Reconfigurable Dual Peptide Tethered Polymer System Offers a Synergistic Solution for Next Generation Dental Adhesives.

Resin-based composite materials have been widely used in restorative dental materials due to their aesthetic, mechanical, and physical properties. However, they still encounter clinical shortcomings mainly due to recurrent decay that develops at the composite-tooth interface. The low-viscosity adhesive that bonds the composite to the tooth is intended to seal this interface, but the adhesive seal is inherently defective and readily damaged by acids, enzymes, and oral fluids. Bacteria infiltrate the resulting gaps at the composite-tooth interface and bacterial by-products demineralize the tooth and erode the adhesive. These activities lead to wider and deeper gaps that provide an ideal environment for bacteria to proliferate. This complex degradation process mediated by several biological and environmental factors damages the tooth, destroys the adhesive seal, and ultimately, leads to failure of the composite restoration. This paper describes a co-tethered dual peptide-polymer system to address composite-tooth interface vulnerability. The adhesive system incorporates an antimicrobial peptide to inhibit bacterial attack and a hydroxyapatite-binding peptide to promote remineralization of damaged tooth structure. A designer spacer sequence was incorporated into each peptide sequence to not only provide a conjugation site for methacrylate (MA) monomer but also to retain active peptide conformations and enhance the display of the peptides in the material. The resulting MA-antimicrobial peptides and MA-remineralization peptides were copolymerized into dental adhesives formulations. The results on the adhesive system composed of co-tethered peptides demonstrated both strong metabolic inhibition of S. mutans and localized calcium phosphate remineralization. Overall, the result offers a reconfigurable and tunable peptide-polymer hybrid system as next-generation adhesives to address composite-tooth interface vulnerability.

Bioinspired multifunctional adhesive system for next generation bio-additively designed dental restorations.

Resin-based composite has overtaken dental amalgam as the most popular material for the repair of lost or damaged tooth structure. In spite of the popularity, the average composite lifetime is about half that of amalgam restorations. The leading cause of composite-restoration failure is decay at the margin where the adhesive is applied. The adhesive is intended to seal the composite/tooth interface, but the adhesive seal to dentin is fragile and readily degraded by acids, enzymes and other oral fluids. The inherent weakness of this material system is attributable to several factors including the lack of antimicrobial properties, remineralization capabilities and durable mechanical performance - elements that are central to the integrity of the adhesive/dentin (a/d) interfacial seal. Our approach to this problem offers a transition from a hybrid to a biohybrid structure. Discrete peptides are tethered to polymers to provide multi-bio-functional adhesive formulations that simultaneously achieve antimicrobial and remineralization properties. The bio-additive materials design combines several functional properties with the goal of providing an adhesive that will serve as a durable barrier to recurrent decay at the composite/tooth interface. This article provides an overview of our multi-faceted approach which uses peptides tethered to polymers and new polymer chemistries to achieve the next generation adhesive system - an adhesive that provides antimicrobial properties, repair of defective dentin and enhanced mechanical performance.

Antimicrobial Peptide-Polymer Conjugates for Dentistry.

Bacterial adhesion and growth at the composite/adhesive/tooth interface remain the primary cause of dental composite restoration failure. Early colonizers, including Streptococcus mutans, play a critical role in the formation of dental caries by creating an environment that reduces the adhesive's integrity. Subsequently, other bacterial species, biofilm formation, and lactic acid from S. mutans demineralize the adjoining tooth. Because of their broad spectrum of antibacterial activity and low risk for antibiotic resistance, antimicrobial peptides (AMPs) have received significant attention to prevent bacterial biofilms. Harnessing the potential of AMPs is still very limited in dentistry-a few studies have explored peptide-enabled antimicrobial adhesive copolymer systems using mainly nonspecific adsorption. In the current investigation, to avoid limitations from nonspecific adsorption and to prevent potential peptide leakage out of the resin, we conjugated an AMP with a commonly used monomer for dental adhesive formulation. To tailor the flexibility between the peptide and the resin material, we designed two different spacer domains. The spacer-integrated antimicrobial peptides were conjugated to methacrylate (MA), and the resulting MA-AMP monomers were next copolymerized into dental adhesives as AMP-polymer conjugates. The resulting bioactivity of the polymethacrylate-based AMP conjugated matrix activity was investigated. The antimicrobial peptide conjugated to the resin matrix demonstrated significant antimicrobial activity against S. mutans. Secondary structure analyses of conjugated peptides were applied to understand the activity differential. When mechanical properties of the adhesive system were investigated with respect to AMP and cross-linking concentration, resulting AMP-polymer conjugates maintained higher compressive moduli compared to hydrogel analogues including polyHEMA. Overall, our result provides a robust approach to develop a fine-tuned bioenabled peptide adhesive system with improved mechanical properties and antimicrobial activity. The results of this study represent a critical step toward the development of peptide-conjugated dentin adhesives for treatment of secondary caries and the enhanced durability of dental composite restorations.

