Optimized growth conditions for HIV protease production showed that the use of chemically defined media resulted in higher yields of the enzyme. The spectrum illustrates the absorption of ultraviolet light by the side chains of the aromatic amino acids of the protein at a wavelength of 280 nm.
INTRODUCTION AND PROJECT AIMS
The field of drug discovery is one of the most interesting and complex fields of research. As such, this study aims to predict the permeability capabilities of the chemically synthesized HIV-1 protease inhibitors using cell-based and artificial membrane techniques.
LITERATURE REVIEW
The transport of drugs through the human body
- Intestinal transport of drugs
- Passive transport
- Active transport
- Hepatic clearance
It has been reported that the most common mode of drug transport is through the transcellular route, because the surface area of the intestinal epithelium is 1000 times larger than the paracellular spaces [18]. Once the drug has escaped metabolism in the intestine, it would have crossed the basolateral membrane of the enterocyte.
Multi-drug resistance
It will then be transported, via the portal vein, to the liver, which is the main site of metabolism in the human body [15]. The current model of the mechanism of action of P-glycoprotein is that drugs can be detected and expelled when they enter the plasma membrane in the manner of a.
Unstirred water layer (UWL)
Additional MDR protein efflux pumps found in enterocytes include the breast cancer resistance protein (BCRP) and the multidrug resistance family (MDRP 1–6). P-gp is recognized as a member of the ATP-binding cassette (ABC) membrane transport protein superfamily.
Properties of potential drugs for optimum absorption
- Solubility and dissolution
- Ionization (pK a )
- Particle size
Therefore, there is a trade-off between solubility and permeability due to the effects of ionization. Another aspect for determining “drug-like” moieties is the framework of the specific drug.
In vitro tools for predicting drug permeability
- Cell-based methods
- The Caco-2 cell line
- The MDCK cell line
- The HT29 cell line
- Methods utilizing artificial membranes
- Immobilized Artificial Membrane (IAM) Columns
- Parallel Artificial Membrane Permeability Assay (PAMPA)
As time progresses, passive diffusion is verified by the transfer of the compound from the apical to the basolateral side through the cell monolayer. Caco-2 cells produce tight junctions between their cells and contain many of the transporters found within the intestinal barrier. In addition, a characteristic feature of this cell line is the expression of the P-gp protein.
Although in some research results where the focus is solely on permeability, this does not represent a positive characteristic of the cell line. This is due to the fact that it interacts with the stationary lipid phase of the column.
![Figure 2.7 Schematic representation of the PAMPA system. Adapted from Manson [75].](https://thumb-ap.123doks.com/thumbv2/pubpdfnet/10643762.0/44.892.119.803.779.909/figure-2-schematic-representation-pampa-adapted-manson-75.webp)
HIV Protease (HIV-PR)
- HIV protease structure
- Mechanism of action
- Substrate binding
- Novel chemically designed protease inhibitors
There are many reports on the mechanism of action of the HIV-1 protease, but most refer to the protease having an acid-base mechanism of action (similar to most enzymes in the aspartic protease family). The most promising mimic of the transition state was hydroxyethylamine, which led to the discovery of the first protease inhibitor, saquinavir. There are reports of different subtypes of HIV-1 occurring in different parts of the world [88].
One of the most remarkable features of the addition of cage parts is that it is able to impart greater membrane permeability properties to the drug. The natural HIV protease substrate FEAIS [105] was chemically modified to replace the F with the cage group to ensure the high specificity of the proposed inhibitor for the active site.
Conclusion
Pinto, M., et al., Enterocyte-like differentiation and polarization of the human colon carcinoma cell line. Wang, Q., et al., Evaluation of the MDR-MDCK cell line as a blood-brain barrier permeability screen. McQuade, T.J., et al., A synthetic HIV-1 protease inhibitor with antiviral activity arrests HIV-like particle maturation.
Darke, P.L., et al., Human immunodeficiency virus protease - bacterial expression and characterization of a purified aspartic protease. Pawar, S.A., et al., Synthesis and molecular modeling studies of novel carbapeptide analogs for HIV-1 protease inhibition.
