resistant Acinetobacter baumannii resists reactive oxygen species and survives in macrophages.

We investigated the intracellular survival of multidrug-resistant Acinetobacter baumannii (MDRAB) clinical isolates in macrophages, after phagocytosis, to determine their virulence characteristics. After ATCC 19606 and 5 clinical isolates of MDRAB were phagocytosed by mouse and human macrophages, the bacterial count of MDRAB strains, R4 and R5, increased in the mouse macrophages, 24 hours after phagocytosis. Bacterial count of the strains, R1 and R2, was almost equal 4 and 24 hours after phagocytosis. Intracellular reactive oxygen species was detected in the macrophages after phagocytosis of these bacteria. Further, the strains R1, R2, R4, and R5 showed higher catalase activity than ATCC 19606. Additionally, strains R1, R4, and R5 grew more efficiently than ATCC 19606 in the presence of H2O2, whereas growth of strains R2 and R3 was marginally more than that of ATCC 19606 in the presence of H2O2. The MDRAB clinical isolates altered the expression of TNF-α, IL-1ß, IL-6, and MIP-2 mRNA induced in J774A.1 cells, 24 hours after phagocytosis. These results provide insights into the renewed virulence characteristics of MDRAB clinical isolates. Finally, tigecycline killed MDRAB phagocytosed by the macrophages more effectively than colistin, although colistin and tigecycline are both considered effective antibiotics for the treatment of MDRAB.

Sub-minimum inhibitory concentrations of colistin and polymyxin B promote Acinetobacter baumannii biofilm formation.

We investigated the numbers of planktonic and biofilm cells and the expression levels of genes encoding efflux pumps and biofilm-related proteins in 10 clinical isolates of multi-drug resistant Acinetobacter baumannii (MDRA) as well as in its standard strain ATCC 19606 in the presence of colistin (CST), polymyxin B (PMB), minomycin (MIN), and tigecycline (TGC) at their respective sub-MICs. The number of planktonic and biofilm cells of ATCC 19606 decreased in the presence of all aforementioned antibiotics in a dose-dependent manner. Cell number also decreased in two representative MDRA strains, R2 and R3, in the presence of MIN and TGC in a dose-dependent manner. In contrast, the number of biofilm cells in these two strains increased in the presence of CST, while they increased significantly in the presence of PMB in R2 only. Pearson correlation analysis revealed that the number of biofilm cells was positively and significantly correlated with the mRNA levels of genes encoding efflux pumps (adeB and adeG) and autoinducer synthase (abaI) in strain R2 and adeB, adeG, adeJ, poly-acetyl-glucosamine-porin (pgaA), and abaI in strain R3 in the presence of CST. It was positively and significantly correlated with the mRNA levels of genes encoding adeB in strain R2 and an outer membrane protein A (ompA) and biofilm-associated protein (bap) in strain R3 in the presence of PMB. These results provide valuable insights into the biofilm formation potency of clinical isolates of MDRA that depends on efflux pumps and biofilm-related genes and its regulation by antibiotics.

Resolvin E1, but not resolvins E2 and E3, promotes fMLF-induced ROS generation in human neutrophils.

E-series resolvins are biosynthesized from eicosapentaenoic acid during the resolution phase of acute inflammation and enhance inflammation resolution. However, the role of E-series resolvins in inflammation resolution is not yet known. Herein, we show that in human polymorphonuclear neutrophils (PMNs), resolvin E1 (RvE1) selectively enhances reactive oxygen species (ROS) generation induced by N-formylmethionyl-leucyl-phenylalanine. The RvE1-mediated enhancement is eliminated by a pan-antagonist of leukotriene B4 (LTB4) receptors, LY255283, or an NADPH oxidase inhibitor, diphenyleneiodonium. Thus, RvE1 enhances NADPH oxidase-mediated ROS generation via LTB4 receptors. Unlike RvE1, resolvins E2 and E3 do not show such activation of PMNs. Our findings suggest that RvE1 contributes to regulation of ROS generation, in accordance with the inflammatory state of the host.

Virulence characteristics of Acinetobacter baumannii clinical isolates vary with the expression levels of omps.

PURPOSE: We investigated the expression levels of virulence factors (ompA, omp33-36 and carO) in five clinical isolates and in a standard ATCC 19606 strain of Acinetobacter baumannii to determine their effect on the virulence characteristics of the isolates. METHODOLOGY: The mRNA levels of omps and proinflammatory cytokines were analyzed by quantitative real-time PCR. For adherence assay, after human lung epithelial cells (A549) were co-cultured with A. baumannii at 37 °C for 2 h, the cell-adherent bacteria was counted. Pearson correlation analysis was used to compare the omps mRNA levels, the proinflammatory cytokines and the number of adherent bacteria. RESULTS: The mRNA levels of ompA in the clinical isolates were higher and similar compared with those in ATCC 19606, whereas the mRNA levels of omp33-36 in the clinical isolates were lower and similar compared with those in ATCC 19606. The mRNA levels of carO in the clinical isolates were significantly higher than those in ATCC 19606. The number of cell-adherent clinical isolates was higher than that of cell-adherent ATCC 19606. Furthermore, the number of cell-adherent clinical isolates was positively and significantly correlated with ompA mRNA level. The mRNA levels of TNF-α, IL-6 and IL-8 in A549 cells co-cultured with the clinical isolates were lower than those in A549 cells co-cultured with ATCC 19606. Moreover, the mRNA levels of TNF-α, IL-6 and IL-8 were negatively and significantly correlated with those of carO in the isolates. CONCLUSION: These results provide insights into the renewed virulence characteristics of A. baumannii clinical isolates that depend on cell adherence capacity and the expression level of omp mRNAs.

