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Pharmacological Reports Copyright © 2013 2013, 65, 1102–1111 by Institute of Pharmacology ISSN 1734-1140 Polish Academy of Sciences Review Biotechnology and genetic engineering in the new drug development. Part III. Biocatalysis, metabolic engineering and molecular modelling Agnieszka Stryjewska1, Katarzyna Kiepura1, Tadeusz Librowski2, Stanis³aw Lochyñski3 1 Department of Bioorganic Chemistry, Faculty of Chemistry, Wroc³aw University of Technology, Wyb. Wyspiañskiego 27, PL 50-370 Wroc
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  Review Biotechnology and genetic engineeringin the new drug development.Part III. Biocatalysis, metabolic engineeringand molecular modelling Agnieszka Stryjewska 1 , Katarzyna Kiepura 1 , Tadeusz Librowski 2 ,Stanis³aw Lochyñski 3            Correspondence:  Abstract: Industrial biotechnology has been defined as the use and application of biotechnology for the sustainable processing and productionof chemicals, materials and fuels. It makes use of biocatalysts such as microbial communities, whole-cell microorganisms or puri-fied enzymes. In the review these processes are described.Drugdesignisaniterativeprocesswhichbeginswhenachemistidentifiesacompoundthatdisplaysaninterestingbiologicalprofileand ends when both the activity profile and the chemical synthesis of the new chemical entity are optimized. Traditional approachesto drug discovery rely on a stepwise synthesis and screening program for large numbers of compounds to optimize activity profiles.Overthepasttentotwentyyears,scientistshaveusedcomputermodelsofnewchemicalentitiestohelpdefineactivityprofiles,geome-triesandrelativities.Thisarticleintroduces interalia theconceptsofmolecularmodellingandcontainsreferencesforfurtherreading. Key words:  biotechnology, metabolic engineering, gene therapy, viral vectors, non-viral vectors, artemisinin, lovastatin Introduction Given the different approaches existing on the defini-tion of ‘biotechnology’, and the plurisemic use of theterm, it seems necessary to briefly introduce its poten-tial different meanings. Biotechnology makes refer-ence to the activity consisting of the utilization or ma-nipulation of living organisms for obtaining productsor implementing processes, generally by means of theintegration of natural and engineering sciences [25].Similarly, diverging approaches exist also in re-spect of the meaning of certain bioproducts, such as biopharmaceuticals. Although biopharmaceutical is 1102               a widely used term, it is not always employed with thesame meaning. There are several possible notions of what a biopharmaceutical is.The first definition, which is the most widely ac-cepted, alludes to biopharmaceuticals as medicinal products, therapeutics, prophylactics and  in vivo  diag-nostics with active ingredients inherently biological innature and manufactured using biotech.A second definition limits biopharmaceutical prod-ucts to those fulfilling the first definition and involv-ing genetic engineering. This corresponds to what has been named “new or modern biotech”, which is a sub-set of the abovementioned notion. Since the earlyeighties, when recombinant DNA and hybridomatechnology were developed, the recourse to this no-tion has become more and more usual. This was, for instance, the definition used by the Federal TradeCommission in its 2009 report on biosimilars. Ac-cording to the Federal Trade Commission, “biologicdrugs are protein-based and derived from living mat-ter or manufactured in living cells using recombinantDNAbiotechnologies”. As it can be observed, this ap- proach limits the concept of ‘biologic drugs’.Another definition of ‘biopharmaceutical’ impliesa contagious use of the term. This can be observedwhen any health-care product that is loosely related to biotechnology is deemed to be a ‘biopharmaceutical’.For instance, all products manufactured by a companythat produces biopharmaceuticals would be consid-ered biopharmaceutical products.Finally, another possible approach, widely usedamong those working in the commercial and mediaareas of the pharmaceutical industry, employs theterm ‘biopharmaceutical’ as a synonym of anythingthat is pharma-related.Therefore, although references are made to other  biopharmaceuticals that fall under the first definition,most problems arise in relation to modern biotechno-logical products which, hence, frequently are the fo-cus of attention [9].In