Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1038

Exploring Inhibitorsof HIV-1 Protease

Interaction Studies with Applicationsfor Drug Discovery

BY

MARIA T. LINDGREN

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2004

7 p

Visst finns det mål och mening med vår färd,men det är ändå resansom är mödan värd. Karin Boye

Till Amanda, Klara &Axel

List of Papers

I Lindgren, M. T., Markgren, P.-O., Nillroth, U., Danielson, U. H. Inhibition of HIV-1 protease by Cu2+ occurs by a reversible allos-teric mechanism involving His-69. Submitted.

II Ahlsén, G., Hultén, J., Shuman, C. F., Poliakov, A., Lindgren, M. T., Alterman, M., Samuelsson, B., Hallberg, A. and Danielson U. H. Resistance profiles of cyclic and linear inhibitors of HIV-1 protease. Antiviral Chemistry and Chemotherapy 13, 27-37 (2002).

III Markgren, P.-O., Lindgren, M. T., Gertow, K., Karlsson, R., Hämäläinen, M. and Danielson, U. H. Determination of interaction kinetic constants for HIV-1 protease inhibitors using optical biosensor technology. Analytical Biochemistry 291, 207-218 (2001).

IV Lindgren, M. T., Shuman, C. F., Hämäläinen, M., Karlsson, R. and Danielson, U. H. Identification of complexities in HIV-1 protease–ligand interactions using biosensor technology. Manuscript.

V Cimitan, S., Lindgren, M. T., Bertucci, C. and Danielson, U. H. Early ADME analysis of anti-tumour and anti-AIDS drugs: Lipid membrane and plasma protein interactions. Submitted.

Reprints of the articles were made with permission from the publishers.

Related work (not included in the thesis):

Danielson, U. H., Lindgren, M. T., Markgren, P.-O. and Nillroth, U. Investigation of an allosteric site of HIV-1 proteinase involved in inhibition by Cu2+. In "Aspartic proteinases" (Ed James) pp 99-103, Plenum Press, NY (1998).

Contents

Introduction...................................................................................................11Background ..............................................................................................11Human Immunodeficiency Virus .............................................................11HIV-1 protease .........................................................................................14

Structure...............................................................................................14Hydrolysis mechanism.........................................................................16Substrate specificity.............................................................................16

HIV-1 protease inhibitors.........................................................................17HIV drug resistance..................................................................................18Interaction kinetics ...................................................................................19

Present investigation .....................................................................................21Aim...........................................................................................................21Strategy ....................................................................................................21Methodology ............................................................................................22

Cloning, expression and purification of HIV-1 protease .....................22Mutagenesis .........................................................................................22Immobilized metal affinity chromatography .......................................23Spectrophotometric and fluorometric assays.......................................23Biosensor based interaction analysis ...................................................24

Results and discussion..............................................................................25Inhibition of HIV-1 protease by Cu2+ ..................................................25

Mutants ...........................................................................................25Immobilized metal affinity chromatography ..................................26Inhibition measurements .................................................................27Fluorescence spectroscopy..............................................................28Conclusions.....................................................................................28

Resistance ............................................................................................29Resistant mutants ............................................................................29Cyclic and linear inhibitors of HIV-1 protease ...............................30

Interaction kinetics...............................................................................31Stabilized surface ............................................................................31Kinetic analysis ...............................................................................32

Binding models....................................................................................32ADME .................................................................................................34

HIV-1 protease inhibitors................................................................35Plasma proteins ...............................................................................35Lipid membranes.............................................................................36Conclusions.....................................................................................37

Conclusions ..............................................................................................37

Acknowledgements.......................................................................................38

Summary in Swedish ....................................................................................40AIDS och HIV..........................................................................................40HIV-1 proteas...........................................................................................40Mål ...........................................................................................................40Studier ......................................................................................................41

Hämning med kopparjoner ..................................................................41Resistens ..............................................................................................41Interaktionskinetik och bindningsmodeller .........................................41ADME .................................................................................................42

Slutsatser ..................................................................................................42

References.....................................................................................................43