Peptide Mediated Antimicrobial Dental Adhesive System.

The most common cause for dental composite failures is secondary caries due to invasive bacterial colonization of the adhesive/dentin (a/d) interface. Innate material weakness often lead to an insufficient seal between the adhesive and dentin. Consequently, bacterial by-products invade the porous a/d interface leading to material degradation and dental caries. Current approaches to achieve antibacterial properties in these materials continue to raise concerns regarding hypersensitivity and antibiotic resistance. Herein, we have developed a multi-faceted, bio-functionalized approach to overcome the vulnerability of such interfaces. An antimicrobial adhesive formulation was designed using a combination of antimicrobial peptide and a ε-polylysine resin system. Effector molecules boasting innate immunity are brought together with a biopolymer offering a two-fold biomimetic design approach. The selection of ε-polylysine was inspired due to its non-toxic nature and common use as food preservative. Biomolecular characterization and functional activity of our engineered dental adhesive formulation were assessed and the combinatorial formulation demonstrated significant antimicrobial activity against Streptococcus mutans. Our antimicrobial peptide-hydrophilic adhesive hybrid system design offers advanced, biofunctional properties at the critical a/d interface.

Modulating pH through lysine integrated dental adhesives.

OBJECTIVES: The objective of this study was to explore the effect of lysine integration to dental adhesives with respect to the polymerization kinetics, neutralization capacities in the acidic microenvironment, dynamic mechanical properties, and thermal properties. MATERIALS AND METHOD: Lysine was incorporated into liquid resin formulations at 2.5 and 5.0wt % with additional water/ethanol co-solvents. The co-monomer system contained 2-hydroxyethyl-methacrylate (HEMA) and Bisphenol A glycerolate dimethacrylate (BisGMA) with a mass ratio of 45/55. The kinetics of photopolymerization, neutralization capacities, lysine-leaching, dynamic mechanical properties and thermal properties of the control and experimental adhesives were analyzed. RESULTS: The degree of conversion of the experimental adhesive was increased substantially at 2.5wt% lysine as compared to the control. The experimental polymers provided acute neutralization of the acidic microenvironment. Approximately half of the lysine was released from the polymer network within one month. Under dry conditions and physiologic temperatures, the incorporation of lysine did not compromise the storage modulus. Comparison of the thermal properties suggests that the more compact structure of the control adhesive inhibits movement of the polymer chains resulting in increased Tg. SIGNIFICANCE: Incorporating lysine in the adhesive formulations led to promising results regarding modulating pH, which may serve as one aspect of a multi-spectrum approach for enhancing the durability of composite restorations. The results provide insight and lay a foundation for incorporating amino acids or peptides into adhesive formulations for pH modulation or desired bioactivity at the interfacial margin between the composite and tooth.

Calcium enhances gene expression when using low molecular weight poly-l-lysine delivery vehicles.

Four molecular weights of poly-l-lysine (PLL) [1000-5000 Da, 1500-8000 Da, 4000-15,000 Da and 15,000-30,000 Da] and three molecular weights of polyethyleneimine (PEI) [800 Da, 2,000 Da, and 25,000 Da] were used to systematically study the effect of calcium (Ca2+) to improve transfection efficiency of polyelectrolyte complexes. Complexes made using different molecular weights of PLL or PEI polymer showed clear differences in the levels of gene expression in the presence and absence of calcium chloride when tested using A549 human lung carcinoma cells. Complexes formed from PLL or PEI 800 Da were exhibited negligible expression of pDNA according to a luciferase reporter assay. Low molecular weight PLL and PEI 800 Da, however, became highly efficient gene delivery vehicles when calcium was added to the nascent complexes while maintaining the low cytotoxicity of low molecular weight polyamines. Additional analyses indicated that the most effective formulations utilized a threshold level of calcium, which created small, stable particles, but also facilitated unpackaging of the gene complexes and release of pDNA.