the years 1971–1973, new technology was brought into effect, which became a great scientificturning point. It was recombinant DNA technology,also known as genetic engineering, which until todayhas formed the base of many biotechnological pro-cesses. Polymerase Chain Reaction is one of elementsof the technology of recombined DNA. The techniquediscovered in 1985 by Mullis enables researchers to produce millions of copies of a specific DNA se-quence in approximately two hours. This automated process bypasses the need to use bacteria for amplify-ing DNA[4].The basic biotechnological processes used mostwidely in the pharmaceutical industry apart from re-combined DNA technology, also including directedmutagenesis, are biocatalysis, technology of mono-clonal antibodies, technology of vaccines, metabolicengineering, and, only recently, gene therapy [9, 13,25] as well. However, these processes are largely basedon the tools of gene engineering. Biotechnology hasa major impact on the pharmaceutical industry. Biocatalysis Biocatalysis is a process used for the transformationor manufacturing of certain products using a biocata-lyst, which may be an enzyme, an enzyme complex,organelle, or an entire cell, that is either growing or not. Biocatalysts are used as free and immobilizedforms. They may be of microbial srcin, or from ananimal or plant. The substrate combines with them in bioreactors, where suitable reaction conditions are setup [1]. Biocatalysts also accelerate the reaction thatoccurs repeatedly. The main advantage of biocatalysisis the possibility to obtain chiral products, essential inthe pharmaceutical industry [21].Biocatalysis carried out in the forms listed below:1. Fermentation – the use of living cells in bioreactorsfor the transformation of simple substrates, such assugar or methanol, into the desired product.2. Fermentation of precursors – similar to the above,using living cells for the transformation of more com- plex substrates, or intermediates.3. Biotransformation – the transformation of the pre-cursor into the product by enzymes or an undividedcell, in several stages.4. Enzyme catalysis – crude extracts or partially puri-fied enzymes are used for the transformation.5. Purified enzymes – used mainly in the pharmaceu-tical industry [6].The process of biocatalysis depends on the stabilityof the protein and enzyme kinetics as well as other  properties associated with the ongoing reaction. To perform efficient biocatalysis, a suitable biocatalystmust first be obtained. It is also necessary to establishthe process and determine whether it will be efficient      1103 Molecular modelling in the new drug development   and profitable. For reactions that do not require regen-eration of the coenzyme (e.g., hydrolysis), either anenzyme or an entire cell can be used. However, if a cofactor is required, the cells are used that way; withthe cofactor being then naturally regenerated by them.For the sake of the process, modifications and ar-rangements are made on every level. Through proteinengineering, the properties of the enzyme are im- proved and its stability is increased. Inside the cell,the import and export of substances is facilitated andunnecessary responses are eliminated. An appropriatemedium is selected and the preferred process condi-tions are set [23].Biocatalysis has also to deal with the toxicity of certain substrates or products of the transformation,which are mostly non-polar compounds. There areseveral methods deployed to avoid harming the cell.The first one uses a two-phase system as the environ-ment of transformation. The non-polar phase containsthe substrate and the product; and the water holds thecell. A different method is to place the enzymes or cells in the solid phase, while the substrates and prod-ucts are present in the gas phase, provided that this is possible. The third method is to add the respectivequantity of the toxic substrate that then becomes usedup immediately and is not accumulated. The productsare gradually discharged [6]. Enzymes Enzymes, proteins with catalytic properties, performthe reactions of stereo-, regio- and chemoselective or a specific nature [1]. Enzymes are qualified into sixdifferent groups: hydrolases, oxidoreductases, trans-ferases, lyses, isomerases, and ligases. In as many as60% of all biotransformations, hydrolases are used,while oxidoreductases are used in 20% of the cases.In the industry, the most commonly used are: prote-ases, lipases, esterases, amylases