Abbreviations

ADME absorption, distribution, metabolismand excretion

AGP 1-acid glycoprotein AIDS acquired immunodeficiency syn-

drome DABCYL 4-(4-dimethylaminophenylazo) ben-

zoic acid EDANS 5-[(2-aminoethyl)amino] naphtha-

lene-1 sulfonic acid EDTA ethylenediamine tetraacetic acid FDA U. S. food and drug administration HAART highly active antiretroviral therapy HIV human immunodeficiency virus HSA human serum albumin IDA iminodiacetic acid IMAC immobilized metal affinity chroma-

tography kcat turnover number, rate of catalysis KD affinity, equilibrium dissociation

constantKi inhibition constant, equilibrium dis-

sociation constant KM Michaelis-Menten constant koff dissociation rate constant kon association rate constant kt mass transport rate constant MDR multi drug resistance PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction RI refractive index RU resonance units SDS sodium dodecylsulfate SPR surface plasmon resonance

11

Introduction

BackgroundHuman immunodeficiency virus (HIV) is the cause of acquired immunodefi-ciency syndrome (AIDS), a devastating illness that has spread all around the world and caused pain and death wherever it has struck. HIV acts by break-ing down the immune defense of its victims. The infected cells of the im-mune system are manipulated to produce their own enemy, HIV, and they are destroyed in this process. When the immune system has been diminished by the viral attack, the body is open for invasion of bacteria and viruses that otherwise would be easy to defeat. This state is called AIDS.

This thesis deals with HIV-1 protease and inhibitors of this viral enzyme. The enzyme is an attractive target for the design of drugs against AIDS since inhibition of the protease prevents viral replication (McQuade et al. 1990). Finding a drug against HIV has been a hot topic since the virus, HIV-1, was first discovered in 1983 (Barré-Sinoussi et al. 1983; Gallo et al. 1984). In 1986 a second similar virus, HIV-2, was isolated from patients in West Af-rica (Clavel et al. 1986). Since then the virus has been a challenge to the scientific society. Numerous attempts to find a drug or vaccine that would exterminate the virus have failed. But today there are several drugs in clini-cal use (De Clercq 2004b) and additional compounds (De Clercq 2004a) that prolong the lives of many HIV infected persons are in development. However, one of the problems is that the virus becomes resistant to the drugs. Another major problem is the inaccessibility of drugs in the develop-ing countries where the majority of the HIV-infected population of the world live.

Human Immunodeficiency Virus To understand how to design drugs against HIV it is important to understand the life cycle of the virus (Fig. 1). HIV is a retrovirus that retains its genome in the form of RNA. When the viral RNA has entered the target cell it is reverse transcribed to DNA by the viral enzyme reverse transcriptase(Hansen et al. 1987; Fujiwara and Craigie 1989). The viral DNA is then incorporated into the DNA of the target cell by the viral enzyme integrase(Grandgenett and Mumm 1990). Here it can stay dormant for years before

12

the host cell machinery transcribes the viral genome back into RNA and new viral particles are formed and spread to new target cells. For this reason, the infection can be latent and the viral load can be low or not even detectable for years after the time of infection. The viral proteins are the building blocks of new viruses that assemble inside the host cell, start to bud off and in this process kill the host cell. After the virus has broken free of the in-fected cell, the process of maturation starts. To make a mature and functional virus polyproteins has to be cleaved into the functional proteins by the viral enzyme protease (Lightfoote et al. 1986; Farmerie et al. 1987; Kräusslich et al. 1988; Le Grice et al. 1988b; Darke et al. 1988).

Fig. 1. Life cycle of HIV. 1. Free virus 2. Binding and fusion 3. Infection 4. Reverse transcription 5. Integration 6. Transcription 7. Assembly 8. Budding 9. Immature virus breaks free of the infected cell 10. Maturation.

The viral enzymes are essential to the virus and are thus good targets for AIDS drugs. If the enzymes are inhibited the viral infection is stopped. Sev-eral drugs targeting the HIV-1 reverse transcriptase and HIV-1 protease are in clinical use today.

A major problem with current AIDS treatment is the emergence of drug resistance. The reverse transcriptase has no proofreading activity and is thus not very accurate, which results in errors in the reverse transcription of viral RNA to DNA. This, in turn, gives many viral proteins that are disabled and

13

incapable of forming new viruses. But the inaccuracy is not only disadvanta-geous to the virus. It also gives the virus flexibility and the possibility to survive in a changing environment. When the host cell is exposed to a drug against HIV, the viruses that survive are the ones with altered proteins (e. g., enzymes) which are not efficiently inhibited by the drug but still work well enough to give a functioning virus. The selection of viruses with favourable properties in the present environment abides by the Darwinian rule of the survival of the fittest. The selected viruses will replicate and infect new tar-get cells. This drug resistance makes the drug in use ineffective, which gives a demand for new drugs designed to be effective against the strains of HIV that are resistant to the clinical drugs used today.

