Добірка наукової літератури з теми "Enzyme; inhibitor design; biotin protein ligase"

Biotin protein ligase (BPL) inhibitors are a novel class of antibacterial that target clinically important methicillin-resistant Staphylococcus aureus (S. aureus). In S. aureus, BPL is a bifunctional protein responsible for enzymatic biotinylation of two biotin-dependent enzymes, as well as serving as a transcriptional repressor that controls biotin synthesis and import. In this report, we investigate the mechanisms of action and resistance for a potent anti-BPL, an antibacterial compound, biotinyl-acylsulfamide adenosine (BASA). We show that BASA acts by both inhibiting the enzymatic activity of BPL in vitro, as well as functioning as a transcription co-repressor. A low spontaneous resistance rate was measured for the compound (<10−9) and whole-genome sequencing of strains evolved during serial passaging in the presence of BASA identified two discrete resistance mechanisms. In the first, deletion of the biotin-dependent enzyme pyruvate carboxylase is proposed to prioritize the utilization of bioavailable biotin for the essential enzyme acetyl-CoA carboxylase. In the second, a D200E missense mutation in BPL reduced DNA binding in vitro and transcriptional repression in vivo. We propose that this second resistance mechanism promotes bioavailability of biotin by derepressing its synthesis and import, such that free biotin may outcompete the inhibitor for binding BPL. This study provides new insights into the molecular mechanisms governing antibacterial activity and resistance of BPL inhibitors in S. aureus.

Abstract Abstract 1415 A growing number of compounds with proteasome and E3-ligase inhibitory activity have demonstrated clinical activity against multiple myeloma (MM) and mantle cell lymphoma. Some of these compounds are now part of the therapeutic regimen. In addition, there is a growing body of evidence that specific deubiquitinases (DUBs) are overexpressed in hematologic malignancies and may be appropriate myeloma/lymphoma therapeutic targets. However, only a limited number of compounds have been described with cell-permeant and selective activity for DUBs. We previously reported the DUB inhibitory activity of small molecule WP1130. This compound caused rapid inactivation of Usp9x, a DUB reported to be overexpressed in MM and some lymphoma primary specimens. Usp9x associates with the anti-apoptotic protein Mcl-1 and protects it from proteasomal destruction by deubiquitination. We have shown that WP1130 inhibits Usp9x activity (and some additional DUBs) and stimulates proteasomal destruction of Mcl-1. However, the mechanism of Usp9x inhibition by WP1130 has not been described. To investigate this, we synthesized a biotinylated derivative of WP1130 that retains cell-penetrant Usp9x DUB inhibitory activity. Using this as a probe, we demonstrate that WP1130 directly and stably interacts with Usp9x in high salt and ionic detergent, suggesting high affinity complex formation. Proteomic assessment revealed that other previously defined cellular targets of WP1130 (Usp5, Usp14, UCH-L5) also formed high affinity complexes with WP1130-biotin. Additional studies suggested that WP1130-biotin covalently modifies Usp9x through a Michael addition reaction with critical Usp9x cysteine residues that result in the inhibition of DUB activity. To further map the sites of the Usp9x-WP1130 interaction, we produced a recombinant catalytic domain of Usp9x (Usp9xCD) and verified that it retained enzymatic activity that could be inhibited by WP1130, albeit at higher concentrations than that required to inhibit the enzyme in intact cells. Mass spectral analysis of Usp9xCD/WP1130 complexes demonstrated formation of covalent WP1130 adducts with a concentration and time dependent stoichiometry. The catalytic domain was also used to determine the affinity and reversibility of WP1130 binding to Usp9x by solution-phase analysis using Bioforte instrumentation. These studies suggested a slow but high-affinity (<1 μM), irreversible binding of WP1130 to the catalytic domain in addition to a second, low-affinity, reversible binding mode. Molecular simulation was used to identify accessible and energetically favorable Cys residues that could represent the location(s) of high-affinity WP1130 binding. These sites are being