Randomized clinical trial of BiClamp forceps versus clamp-crushing technique in open liver resection.

BACKGROUND: The aim of this trial was to compare the efficacy and safety of BiClamp forceps with the "gold-standard" clamp-crushing technique for open liver resection. METHODS: From October 2014 to May 2016, 86 consecutive patients scheduled to undergo hepatic resection were randomized to a BiClamp forceps group (n = 43) or to a clamp-crushing technique group (n = 43). RESULTS: Background characteristics of the two groups were closely matched. There were no significant differences between the BiClamp forceps group and clamp-crushing group in total intraoperative blood loss (339.81 ± 257.20 ml vs. 376.73 ± 303.67 ml, respectively; P = 0.545) or blood loss per transection area (5.35 ± 3.27 ml/cm2 vs. 5.44 ± 3.02 ml/cm2 , respectively; P = 0.609). Liver transection speed, the need of blood transfusion, morbidity, length of postoperative hospital stay, total hospitalization cost and liver function recovery were similar in the two groups. Multivariate logistic regression analysis identified major hepatectomy, multiple resections and liver transection time ≥30 min as significantly unfavorable factors for decreased intraoperative blood loss. CONCLUSIONS: Liver parenchymal transection with BiClamp forceps is as safe and feasible as the gold-standard clamp-crushing technique.

One-layer versus two-layer duct-to-mucosa pancreaticojejunostomy after pancreaticoduodenectomy: study protocol for a randomized controlled trial.

BACKGROUND: Although various pancreaticojejunal duct-to-mucosa anastomosis methods have been developed to reduce the postoperative risks of pancreaticoduodenectomy, pancreatic fistula remains the most serious complication with a high incident rate. The aim of this study is to compare the safety and effectiveness of one-layer and two-layer duct-to-mucosa pancreaticojejunostomy in patients undergoing pancreaticoduodenectomy. METHODS/DESIGN: In this study, adult patients who sign consent forms will be recruited and scheduled for elective pancreaticoduodenectomy. One hundred and fourteen patients will be included and randomized before pancreaticojejunal reconstruction and after resection of the lesion from the pancreatic or periampullary region. The primary efficacy endpoint is the incident rate of postoperative pancreatic fistula. Statistical analysis will be based on the intention-to-treat population. Patients will be followed up for 3 months by monitoring for complications and other adverse events. DISCUSSION: This prospective, single-center, randomized, single-blinded, two-group parallel trial is designed to compare one-layer with two-layer duct-to-mucosa anastomosis for pancreaticojejunal anastomosis during elective pancreaticoduodenectomy. TRIAL REGISTRATION: Clinical Trials.gov: NCT02511951 . Registered on 29 July 2015.

BiClamp® forcep liver transection versus clamp crushing technique for liver resection: study protocol for a randomized controlled trial.

BACKGROUND: Blood loss and the requirement of blood transfusions during liver transection have been shown to correlate well with higher morbidity and mortality rates and a worse prognosis. Various devices for liver parenchymal transection have been developed to reduce intraoperative blood loss. The goal of this study is to evaluate the safety and effectiveness of BiClamp® forcep transection compared to a clamp crushing technique in patients undergoing liver resection. METHODS/DESIGN: This study will include patients 18 years and older scheduled for hepatectomy with hepatic vascular exclusion who give informed consent. A sample size of 48 patients in each randomization arm will be calculated to detect a difference in the reduction of blood loss of approximately 200 ml (90% power and α = 0.05 (two-tailed)). The primary efficacy endpoint of the trial will be the total intraoperative blood loss based on the randomized dissection technique. The statistical analysis is based on the intention-to-treat population. Patients will be followed up on for three months for complications and adverse events. DISCUSSION: This prospective, single-center, randomized controlled, single-blinded, two-group parallel trial is designed to assess the efficacy and safety of BiClamp forcep hepatectomy versus clamp crushing for parenchymal transection during elective hepatic resection. TRIAL REGISTRATION: This trial was registered with Clinicaltrials.gov (identifier: NCT02197481 ) on 15 July 2014.