and amidases [14].With genetic engineering, changes at the level of theenzyme can be made, altering its properties and lead-ing to the formation of other varieties of the product.In addition, enzymatic engineering allows for the pro-duction of enzymes effective in a non-aqueous envi-ronment. This kind of environment is used in bio-catalysis due to its interesting properties such as in-creased solubility of the substrate or hydrolyticreaction reversibility. Despite this, the enzymes ex-hibit lower activity in a non-aqueous environmentthan in water. The addition of salt to the protein solu-tion stabilizes its structure, which causes its greater activity. In this way, subtilisin can be activated as wellas many other enzymes. In addition to salt, crownethers, transition analogues and substrates, plus their copies, have an activating effect. This method ismainly used in the pharmaceutical industry [6].Enantiomers are very important due to the fact thateach variety has different characteristics. Thalido-mide, a drug used by pregnant women in the ‘60s withantiemetic and analgesic qualities, was used as a race-mic mixture meaning  R  and  S   enantiomers. The  R form had a therapeutic effect, but  S   (Fig. 1) inducedmutations and caused teratogenic effects. As such, theappropriate enzymes in the processes of production of  pharmaceuticals are very important. Chiral productsare obtained by the transformation of natural, pureenantiomers, asymmetric synthesis, or the separationof a racemate [1]. Cells The biocatalysis processes carried out by cells arealso stereo- and regioselective. The use of whole cells provides an opportunity to add a new function to thereactant and permits complex molecules without pro-tecting them from decay. In addition, the cells as bio-catalysts do not require complicated reaction condi-tions, and when used in an immobilized form, theycan be recovered and used in subsequent transforma-tions.The use of whole cells is of course mainly the useof their enzymes. For example, amino alcohols andamino acids are formed by oxidoreductases and ami-notransferases, while enantioselective hydroxylationand epoxidation is catalyzed by cellular monooxyge-nases, and many chiral products are synthesized bythe action of hydrolases. However, the pharmaceuticalindustry increases the use of enzymes by themselves, because the complexity of processes occurring in the 1104      Fig. 1.  S     cell is more difficult to control when compared to therelative simplicity of pure enzyme action [7, 10, 12].Before using the cells as biocatalysts, they can also be modified earlier. Recombinant DNA technologycan be applied here, leading to increased productionof the desired enzymes [17]. Production of cephalosporin C  -Lactam antibiotic, belonging to antibodies, cepha-losporin is formed on the basis of three amino acids,the same as other antibiotics in this group. Theseamino acids are: L-  -aminoadipic acid, L-lysine andL-valine.During the synthesis of biocatalyzed cephalosporin by means of   Acrimonious chrysogenum , three basicamino acids are converted into   -(L-  -amino-adipyl)-L-cysteinyl-D-valine (ACV) in a reactioncatalyzed by ACV synthetase. Another key step is thecyclization of the linear ACV. This way, isopenicillin N (IPN) is created, which is a bioactive intermediatewhose synthesis reaction is catalyzed by isopenicil-line N synthetase (IPNS). Epimerases converts IPNinto penicillin N, which is a precursor for the synthe-sis of cephalosporins and cefamycins. Then, added tothe biosynthesis is the synthetase of deacetoxycefalo-sporin C, an enzyme catalyzing the reaction of con-version of penicillin N in deacetoxycefalosporin C(DAOC) and DAOC hydroxylase creates deacetylce-falosporine C (DAC). The last reaction and simulta-neously at the same time the limiting step is the acety-lation by acetyl-CoA DAC: DAC, leading to the for-mation of active cephalosporin C [8] (Fig. 2).The synthesis of this antibiotic is carried out by thefermentation of “fed-batch”, powered initially by car- bohydrates changed in the later stages into oils. Thegrowth of fungi on oils is slow, thus the mycelia beginto form multicellular artrospores. This facilitates theaccess of air, which increases the synthesis of cepha-losporins. The pH is maintained at 6.2–7.0 and thetemperature at 24–28°C [10].      1105 Molecular modelling in the new drug development  Fig. 2.  

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