confirmed by proteomic assessment of WP1130-biotin-retaining peptides derived from trypsinization of Usp9x/WP1130 adducts. Further structural simulation of Usp9x using homology modeling suggested that the catalytic domain of Usp9x contains a unique lobe (aa1576–1642), which lies close to recently described sites of mTOR phosphorylation. Our initial studies suggest that inhibition of mTOR (with Torin-2) in MM cells results in activation of Usp9x activity. The role of phosphorylation in the regulation of Usp9x activity and stability is currently being investigated but our initial observations suggest that Usp9x plays an important regulatory role in the mTOR/5'-AMP kinase cascade. Collectively, our studies suggest that WP1130 inhibits Usp9x activity through covalent modification of critical Cys residues important for Usp9x catalytic activity. Identification of these sites and how phosphorylation influences Usp9x catalytic activity are critical for developing more potent and selective Usp9x inhibitors. Since Usp9x expression has been associated with short survival and poor prognosis in MM patients, potent and selective Usp9x inhibitors could show therapeutic promise for MM patients and other cancers with up-regulated Usp9x activity. Disclosures: Jakubowiak: Ortho Biotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Exelixis: Consultancy, Honoraria. Talpaz:ARIAD: Research Funding.

Abstract Notch signaling represents a central pathway regulating hematopoiesis, stem cell differentiation, and malignant transformation in human cancer. Activation of highly conserved Notch1 receptors results in cleavage and release of an intracellular domain (ICN1). Following translocation to the nucleus, ICN1 forms a ternary complex with the transcriptional repressor CSL (CBF-1, Suppressor of Hairless and Lag-1) bound to cognate DNA. This event triggers a repressor-to-activator switch, as an interfacial groove is formed which recruits the Mastermind-Like (MAML1) co-activator protein. Activating mutations in NOTCH1 are found in more than 50% of patients with T-Cell Acute Lymphoblastic Leukemia (T-ALL), promoting protein stability and establishing a direct link to disease pathogenesis. Pharmacologic efforts to target the Notch pathway in T-ALL have been directed at gamma secretase, a regulatory enzyme in Notch activation. Recently, the observed clinical resistance to gamma secretase inhibitors has been explained, in part, by additional mutations in the Notch-targeting ubiquitin ligase, Fbxw7, which further increases oncoprotein stability. Therefore, direct inhibitors of ICN1 function are highly desirable. Drawing upon insights afforded by the resolved crystal structure of the DNA-bound ICN1:MAML1:CSL complex, we synthesized a series of hydrocarbon stapled alpha-helical peptides targeting Notch (SAHNs) based on minimal motifs of the MAML protein predicted to engage the composite ICN1:CSL interface. Direct, high-affinity binding to purified components of the Notch complex was confirmed using surface plasmon resonance (SPR). Nuclear access of SAHN1 was confirmed using quantitative epifluorescent and confocal microscopy. Intracellular association with ICN1 and CSL was established using bidirectional affinity chromatography. Using a novel CSL-responsive reporter construct, we observed inhibition of endogenous Notch transactivation by SAHN1 in T-ALL cell lines. Furthermore, SAHN1 induces a dose-dependent knockdown of endogenous Notch1 target genes including HES1, HEY1 and cMYC in T-ALL cell lines. Remarkably, inhibition of Notch signaling by SAHN1 confers selective cytotoxicity at 48 hours in a panel of T-ALL cell lines with known mutations in NOTCH, including those resistant to gamma secretase inhibitors. Supporting an on-target mechanism of action, we have prepared a damaged analogue of SAHN1 containing a two-residue rearrangement (SAHN1D). SAHN1D possesses reduced binding affinity for the Notch complex and despite comparable intracellular access, SAHN1D lacks both transcriptional and cytotoxic effects on cultured T-ALL cell lines in vitro. Efficacy studies have also been performed in vivo using a novel murine model of T-ALL. In summary, we report here the design, biochemical characterization and translational rationale supporting the first direct inhibitor of the Notch transactivation complex in T-ALL.