Single-incision versus conventional three-port laparoscopic appendectomy: A meta-analysis of randomized controlled trials.

PURPOSE: The aim of this article was to compare the advantages and disadvantages of single-incision laparoscopic appendectomy (SILA) and conventional three-port laparoscopic appendectomy (CTLA). MATERIAL AND METHODS: A meta-analysis was performed by analyzing all randomized controlled trials (RCTs) published in English that compared SILA and CTLA for appendicitis in adults and children. These studies compared these two methods from different angles including outcomes of interest, patient characteristics, operative time, pain visual analogue scales scores (VAS scores), length of hospital stay, time to return to full activity, resumption of diet, postoperative complications and cosmetic results The risk ratios (RR) and mean difference (MD) with 95% confidence intervals (CIs) were employed to assess the outcome. RESULTS: Seven recent RCTs encompassing 1170 patients (586 SILA and 584 CTLA cases) were included in this meta-analysis. The pooled results demonstrated that conversion rate, drain inserted, reoperation, length of hospital stay, resumption of normal diet and postoperative complications were statistically comparable between the two groups. The postoperative abdominal pain within 24 h was -0.57 in favor of the SILA technique (p = 0.05). Compared with CTLA, SILA showed a better cosmetic satisfaction score (SMD, 0.58; 95% CI, 0.32-0.83; p < 0.0001) and shorter time to recover normal activity (WMD, -0.69; 95% CI, -1.11-0.26; p = 0.001). However, SILA has a longer operative time (WMD, 5.38; 95% CI, 2.94-7.83; p < 0.0001). CONCLUSIONS: In selected patients, SILA was confirmed to be as safe and effective as CTLA. Despite the longer operative time, SILA has higher cosmetic satisfaction and shorter recovery time to normal activity. Due to the limitations of the available data, further research is needed.

Pancreaticogastrostomy is associated with significantly less pancreatic fistula than pancreaticojejunostomy reconstruction after pancreaticoduodenectomy: a meta-analysis of seven randomized controlled trials.

OBJECTIVES: This study aimed to compare pancreaticojejunostomy (PJ) with pancreaticogastrostomy (PG) after pancreaticoduodenectomy (PD). METHODS: A literature search of PubMed and the Cochrane Central Register of Controlled Trials for studies comparing PJ with PG after PD was conducted. The primary outcome for meta-analysis was pancreatic fistula. Secondary outcomes were morbidity, mortality, biliary fistula, intra-abdominal fluid collection, hospital length of stay (LoS), postoperative haemorrhage and reoperation. Outcome measures were odds ratios (ORs) and mean differences with 95% confidence intervals (CIs). RESULTS: Seven recent RCTs encompassing 1121 patients (559 PJ and 562 PG cases) were involved in this meta-analysis. Incidences of pancreatic fistula (10.6% versus 18.5%; OR 0.52, 95% CI 0.37-0.74; P = 0.0002), biliary fistula (2.3% versus 5.7%; OR 0.42, 95% CI 0.03-3.15; P = 0.03) and intra-abdominal fluid collection (8.0% versus 14.7%; OR 0.50, 95% CI 0.34-0.74; P = 0.0005) were significantly lower in the PG than the PJ group, as was hospital LoS (weighted mean difference: -1.85, 95% CI -3.23 to -0.47; P = 0.008). Subgroup analysis indicated that severe pancreatic fistula (grades B or C) occurred less frequently in the PG than the PJ group (8.3% versus 20.5%; OR 0.37, 95% CI 0.23-0.59; P < 0.00001). However, there was no significant difference in morbidity (48.9% versus 51.0%; OR 0.90, 95% CI 0.70-1.16; P = 0.41), mortality (3.2% versus 3.5%; OR 0.82, 95% CI 0.43-1.58; P = 0.56), delayed gastric emptying (16.6% versus 14.7%; relative risk: 1.02, 95% CI 0.62-1.68; P = 0.94), postoperative haemorrhage (9.6% versus 11.1%; OR 0.82, 95% CI 0.54-1.24; P = 0.35) or reoperation (9.9% versus 9.8%; OR 0.93, 95% CI 0.60-1.43; P = 0.73). CONCLUSIONS: Pancreaticogastrostomy provides benefits over PJ after PD, including in the incidences of pancreatic fistula, biliary fistula and intra-abdominal fluid collection and in hospital LoS. Therefore, PG is recommended as a safer and more reasonable alternative to PJ reconstruction after PD.