Fig. 2. Human immunodeficiency virus. The mature viral particle and the viral ge-nome.

The genome of HIV contains three major genes, gag-pol-env (Wain-Hobsonet al. 1985)(Fig. 2), where gag codes for the proteins of the viral internal coat, pol codes for the viral enzymes and env codes for the envelope pro-teins. In addition to the gag, pol and env genes, the virus contains genes which encode proteins involved in regulation of gene expression and the infection process. The viral envelope contains the viral envelope proteins

14

and lipids from the host cell membrane that the virus brings with it when it buds off from the host cell.

HIV-1 protease The viral protease is essential for viral infectivity (Kohl et al. 1988). Tran-scription of viral DNA in the host cell results in several polyproteins, includ-ing the gag and the gag-pol polyproteins, both of which have to be cleaved into the functional proteins by the protease (Le Grice et al. 1988a). This cleavage is performed in a process called maturation. After maturation the virus is ready to infect a new target cell.

StructureThe crystal structure of native HIV-1 protease was first reported in 1989 (Lapatto et al. 1989; Navia et al. 1989; Wlodawer et al. 1989). HIV-1 prote-ase (Fig. 3) consists of two identical subunits forming a homodimer where each monomer is a 99-residue chain, giving the dimer a molecular weight of 22 kDa.

Fig. 3. HIV-1 protease with an inhibitor bound in the active site. The two aspartic acid residues performing the catalytic reaction are shown in the active site.

The active site is situated in a cleft between the two subunits and it is flanked by two loops, called flaps, which have to open in order to let the substrate in. The flaps then close for catalysis to take place, after which they open again

15

to release the products. The protease structure is thus very flexible and struc-tural changes occur in regions far away from the active site during catalysis

Two aspartic acid residues, Asp25 and Asp25’, one from each subunit, are situated at the bottom of the active site cleft. These residues are respon-sible for the catalytic activity of the protease, making it a member of the aspartic protease family. The region where the two subunits meet, called the dimerization interface, is responsible for holding the two subunits together, the protease is only active as a dimer.

H H

H HNH2

NN

NN

OH

O

O

O

O

O

OHH

O OOHO

P2

P1

P1'

P2'

P3'

Asp25'Asp25

H H

H HNH2

NN

NN

OH

O

O

O

O

OOH

HO OOHO

Asp25'Asp25

P2

P1

P1'

P2'

P3'

A)

B)

Fig. 4. Schematic presentation of peptide hydrolysis performed by aspartic prote-ases. A) Formation of transition-state. B) Breakage of transition-state.

16

Hydrolysis mechanism The hydrolysis mechanism for HIV protease (Hyland et al. 1991; Rodriguezet al. 1993), like all aspartic proteases, is dependent on the catalytic activity of two aspartic acid residues (Asp’s), one in each subunit. They activate a substrate water molecule in the active site that performs an attack on a car-bonyl atom in the peptide bond. The water molecule is polarized into a nu-cleophilic state by one of the aspartic acids and attacks the polarized car-bonyl at the scissile bond. During catalysis a tetrahedral intermediate, the transition-state (Fig. 4), is formed and broken.

HIV protease also contains a structural water molecule in the active site, which forms a bridge between the substrate and the flaps. This water mole-cule donates its hydrogens to a carbonyl atom in the substrate and hydrogen bonds to the amide hydrogens in residues Ile50 and Ile50’ (Baca and Kent 1993), thus stabilizing the closed conformation of the enzyme.

Substrate specificity The natural substrates for HIV-1 protease are the gag and gag-pol polypro-teins. The polyproteins are cleaved at ten different sites. Substrate side chains and the corresponding interacting groups of an inhibitor are desig-nated P4, P3, P2 and P1 on the amino-terminal side of the scissile bond and P1’, P2’, P3’ and P4’ on the carboxy-terminal side (Schechter and Berger 1967). These correspond to the subsites of the binding site in the protease; S4–S4’. To ensure specific and efficient cleavage of a synthetic substrate at least seven amino acid residues occupying the P4–P3’ positions are neces-sary. To determine the preference for certain amino acids at a given position, specific sites on the substrate have been varied (Konvalinka et al. 1990; Tözsér et al. 1991, 1992, 1997, 2000; Griffiths et al. 1992) (Wlodawer and Erickson 1993). One of the conclusions is that substrate specificity is de-pendent on the overall sequence and not just the residues linked by the scis-sile bond.The cleavage sites can be divided into two main classes (Griffiths et al.1992):

Class 1: Contains a Tyr/Phe-Pro Class 2: Between two hydrophobic amino acids

Hydrophobicity is the general common feature at the cleavage sites, but the enzyme does not have a stringent specificity and charged residues can also occur.

