High Yield Expression of Human BACE Constructs in Eschericia coli for Refolding, Purification, and High Resolution Diffracting Crystal Forms

Tomasselli, A. G.; Paddock, Donna J.; Emmons, Thomas L.; Mildner, Ana M.; Leone, Joseph W.; Lull, June M.; Cialdella, J. I.; Prince, D. Bryan; Fischer, H.; Heinrikson, Robert L.; Benson, Timothy E.

doi:10.2174/092986608783489553

Cited by 14 publications

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“…This study has been performed with the aim of obtaining information about the structural characteristics responsible for the anti‐HIV activity of this class of compounds, that is, to understand the effect of substitution on their inhibitory activity using Kier‐Hall E‐state indices. Also, the study is aimed at determining QSAR of HIV‐1 NNRTIs for the class of TIBO1, 4 and its derivatives 18. E‐state indices for various functional groups (namely, E X‐8 , E Z‐O , E Z‐S, E R , E X′‐7 , E X′‐di , E N , E NR ) are calculated using e‐calc 40.…”

Section: Methodsmentioning

confidence: 99%

“…The human immunodeficiency virus (HIV‐1)1–5 is the causative agent of the dreaded global disease, acquired immunodeficiency syndrome (AIDS), which was first detected in mid‐1980s 6. It is now recognized that the immune deficiency is a consequence of continuous high‐level HIV replication leading to an increased impairment of cell‐mediated immunity due to gradual attrition of the key immune effector cell, the CD4 lymphocyte 7.…”

Section: Introductionmentioning

confidence: 99%

“…Till now, a wide range of compounds have been synthesized and tested to combat the pandemic disease. Tetrahydroimidazo‐[4,5,1‐jk]‐benzodiazepinone (TIBO)1, 4 and its derivatives reported by Pauwels et al18 is one of the most potent and selective inhibitor exhibiting vital activity against the reverse transcriptase enzyme (HIV‐1 RT). Following the discovery of the TIBO18 and a‐APA,19 compounds from a broad screen of the Janssen compound library, systematic lead optimization yielded the NNRTIs tivirapine (R86183), a TIBO derivative,20 and loviride (R90385), an a‐APA derivative.…”

Section: Introductionmentioning

confidence: 99%

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Computational modeling of tetrahydroimidazo‐[4,5,1‐jk][1,4]‐benzodiazepinone derivatives: An atomistic drug design approach using Kier‐Hall electrotopological state (E‐state) indices

Sapre¹,

Pancholi²,

Gupta³

et al. 2008

J Comput Chem

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Quantitative structure-activity relationships (QSAR), based on E-state indices have been developed for a series of tetrahydroimidazo-[4,5,1-jk]-benzodiazepinone derivatives against HIV-1 reverse transcriptase (HIV-1 RT). Statistical modeling using multiple linear regression technique in predicting the anti-HIV activity yielded a good correlation for the training set (R(2) = 0.913, R(2)(adj) = 0.897, Q(2) = 0.849, MSE = 0.190, F-ratio = 59.97, PRESS = 18.05, SSE = 0.926, and p value = 0.00). Leave-one-out cross-validation also reaffirmed the predictions (R(2) = 0.850, R(2)(adj) = 0.824, Q(2) = 0.849, MSE = 0.328, and PRESS = 18.05). The predictive ability of the training set was also cross-validated by a test set (R(2) = 0.812, R(2)(adj) = 0.799, Q(2) = 0.765, MSE = 0.347, F-ratio = 64.69, PRESS = 7.37, SSE = 0.975, and p value = 0.00), which ascertained a satisfactory quality of fit. The results reflect the substitution pattern and suggest that the presence of a bulky and electropositive group in the five-member ring and electron withdrawing groups in the seven-member ring will have a positive impact on the antiviral activity of the derivatives. Bulky groups in the six-member ring do not show an activity-enhancing impact. Outlier analysis too reconfirms our findings. The E-state descriptors indicate their importance in quantifying the electronic characteristics of a molecule and thus can be used in chemical interpretation of electronic and steric factors affecting the biological activity of compounds.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Computational modeling of tetrahydroimidazo‐[4,5,1‐jk][1,4]‐benzodiazepinone derivatives: An atomistic drug design approach using Kier‐Hall electrotopological state (E‐state) indices