Prostate-targeted biodegradable nanoparticles loaded with androgen receptor silencing constructs eradicate xenograft tumors in mice.

BACKGROUND: Prostate cancer is the major cause of cancer death in men and the androgen receptor (AR) has been shown to play a critical role in the progression of the disease. Our previous reports showed that knocking down the expression of the AR gene using a siRNA-based approach in prostate cancer cells led to apoptotic cell death and xenograft tumor eradication. In this study, we utilized a biodegradable nanoparticle to deliver the therapeutic AR shRNA construct specifically to prostate cancer cells. MATERIALS & METHODS: The biodegradable nanoparticles were fabricated using a poly(dl-lactic-co-glycolic acid) polymer and the AR shRNA constructs were loaded inside the particles. The surface of the nanoparticles were then conjugated with prostate-specific membrane antigen aptamer A10 for prostate cancer cell-specific targeting. RESULTS: A10-conjugation largely enhanced cellular uptake of nanoparticles in both cell culture- and xenograft-based models. The efficacy of AR shRNA encapsulated in nanoparticles on AR gene silencing was confirmed in PC-3/AR-derived xenografts in nude mice. The therapeutic property of A10-conjugated AR shRNA-loaded nanoparticles was evaluated in xenograft models with different prostate cancer cell lines: 22RV1, LAPC-4 and LNCaP. Upon two injections of the AR shRNA-loaded nanoparticles, rapid tumor regression was observed over 2 weeks. Consistent with previous reports, A10 aptamer conjugation significantly enhanced xenograft tumor regression compared with nonconjugated nanoparticles. DISCUSSION: These data demonstrated that tissue-specific delivery of AR shRNA using a biodegradable nanoparticle approach represents a novel therapy for life-threatening prostate cancers.

Over-expression of angiotensin II type 2 receptor gene induces cell death in lung adenocarcinoma cells.

The endogenous angiotensin II (Ang II) type 2 receptor (AT 2) has been shown to mediate apoptosis in cardiovascular tissues. Thus, the aim of this study was to explore the anti-cancer effect of AT 2 over-expression on lung adenocarcinoma cells in vitro using adenoviral (Ad), FuGENE, and nanoparticle vectors. All three gene transfection methods efficiently transfected AT 2 cDNA into lung cancer cells but caused minimal gene transfection in normal lung epithelial cells. Ad-AT 2 significantly attenuated multiple human lung cancer cell growth (A549 and H358) as compared to the control viral vector, Ad-LacZ, when cell viability was examined by direct cell count. Examination of annexin V by flow cytometry revealed the activation of the apoptotic pathway via AT 2 over-expression. Western Blot analysis confirmed the activation of caspase-3. Similarly, poly (lactide-co-glycolic acid) (PLGA) biodegradable nanoparticles encapsulated AT 2 plasmid DNA were shown to be effectively taken up into the lung cancer cell. Nanoparticle-based AT 2 gene transfection markedly increased AT 2 expression and resultant cell death in A549 cells. These results indicate that AT 2 over-expression effectively attenuates growth of lung adenocarcinoma cells through intrinsic apoptosis. Our results also suggest that PLGA nanoparticles can be used as an efficient gene delivery vector for lung adenocarcinoma targeted therapy.

"Soft" calcium crosslinks enable highly efficient gene transfection using TAT peptide.