Peroxynitrite induces formation of N( epsilon )-(carboxymethyl) lysine by the cleavage of Amadori product and generation of glucosone and glyoxal from glucose: novel pathways for protein modification by peroxynitrite.

Accumulation of advanced glycation end products (AGEs) on tissue proteins increases with pathogenesis of diabetic complications and atherosclerosis. Here we examined the effect of peroxynitrite (ONOO(-)) on the formation of N( epsilon )-(carboxymethyl)lysine (CML), a major AGE-structure. When glycated human serum albumin (HSA; Amadori-modified protein) was incubated with ONOO(-), CML formation was detected by both enzyme-linked immunosorbent assay and high-performance liquid chromatography (HPLC) and increased with increasing ONOO(-) concentrations. CML was also formed when glucose, preincubated with ONOO(-), was incubated with HSA but was completely inhibited by aminoguanidine, a trapping reagent for alpha-oxoaldehydes. For identifying the aldehydes that contributed to ONOO(-)-induced CML formation, glucose was incubated with ONOO(-) in the presence of 2,3-diaminonaphthalene. This experiment led to identification of glucosone and glyoxal by HPLC. Our results provide the first evidence that ONOO(-) can induce protein modification by oxidative cleavage of the Amadori product and also by generation of reactive alpha-oxoaldehydes from glucose.

Glycolaldehyde-modified bovine serum albumin downregulates leptin expression in mouse adipocytes via a CD36-mediated pathway.

Previous observations by us have clarified that proteins modified by advanced glycation end products (AGEs) are recognized as effective ligands by CD36-overexpressed CHO cells and undergo receptor-mediated endocytosis. CD36, a member of the class B scavenger receptor family, also acts as a fatty acid transporter in adipocytes. Oxidized low-density lipoprotein (Ox-LDL), a ligand for CD36, is known to upregulate CD36 by activating peroxisome proliferator-activated receptor gamma (PPAR-gamma) in macrophages, whereas PPAR-gamma ligands such as troglitazone and 15-deoxy-delta12,14-prostaglandin J2 decrease leptin secretion from adipocytes. The purpose of this study was to examine effects of AGE ligands on leptin expression in adipocytes. Glycolaldehyde-modified bovine serum albumin (GA-BSA) decreased leptin expression at both the protein and mRNA levels in 3T3-L1 adipocytes and mouse epididymal adipocytes. The binding to and subsequent endocytic degradation of GA-BSA by 3T3-L1 adipocytes were effectively inhibited by a neutralizing anti-CD36 antibody. These results indicate that the ligand interaction of GA-BSA with CD36 leads to downregulation of leptin expression in 3T3-L1 adipocytes, suggesting that AGE-induced leptin downregulation is linked to reduction of the insulin sensitivity in metabolic syndrome.

Pathological roles of advanced glycation end product receptors SR-A and CD36.

The pathological significance of advanced glycation end product (AGE)-modified proteins deposited in several lesions is generally accounted for by their cellular interaction via the AGE receptors and subsequent acceleration of the inflammatory process. In this study, we focused on two AGE receptors-specifically, the role of SR-A in pathogenesis of diabetic nephropathy and the role of CD36 in AGE-induced downregulation of leptin by adipocytes. In terms of SR-A, diabetic wild-type mice exhibited increased urinary albumin excretion, glomerular hypertrophy, and mesangial matrix expansion, whereas SR-A-knockout mice showed reduced glomerular size and mesangial matrix area. In these diabetic SR-A-knockout mice, the number of macrophages that infiltrated into glomeruli was remarkably reduced (P < 0.05), suggesting that SR-A-dependent glomerular migration of macrophages plays an important role in the pathogenesis of diabetic nephropathy. In terms of CD36, incubation of glycolaldehyde-modified bovine serum albumin (GA-BSA) with 3T3-L1 adipocytes reduced leptin secretion by these cells. The binding of GA-BSA to these cells and subsequent endocytic degradation were effectively inhibited by a neutralizing anti-CD36 antibody. AGE-induced downregulation of leptin was protected by N-acetyl-cysteine, an antioxidant. These results indicate that the interaction of AGE ligands with 3T3-L1 adipocytes via CD36 induces oxidative stress and leads to inhibition of leptin expression by these cells, suggesting a potential link of this phenomenon to exacerbation of the insulin sensitivity in metabolic syndrome.

Structurally novel highly potent proteasome inhibitors created by the structure-based hybridization of nonpeptidic belactosin derivatives and peptide boronates.

We previously developed highly potent proteasome inhibitor 1 (IC50 = 5.7 nM) and its nonpeptide derivative 2 (IC50 = 29 nM) by systematic structure-activity relationship studies of the peptidic natural product belactosin A and subsequent rational topology-based scaffold hopping, respectively. Their cell growth inhibitory activities, however, were only moderate (IC50 = 1.8 µM (1) and >10 µM (2)). We therefore planned to replace the unstable ß-lactone warhead with a more stable boronic acid warhead. Importantly, belactosin derivatives bind mainly to the proteasome binding site, which is different from that occupied by known peptide boronate proteasome inhibitors such as bortezomib, suggesting that their hybridization might lead to the development of novel potent inhibitors. Here we describe design, synthesis, and biological activities of the newly developed potent hybrid proteasome inhibitors. Interestingly, these hybrids, unlike bortezomib, were highly selective for proteasomes and have long residence times despite having the same boronic acid warhead.