Background: Buruli ulcer (BU), caused by Mycobacterium ulcerans is a neglected tropical disease characterized by necrotic skin lesions. Antibiotic therapy and excision of the lesions are the treatments for this chronic disease. During the management of the disease, the emergence of drug resistance in these bacilli is a major challenge. Therefore, there is a need to identify new drug targets against this important pathogen. Objective: The study aimed to investigate novel drug targets exploring virulence factors of M. ulcerans by in silico analysis. Method: Virulence proteins encoded by the chromosome of Mycobacterium ulcerans strain Agy99 were retrieved and analyzed for their cellular localization, human non-homology and essentiality. Further, proteins were analyzed for their physio-chemical characterization, drug resistance analysis, protein interaction analysis, metabolic pathway prediction, and druggability prediction by various databases and online software to find their suitability as drug targets. The structure of the predicted drug targets was also modeled and validated. Among three predicted drug targets, MUL_4536 was subjected to molecular docking with some known inhibitor compounds also. Receptor-ligand complex with the highest binding energy was selected for molecular dynamic (MD) simulation to determine the structural stability of the complex. Results: Three virulence proteins MUL_4536, MUL_3640, and MUL_2329 encoding enzymes iso-citrate lyase, lysine-N-oxygenase, pup-protein ligase, respectively were predicted as a drug target against M. ulcerans. Isocitrate lyase has been identified as a potential drug target in many other mycobacterial and non-mycobacterial diseases. Lysine-N-oxygenase is the enzyme of mycobactin biosynthesis pathway and pup-protein ligase is associated with the pup-proteasome system. Proteins of these pathways have been studied as attractive drug targets in previous research works, which further support our predictions. Conclusion: Our computational analysis predicted new drug targets, which could be used to design drugs against M. ulcerans. However, these predicted proteins require further experimental validation for their potential use as drug targets. other: NA

There is a well-documented need to replenish the antibiotic pipeline with new products to combat the rise of drug resistant bacteria, such as the superbug methicillin resistant Staphylococcus aureus (MRSA). One strategy to combat drug resistance is to identify new chemical classes with novel mechanisms of action and that are not subject to existing resistance mechanisms. As most of the obvious bacterial drug targets with no equivalents in mammals have been well explored, targets with a closely related human homologue represent a new frontier in antibiotic discovery. However, to avoid potential toxicity to the host, these inhibitors must have extremely high selectivity for the bacterial target over the human equivalent. This thesis is focused upon exploiting the ubiquitous enzyme biotin protein ligase (BPL), which is involved in the essential cellular process of attaching biotin onto biotin-dependent enzymes. Due to the pivotal metabolic roles played by biotin-dependent enzymes in bacteria, BPL has been proposed as a promising new antibiotic target. Hence, BPL inhibitors with selectivity for the bacterial isozyme over the human equivalent promise a new class of antibiotic to combat MRSA. The aim of this project was to provide proof of concept data demonstrating that BPL from a pathogen could be selectively targeted for inhibition over the human equivalent. Here I employed a combination of structure-guided drug design and fragment-based approaches to discover novel BPL inhibitors. The X-ray crystal structure of S. aureus BPL (SaBPL) shows two adjacent binding sites for the ligands biotin and ATP, making it an ideal candidate for a fragment-based approach to drug discovery. Although the residues at the biotin-binding site are highly conserved, the nucleotide pocket shows a high degree of variability that can be exploited to create compounds selective towards BPLs from pathogens. The biotin 1,2,3 triazole analogues identified in this work yielded our most potent and selective inhibitor (Ki = 90 nM) [i