PURPOSE: Typically, low molecular weight cationic peptides or polymers exhibit poor transfection efficiency due to an inability to condense plasmid DNA into small nanoparticles. Here, efficient gene delivery was attained using TAT/pDNA complexes containing calcium crosslinks. METHODS: Electrostatic complexes of pDNA with TAT or PEI were studied with increasing calcium concentration. Gel electrophoresis was used to determine DNA condensation. The morphology of the complexes was probed by transmission electron microscopy. Transfection efficiency was assessed using a luciferase reporter plasmid. The accessibility of phosphate and amine groups within complexes was evaluated to determine the effect of calcium on structure. RESULTS: TAT/pDNA complexes were condensed into small, 50-100 nm particles by optimizing the concentration of calcium. Complexes optimized for small size also exhibited higher transfection efficiency than PEI polyplexes in A549 cells. TAT and TAT complexes displayed negligible cytotoxicity up to 5 mg/mL, while PEI exhibited high cytotoxicity, as expected. Probing the TAT-Ca/pDNA structure suggested that calcium interacted with both phosphate and amine groups to compact the complexes; however, these "soft" crosslinks could be competitively disrupted to facilitate DNA release. CONCLUSION: Small and stable TAT-Ca/pDNA complexes were obtained via "soft" calcium crosslinks leading to sustained gene expression levels higher than observed for control PEI gene vectors. TAT-Ca/pDNA complexes were stable, maintaining particle size and transfection efficiency even in the presence of 10% of FBS. TAT-Ca complexes offer an effective vehicle offering potential for translatable gene delivery.

ICAM-1 targeting of doxorubicin-loaded PLGA nanoparticles to lung epithelial cells.

Interaction of leukocyte function associated antigen-1 (LFA-1) on T-lymphocytes and intercellular adhesion molecule-1 (ICAM-1) on epithelial cells controls leukocyte adhesion, spreading, and extravasation. This process plays an important role in leukocyte recruitment to a specific site of inflammation and has been identified as a biomarker for certain types of carcinomas. Cyclo-(1,12)-PenITDGEATDSGC (cLABL) has been shown to inhibit LFA-1 and ICAM-1 interaction via binding to ICAM-1. In addition, cLABL has been shown to internalize after binding ICAM-1. The possibility of using cLABL conjugated nanoparticles (cLABL-NP) as a targeted and controlled release drug delivery system has been investigated in this study. The cLABL peptide was conjugated to a modified Pluronic surfactant on poly (DL-lactic-co-glycolic acid) (PLGA) nanoparticles. The cLABL-NP showed more rapid cellular uptake by A549 lung epithelial cells compared to nanoparticles without peptide. The specificity of ICAM-1-mediated internalization was confirmed by blocking the uptake of cLABL-NP to ICAM-1 using free cLABL peptide to block the binding of cLABL-NP to ICAM-1 on the cell surface. Cell studies suggested that cLABL-NPs targeted encapsulated doxorubicin to ICAM-1 expressing cells. Cytotoxicity assay confirmed the activity of the drug incorporated in nanoparticles. Sustained release of doxorubicin afforded by PLGA nanoparticles may enable cLABL-NP as a targeted, controlled release drug delivery system.

Antitumoral activity of rapamycin mediated through inhibition of HIF-1alpha and VEGF in hepatocellular carcinoma.

Rapamycin (RAPA) inhibits tumor growth and angiogenesis in hepatocellular carcinoma (HCC). The molecular mechanism underlying the antitumoral effects of RAPA remains unclear. Here we established a chemical-induced rat HCC model to investigate the signaling pathways mediating RAPA's antitumor activity. We found that RAPA exposure significantly diminished tumor growth, angiogenesis, and metastasis of HCC. Meanwhile, the antitumor drug dramatically decreased expression of HIF-1alpha and VEGF, either at mRNA or protein levels. Moreover, the low-dose of RAPA (1.5 mg/kg/day) was effective enough to markedly inhibit tumor progression of HCC. The preliminary results suggested that the antitumoral effects of RAPA might be at least partially mediated through downregulation of HIF-1alpha and VEGF, and low-dose RAPA-based regimens exhibited a promising future in treatment of HCC.

Structural analysis of inhibition of E. coli methionine aminopeptidase: implication of loop adaptability in selective inhibition of bacterial enzymes.

BACKGROUND: Methionine aminopeptidase is a potential target of future antibacterial and anticancer drugs. Structural analysis of complexes of the enzyme with its inhibitors provides valuable information for structure-based drug design efforts. RESULTS: Five new X-ray structures of such enzyme-inhibitor complexes were obtained. Analysis of these and other three similar structures reveals the adaptability of a surface-exposed loop bearing Y62, H63, G64 and Y65 (the YHGY loop) that is an integral part of the substrate and inhibitor binding pocket. This adaptability is important for accommodating inhibitors with variations in size. When compared with the human isozymes, this loop either becomes buried in the human type I enzyme due to an N-terminal extension that covers its position or is replaced by a unique insert in the human type II enzyme. CONCLUSION: The adaptability of the YHGY loop in E. coli methionine aminopeptidase, and likely in other bacterial methionine aminopeptidases, enables the enzyme active pocket to accommodate inhibitors of differing size. The differences in this adaptable loop between the bacterial and human methionine aminopeptidases is a structural feature that can be exploited to design inhibitors of bacterial methionine aminopeptidases as therapeutic agents with minimal inhibition of the corresponding human enzymes.