RESEARCH RESULTS I
Optimization of the isolation, purification and enzyme activity of HIV-1 protease subtype C
Abstract
- Strains
- Media culturing conditions
- Protease extraction from recombinant E. coli
- HIV Protease Purification .1 Column purification
- Confirmation of HIV protease expression
- Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)
- Determination of protein concentration
- Determination of HIV-PR CSA Protease Activity
The protease coding region in the transgenic strain was confirmed by DNA sequencing. Sayed of the Protein Structure-Function Research Unit (University of the Witwatersrand, Johannesburg, South Africa). Fractions (5 mL) were collected and subjected to the Bradford assay (Sigma Aldrich, Germany) to determine the presence of protease.
Fractions containing the protease were pooled and formic acid (Sigma Aldrich, Germany) was added to a final concentration of 25 mM, resulting in a decrease in pH to approximately 3.0. Confirmation of gene expression and subsequent purification of the protease enzyme was performed using SDS-PAGE.
Results
- Optimization using different media .1 Growth curve analysis of media
- Purification using different media
- Final HIV-PR CSA purification
- Protease conformation
- Enzyme specificity
54 3.4.2 Optimization of the HIV-PR isolation and purification method using methods other than the Bradford assay. Purification of the protease was optimized by methods described above, where analysis omitted the Bradford assay. Comparison of the results (Figure 3.5) shows that the UV spectrophotometric analysis of the pooled fractions eluted by the column was not reliable due to multiple band formation and loss of protease due to their presence in the "tail" fraction.
As mentioned in the Materials and Methods section above, this substrate mimics the cleavage site of the peptide substrate of HIV-1 protease. The hydrolytic activity of the protease was followed by the degradation of the chromogenic substrate, resulting in a decrease in absorbance.
Discussion
The point of induction of expression is very important, since the inducer, if used too early, would significantly contribute to the metabolic stress of the bacterium and ultimately to low expression of heterologous proteins. The Bradford test has been used for decades to detect protein concentration. Ultraviolet spectrophotometric analysis of the renatured and purified protease (Figure 3.6) confirmed the presence of the protease, as the unique “shoulder” of the fingerprint between 285 and 295 nm is a feature indicative of HIV-1 protease [36, 37]. ].
Therefore, further evidence of pure and active HIV-PR CSA was shown in the enzyme-specific reaction, and the specific activity of the enzyme was revealed to be a positive enzyme reaction. Since HIV protease cleaves a specific sequence of the chromogenic peptide and thus reduces its absorbance, it is safe to assume that the enzyme has an efficient ability to selectively cleave the substrate (Figure 3.7).
Conclusion
Mosebi, S., et al., Active-site mutations in the South African human immunodeficiency virus type 1 subtype C protease significantly influence clinical inhibitor binding: kinetic and thermodynamic study. Ido, E., et al., Kinetic studies of human immunodeficiency virus type 1 protease and its active site hydrogen-bonding mutant A28S. Ido, E., et al., Kinetic studies of human immunodeficiency virus type-1 protease and its active site hydrogen bond mutant A28S.
Mosebi, S., et al., Active site mutations in South African immunodeficiency virus subtype 1 C protease have a significant impact on clinical inhibitor binding: Kinetic and thermodynamic study. Pawar, S.A., et al., Synthesis and molecular modeling studies of novel carbapeptide analogs for inhibition of.
RESEARCH RESULTS II
Membrane permeability of chemically synthesized novel HIV-1 protease inhibitors
Abstract
Data from permeability assays of peptide inhibitors were compared with three drugs (antipyrine, metoprolol, and caffeine) that belong to the list proposed by the FDA for validation of in vitro permeability methods. A comparison of the passive permeation profile of the inhibitors versus Saquinavir, a commercially available HIV-1 protease inhibitor, was established, confirming our hypothesis. It was predicted that both of these modifications would increase the inhibition of the protease as well as the binding capacity to the enzyme.