is subscript] with >1100-fold selectivity for the SaBPL over the human homologue (Chapter 2). The molecular basis for the selectivity was identified using mutagenesis studies with a key arginine residue in the BPL active site necessary for selective binding. Importantly, the biotin triazole inhibitors showed in vivo cytotoxicity against S. aureus, but not cultured mammalian cells (Chapter 2). In an attempt to identify new chemical scaffolds with improved ligand efficiency for chemical development, a series of analogues based on the natural ligand biotin were also designed and tested for enzyme inhibition and antimicrobial activities against clinically relevant strains of S. aureus (Chapter 3). This approach resulted in highly potent compounds (Ki < 100 nM) [i is subscript] with antibacterial activity against MRSA strains (MIC = 2 – 16 μg/mL). Whilst only moderate selectivity over the human enzyme (10 - 20 fold) was observed, the biotin analogues provided a suitable chemical scaffold with high ligand efficiency for further chemical development. One of the compounds identified was biotin acetylene, which forms a long lived complex with SaBPL and is a precursor for in situ click reactions. This target-guided approach to drug discovery relies on the ability of the enzyme to choose its own inhibitors from a range of acetylene and azide building blocks to form specific triazole products. Since a class of biotin-triazole molecules had already been identified as selective inhibitors of SaBPL, we reasoned that this enzyme would provide an ideal candidate for performing in situ click approach to inhibitor discovery. In this work, a protocol for the BPL-catalyzed in situ click reaction was optimized to select the optimum triazole-based inhibitor using biotin acetylene as an anchor molecule to recruit complimentary fragments that could bind in the peripheral ATP pocket (Chapter 4). The in situ reaction was shown to be improved by the use of a SaBPL mutant that promoted diffusion of the triazole product from the active site following synthesis. This novel approach improved efficiency and ease of detection (Chapter 4). Apart from drug discovery, this thesis also focuses on enzymatic characterization of SaBPL and highlighting the key differences between SaBPL and the human homologue. The structure of human BPL is yet to be reported, so structure-function studies were performed to elucidate new information about the bacterial and human enzymes. The oligomeric state of SaBPL was investigated using analytical ultracentrifugation in its apo form and in the presence of ligands (Chapter 5). Unlike human BPL, SaBPL was shown to dimerize in solution. A single amino acid in SaBPL, Phe123, was identified to have a dual key role in dimer formation and inhibitor binding (Chapter 5). One of the major roadblocks to obtaining crystals of the full-length human enzyme is the low yield of protein obtained from recombinant expression and purification. In this thesis, an alternative approach is described that could be used to increase our chances of obtaining structural data about the human BPL. I created a ‘humanized’ chimeric protein in which all seven residues in the nucleotide pocket of SaBPL that are not conserved with the human BPL were mutated to their human equivalents. This ‘humanized’ protein exhibited similar kinetic and inhibition properties to the human enzyme (Chapter 6). Crystal trials have commenced to help direct future drug development efforts. Further studies on the human BPL enzyme will also be described, including the dissection of the binding mechanism using surface plasmon resonance (Chapter 7). The N-terminal domain of this enzyme was shown to play a role in stabilizing the complex between the enzyme and the biotin domain substrate, providing the first molecular explanation for human BPL-deficient patients that do not respond to biotin therapy. In summary, this work demonstrates for the first time that BPL from the clinically important pathogen Staphylococcus aureus can be selectively inhibited. A provisional patent has been filed for the biotin 1,2,3 triazole molecules I have identified. These discoveries will enable further development of a new class of antibiotics.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2013