The HIV-1 protease is susceptible to autoproteolysis; the enzyme cleaves itself at certain positions in the polypeptide chain (Strickler et al. 1989; Rosé

17

et al. 1993; Mildner et al. 1994), which complicates purification and analysis of the enzyme.

HIV-1 protease inhibitors Inhibitors of HIV-1 protease block the activity of the enzyme and prevent viral replication. The mechanisms of protease inhibition are different, de-pending on the type of inhibitor:

Transition-state analogues work by directly blocking the catalytic activ-ity by non-covalent and reversible binding to the active site. Non-peptide active site inhibitors (Moelling et al. 1990; Salto et al.1994) bind covalently and irreversibly to residues in the active site. Allosteric inhibitors interact with the enzyme by binding at a site distant from the active site. The binding induces a change in the overall structure of the protease, e. g., locking the structure in a certain position, thereby preventing catalysis. Dimerization inhibitors (Boggetto and Reboud-Ravaux 2002) that act at the dimerization interface and prevent dimerization. Monoclonal antibodies (Rezacova et al. 2002) bind specifically to non active site epitopes on the protease and prevent catalysis.

The clinical inhibitors (Fig. 5) are all linear transition-state analogues, that bind to the active site. They are asymmetric peptide mimetics with a central secondary hydroxyl group that binds to the catalytic aspartic acid residues and mimics the oxygen in the tetrahedral transition-state for amide cleavage (Wlodawer and Vondrasek 1998). The asymmetry of inhibitors may be of importance for resistance development, since the enzyme is a symmetrical dimer and thus has problems in adapting to an asymmetrical inhibitor. Cur-rently there are seven clinical inhibitors of the HIV-1 protease: amprenavir (VX-478) (Kim et al. 1995), atazanavir (BMS-232632) (Robinson et al.2000), indinavir (L-735, 524) (Dorsey et al. 1994), lopinavir (ABT-378) (Sham et al. 1998), nelfinavir (AG1343) (Kaldor et al. 1997), ritonavir (ABT-538) (Kempf et al. 1998) and saquinavir (Ro 31-8959) (Roberts et al.1990). The first HIV-1 protease inhibitor to be approved by the U. S. Food and Drug Administration (FDA) was saquinavir in 1995.

The recommended therapy of today includes treatment with a combina-tion of inhibitors of both the reverse transcriptase and the protease. The combined drug “cocktail” treatment, also known as highly active antiretrovi-ral therapy (HAART), is a very effective therapy. It has contributed to changing AIDS from being an automatic death sentence to what is now often a chronic, but manageable, disease. But the HAART treatment is not an AIDS cure. Although the viral load may be very low or not even detectable

18

following HAART treatment, the virus is still present, hiding in cellular res-ervoirs of the body (Potter et al. 2004). Continuous treatment for the rest of the patients’ life is necessary. However, the therapy often gives troublesome side effects, which may cause the patient to discontinue the medication. The virus will then have the chance to start replicating again and also to develop resistance against the inhibitors.

NH2

OH

OO

SNN

HO O

ONH

O

OHOH

NH

N

O

N N

O

O NH

O

N

ONH

OHN N

HO

NH

O

O NH

H

H

NOH

NH

NH

O

O

N

ONH2

NH

O

H

H

S

OH

OHO

NNH

OO

NH NO

NH

OH

NH

O

S

NO

O OH

OS

N

ONH

NH

NH

N

1. 2. 3.

5.4.

1.

7.6.

NH2

OH

OO

SNN

HO O

ONH

O

OHOH

NH

N

O

N N

O

O NH

O

N

ONH

OHN N

HO

NH

O

O NH

H

H

NOH

NH

NH

O

O

N

ONH2

NH

O

H

H

S

OH

OHO

NNH

OO

NH NO

NH

OH

NH

O

S

NO

O OH

OS

N

ONH

NH

NH

N

1. 2. 3.

5.4.

1.

7.6.

Amprenavir Indinavir Nelfinavir

Saquinavir Ritonavir

Lopinavir Atazanavir

Fig. 5. Clinical inhibitors of HIV-1 protease.