Inhibition of monometalated methionine aminopeptidase: inhibitor discovery and crystallographic analysis.

Two divalent metal ions are commonly seen in the active-site cavity of methionine aminopeptidase, and at least one of the metal ions is directly involved in catalysis. Although ample structural and functional information is available for dimetalated enzyme, methionine aminopeptidase likely functions as a monometalated enzyme under physiological conditions. Information on structure, as well as catalysis and inhibition, of the monometalated enzyme is lacking. By improving conditions of high-throughput screening, we identified a unique inhibitor with specificity toward the monometalated enzyme. Kinetic characterization indicates a mutual exclusivity in binding between the inhibitor and the second metal ion at the active site. This is confirmed by X-ray structure, and this inhibitor coordinates with the first metal ion and occupies the space normally occupied by the second metal ion. Kinetic and structural analyses of the inhibition by this and other inhibitors provide insight in designing effective inhibitors of methionine aminopeptidase.

Effects of impromidine- and arpromidine-derived guanidines on recombinant human and guinea pig histamine H1 and H2 receptors.

Imidazolylpropylguanidines derived from impromidine and arpromidine are more potent and efficacious agonists at the guinea pig histamine H2 receptor (gpH2R) than at the human H2R (hH2R) in the GTPase assay. Additionally, such guanidines are histamine H1 receptor (H1R) antagonists with preference for the human relative to the guinea pig receptor. The purpose of this study was to examine structure-activity relationships of guanidines at human and guinea pig H1R and H2R species isoforms expressed in Sf9 insect cells. Three impromidine analogues and six arpromidine analogues exhibited agonistic activity at H2R and antagonistic activity at H1R as assessed in the steady-state GTPase assay. Species selectivity of derivatives was similar as compared with the parent compounds. None of the structural modifications examined (different aromatic ring systems and different ring substituents) was superior in terms of H2R potency and efficacy relative to impromidine and arpromidine, respectively. These data point to substantial structural constraints at the agonist binding site of H2R. Guanidines exhibited distinct structure-activity relationships for H1R antagonism in a radioligand competition binding assay and the GTPase assay and for H1R inverse agonism. Our data indicate that it is difficult to obtain guanidine-type agonists with high potency and high efficacy for hH2R, but those compounds may be useful tools for exploring the antagonist binding site and constitutive activity of H1R.

Structural basis of catalysis by monometalated methionine aminopeptidase.

Methionine aminopeptidase (MetAP) removes the amino-terminal methionine residue from newly synthesized proteins, and it is a target for the development of antibacterial and anticancer agents. Available x-ray structures of MetAP, as well as other metalloaminopeptidases, show an active site containing two adjacent divalent metal ions bridged by a water molecule or hydroxide ion. The predominance of dimetalated structures leads naturally to proposed mechanisms of catalysis involving both metal ions. However, kinetic studies indicate that in many cases, only a single metal ion is required for full activity. By limiting the amount of metal ion present during crystal growth, we have now obtained a crystal structure for a complex of Escherichia coli MetAP with norleucine phosphonate, a transition-state analog, and only a single Mn(II) ion bound at the active site in the position designated M1, and three related structures of the same complex that show the transition from the mono-Mn(II) form to the di-Mn(II) form. An unliganded structure was also solved. In view of the full kinetic competence of the monometalated MetAP, the much weaker binding constant for occupancy of the M2 site compared with the M1 site, and the newly determined structures, we propose a revised mechanism of peptide bond hydrolysis by E. coli MetAP. We also suggest that the crystallization of dimetalated forms of metallohydrolases may, in some cases, be a misleading experimental artifact, and caution must be taken when structures are generated to aid in elucidation of reaction mechanisms or to support structure-aided drug design efforts.