Biotin protein ligase (BPL) catalyses the ordered reaction of biotin and ATP to give biotinyl-5’-AMP 1.03, which then activates a number of biotin dependent enzymes that are critical to cell survival. Research undertaken in this thesis highlights strategies to selectively inhibit Staphylococcus aureus biotin protein ligase (SaBPL) over the mammalian equivalent using 1,2,3-triazole and acylsulfonamide isosteres to replace the phosphoroanhydride linker found in biotinyl-5’-AMP 1.03. Chapter one describes the structure and catalytic mechanism of the target enzyme SaBPL, along with an overview of chemical analogues of biotin and biotinyl-5’-AMP 1.03 as BPL inhibitors reported to date. Preliminary studies on the utility of a 1,2,3-triazole as a bioisostere of the phosphoroanhydride linker of biotinyl-5’-AMP 1.03 are also discussed. Chapter two further examines 1,2,3-triazole analogues of lead SaBPL bisubstrate inhibitors 1.22 and 1.23. Specific chemical modifications were carried out at the ribose of biotinyl-5’- AMP 1.03, and a new class of purine analogues was developed to mimic the adenine group as in 1.03. In silico docking experiments using our x-ray structure of SaBPL aided in the design of these analogues by predicting optimal binding conformations. A structure activity relationship for the ribose and adenine mimics was developed and this revealed limited improvement in potency against SaBPL on modification at these two sites. Chapter three reports the first examples of truncated 1,2,3-triazole based BPL inhibitors with a 1-benzyl substituent designed to interact with the ribose binding pocket of SaBPL. In silico docking studies using a crystal structure of SaBPL aided in the selection of benzyl groups that present in the ribose-binding pocket of SaBPL. The halogenated benzyl derivatives 3.20, 3.21, 3.23 and 3.24 provided the most potent inhibitors of SaBPL with the respective Kᵢ value of 0.28, 0.6, 0.39 and 1.1 μM. These compounds also inhibited the growth of S. aureus ATCC49775 (MIC = 4 – 16 μg/ml), while possessing low cytotoxicity against HepG2 cells. Chapter four builds upon the active 1,2,3-triazole based inhibitors of SaBPL described in chapter two and three with an investigation at C5 of the triazole ring to generate 1,4,5- trisubstituted 1,2,3-triazoles. A class of 5-iodo 1,2,3-triazoles was synthesised from 1- iodoacetylene 4.02 and azides using CuAAC. Subsequent halogen exchange reaction allowed conversion of iodide to other halogens. 5-Fluoro-1,2,3-triazole 4.07, the lead compound from this series of inhibitors, proved to be a potent and selective inhibitor of SaBPL (Kᵢ = 0.42 ± 0.06 μM) and it significantly reduced S. aureus growth with no cell growth apparent at 16 μg/mL. Chapter five investigates the use of acylsulfonamide as a bioisostere of the phosphoroanhydride linker as in biotinyl-5’-AMP 1.03. Acylsulfonamide 5.05 was found as the most active and selective inhibitor of SaBPL (Kᵢ = 0.72 x 10⁻³ μM) and MtbBPL (Kᵢ = 0.74 x 10⁻³ μM) reported to date. Antibacterial studies revealed that 5.05 was active against susceptible S. aureus (MIC = 0.5-1.0 μg/mL), methicillin-resistant S. aureus ((MIC = 0.5- 1.0 μg/mL) and Mycobacterial tuberculosis ((MIC = 51 μg/mL). Finally, the x-ray structure 5.05 bound to SaBPL was solved to reveal important molecular interactions critical to the potency of 5.05 and emphasized the acylsulfonamide moiety as an effective bioisostere of phosphoroanhydride linker. Chapter six discusses the use of in situ click chemistry as an alternative approach for the synthesis of 1,2,3-triazoles. The target enzyme SaBPL was directly involved in the selection of its optimum triazole based inhibitor by catalysing the reaction of biotin acetylene and organic azides without copper as a catalyst. The use of high throughput LC/MS provided improved efficiency and sensitivity of detection of triazole-based inhibitors and allowed the in situ approach to be widely applied to BPLs from other bacteria. Chapter seven details the experimental procedures for compounds described in chapter 2 – 6, and the chromatographic analysis of in situ click experiments described in chapter 6.
Thesis (Ph.D.) -- University of Adelaide, School of Physical Sciences, 2016.