Sapre¹,

Pancholi²,

Gupta³

et al. 2008

J Comput Chem

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“…Previously, it was shown that although BACE knockout mice have reduced Ab production, they do not exhibit any abnormal phenotypes (Luo et al 2001). The BACE gene was cloned and characterized in 1999 (Vassar et al 1999), and various BACE constructs have been expressed in insect (Bruinzeel et al 2002), mammalian , and E. coli cells (Sardana et al 2004;Tomasselli et al 2008).…”

Section: Introductionmentioning

confidence: 99%

High-level expression of a human β-site APP cleaving enzyme in transgenic tobacco chloroplasts and its immunogenicity in mice

et al. 2010

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Plastid transformation has to date been applied to the expression of heterologous genes involved in agronomic traits and to the production of useful recombinant proteins. Here, we report a feasibility study for producing the human β-site APP cleaving enzyme (BACE) via transformation of tobacco chloroplasts. Stable integration of human BACE into the plastome was confirmed by PCR. Genomic Southern blot analysis detected the presence of the tobacco aadA and human BACE genes between trnI and trnA in the plastome. Northern blot analysis revealed that the aadA and BACE genes were both properly transcribed into a dicistronic transcriptional unit. Human BACE protein expression in transplastomic tobacco was determined by western blot analysis. ELISA analysis revealed that, based on a dilution series of E. coli-derived BACE as a standard, transplastomic lines accumulated BACE to levels of 2.0% of total soluble proteins. When mice were gavaged with the transplastomic tobacco extracts, they showed an immune response against the BACE antigen. The successful production of plastid-based BACE protein has the potential for developing a plant-based vaccine against Alzheimer disease.

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“…According to the current literature, purifying BACE1 using an E. coli expression system requires either multiple, pHadjusted refolding steps 18 or a particular affinity column for purification. 19 Overall, the protein yield ranges from 1 to 8 mg from 1 L of culture. Here, a relatively simple refolding and purification method.…”

mentioning

confidence: 99%

Development of an Efficient Enzyme Production and Structure-Based Discovery Platform for BACE1 Inhibitors

et al. 2019

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BACE1 (Beta-site Amyloid Precursor Protein (APP) Cleaving Enzyme 1) is a promising therapeutic target for Alzheimer’s Disease (AD). However, efficient expression, purification, and crystallization systems are not well described or detailed in the literature nor are approaches for treatment of enzyme kinetic data for potent inhibitors well described. We therefore developed a platform for expression and purification of BACE1, including protein refolding from E.coli inclusion bodies, in addition to optimizing a reproducible crystallization procedure of BACE1 bound with inhibitors. We also report a detailed approach to the proper analysis of enzyme kinetic data for compounds that exhibit either rapid-equilibrium or tight-binding mechanisms. Our methods allow for the purification of ∼15 mg of BACE1 enzyme from 1 L of culture which is higher than reported yields in the current literature. To evaluate the data analysis approach developed here, a well-known potent inhibitor and two of its derivatives were tested, analyzed, and compared. The inhibitory constants (K i) obtained from the kinetic studies are in agreement with dissociation constants (K d) that were also determined using isothermal titration calorimetry (ITC) experiments. The X-ray structures of these three compounds in complex with BACE1 were readily obtained and provide important insight into the structure and thermodynamics of the BACE1-inhibitor interactions.

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High Yield Expression of Human BACE Constructs in Eschericia coli for Refolding, Purification, and High Resolution Diffracting Crystal Forms

Cited by 14 publications

References 0 publications

Computational modeling of tetrahydroimidazo‐[4,5,1‐jk][1,4]‐benzodiazepinone derivatives: An atomistic drug design approach using Kier‐Hall electrotopological state (E‐state) indices

Computational modeling of tetrahydroimidazo‐[4,5,1‐jk][1,4]‐benzodiazepinone derivatives: An atomistic drug design approach using Kier‐Hall electrotopological state (E‐state) indices

High-level expression of a human β-site APP cleaving enzyme in transgenic tobacco chloroplasts and its immunogenicity in mice

Development of an Efficient Enzyme Production and Structure-Based Discovery Platform for BACE1 Inhibitors

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