When tested against the pure enzyme that was generated in Chapter 3, these inhibitors were effective in inhibiting enzyme activity in test tube-based reactions. Furthermore, both of these inhibitors were able to increase the binding affinity of the inhibitor to the target enzyme.
Materials and methods .1 Cell line
- Cell culture
- Growth and maintenance of cell line
- Cryopreservation of cell line
- Mycoplasma detection and prevention
- Cell-based trans-membrane assay to determine membrane permeability
- Seeding of cells
- Determination of monolayer integrity
- Parallel Artificial Membrane Permeability Assay (PAMPA)
- Preparation of reference drugs
- Generation of standard curves
- Preparation of test compounds
- PAMPA analysis
- Determination of the integrity of PAMPA membrane .1 TEER measurements
- Lucifer yellow (LY) rejection
To facilitate the adhesion of the cells to the flask, 7.5 µl poly-D-Lysine (Sigma Aldrich, Germany) was added. It has long been established that the integrity of monolayers can be determined by measuring the transepithelial electrical resistance (TEER) across the monolayer. 5 µl of PAMPA lipid mixture 1 was carefully added to each well of the donor plate, taking care not to puncture the membrane.
After applying the artificial membrane, 150 µL of drug-containing donor solution (drugs dissolved in PBS) was added to each well of the donor plate. As previously mentioned, trans-epithelial electrical resistance (TEER) is one of the most common means of determining the integrity of a membrane.
Results
- Cell growth and maintenance
- Mycoplasma detection assay
- TEER
- Parallel Artificial Membrane Permeability Assay (PAMPA) .1 Generation of standard curves
As shown in Figure 4.4, the cells were not consistent in their growth patterns and membrane formation was not achieved. The cell-based method was difficult to implement, so an assessment of the permeability characteristics of the inhibitors was obtained using a parallel artificial membrane permeability assay (PAMPA). However, the obtained results indicate minimal permeability through the PAMPA artificial membrane and 0% absorbed fraction (Figure 4.9 and Table 4.7).
It does not allow for in-depth analysis, such as quantification of the inhibitor's ability to permeate the membrane, as a cell-based assay would. The cell-based assay used in this study generated inconclusive data; therefore, an assessment of HIV-1 protease inhibitor permeability was established using the PAMPA assay.
Acknowledgements
Cho, M.J., et al., Madin Darby canine kidney (MDCK) epithelial cell monolayer as a model of a cellular transport barrier. Zhu, C.Y., et al., A comparative study of an artificial membrane permeability assay for high-throughput drug absorption potential profiling. Masungi, C., et al., Parallel artificial membrane permeability assay (PAMPA) combined with 10-day multiscreen Caco-2 cell culture as a tool for evaluating new drug candidates.
Merry, C., et al., Pharmacokinetics of saquinavir alone and in combination with nelfinavir in HIV-infected patients. Kerns, E.H., et al., Combined application of parallel artificial membrane permeability assay and Caco-2 permeability assays in drug discovery.
GENERAL DISCUSSION AND CONCLUSION
It was observed that the use of chemically defined medium promoted a greater biomass yield of the transgenic E. Sufficient amounts of HIV protease were purified and used for testing the inhibitory potential of chemically synthesized novel HIV protease inhibitors. However, this would imply the return to the initial chemical synthetic protocol and it should be noted that changes in the structure of these putative HIV-1 protease inhibitors may slow down their inhibitory activity.
The dynamics of drug design must be approached from a different perspective to bypass the return to the design stages of the process. Both will be extremely beneficial at the start of the drug design process.
![Figure 2.5 Effects of structural properties on solubility and permeability. Adapted from Kerns and Di [36]](https://thumb-ap.123doks.com/thumbv2/pubpdfnet/10643762.0/37.892.249.624.614.804/figure-effects-structural-properties-solubility-permeability-adapted-kerns.webp)
![Figure 2.12 FDA approved HIV-1 protease inhibitors. Adapted from Makatini et al. [96]](https://thumb-ap.123doks.com/thumbv2/pubpdfnet/10643762.0/51.892.111.778.105.966/figure-fda-approved-hiv-protease-inhibitors-adapted-makatini.webp)