HIV drug resistance Resistance development is a process of selection. The high mutation rate of HIV leads to a virus population where drug resistant mutants exist even be-fore any treatment is initiated. When a patient infected with HIV is treated with a HIV-1 protease inhibitor, any viruses that survive must have an al-tered protease. These viral variants are thus resistant towards the inhibitor. There are a number of amino acid residues involved in resistance towards HIV-1 protease inhibitors. Which mutations will be successful for the virus depends on the particular inhibitor. Some mutations give resistance towards

19

several inhibitors, known as cross-resistance. After a period of treatment with different protease inhibitors the virus may have developed multi drug resistance (MDR) and none of the existing drugs will work. There may also occur mutations in the natural substrates of the protease, or compensatory mutations which are changes affecting the catalytic efficiency or stability of the enzyme, so as to overcome possible losses in catalytic efficiency follow-ing mutations in the protease. Loss in catalytic efficiency can also be cir-cumvented by increased expression levels of the protease.

Mutations in the HIV-1 protease gene causing resistance have been re-ported for all of the clinically used protease inhibitors (reviewed in (Boden and Markowitz 1998; Miller 2001)), of which the most common mutations concern Val82, Ile84 and Leu90. Exchange of Gly48 is often associated with resistance following treatment with saquinavir (Jacobsen et al. 1996). The different mutations can occur alone or in combination with other mutations. The residues Val82 and Ile84 are located in the active site, Gly48 in the flaps and Leu90 in the dimerization interface.

The propensity of HIV to develop resistance leads to a continuing need for new drugs with other resistance patterns (Potter et al. 2004). It is also important that the patient continues taking the medication properly to sup-press virus replication and reduce the speed of resistance development.

Interaction kinetics When two molecules interact, for example, an enzyme (E) and an inhibitor (I), they may form a complex (EI). The complex formation can be described by the reaction formula:

E+I EIkon

koff

Where kon (M-1s-1) is the association rate constant and koff (s-1) is the dissocia-tion rate constant.

The affinity of the inhibitor for the enzyme is defined as the equilibrium dissociation constant (KD).

KD=koff/kon=[E][I]/[EI]

Affinity is the relation between the concentrations of the free components ([E] and [I]) and the concentration of complex ([EI]) at equilibrium.

20

Kinetic characterization of an enzyme gives the kinetic parameters, kcatand KM, and the catalytic efficiency (kcat/KM) of the enzymatic reaction where substrate (S) is converted to product (P):

E+S ES E+Pk1 kcat

k-1

The inhibition constant (Ki) for enzyme-inhibitor complex can be determined by measuring the effect of inhibitor on the catalytic activity of the enzyme.

Ki =[E][I]/[EI]

Ki (inhibition constant), like KD (affinity), represent the dissociation constant at equilibrium.

21

Present investigation

AimThe aim of the studies presented herein has been to explore inhibitors that can be used as drugs against AIDS. The investigated inhibitors all target HIV-1 protease.

StrategyFive different sub studies (Fig. 6) were employed for characterizing HIV-1 protease inhibitors and their interactions with the enzyme.

Fig. 6. The different sub studies used in this thesis to explore inhibitors of HIV-1 protease.

The investigated inhibitors included clinical and non-clinical inhibitors, ac-tive site and allosteric inhibitors, transition-state analogues and metal-ions. In addition, different enzyme variants were constructed to investigate the contribution of different amino acid residues to the interaction with different ligands.

22

One interest has been the resistance to clinical and non-clinical anti-viral agents targeting the protease. In order to identify structure-activity relation-ships of relevance for resistance, the interactions and kinetics among differ-ent enzyme variants and inhibitors were studied. Such relationships could not be identified by standard inhibition studies. A biosensor based approach was therefore taken. Methodological improvements involving stabilization of the enzyme surface and selection of suitable interaction model were re-quired. In an ADME study the absorption and distribution of clinical drugs against AIDS were studied in a simplified model system for improvement of the earlier stages of drug development.

This strategy thus encompassed a wide range of techniques and scientific problems that contributed to a better understanding of the characteristics of HIV protease inhibitors and how they can be improved.

MethodologyThe experimental methods used in the studies are primarily:

Cloning, expression and purification of HIV-1 protease MutagenesisImmobilized metal affinity chromatography Spectrophotometric and fluorometric assays Biosensor based interaction analysis

The methods are briefly described in the following sections and in more detail in the papers (papers I-V).

Cloning, expression and purification of HIV-1 protease The HIV-1 protease gene was isolated from the HXB strain of HIV-1 and inserted into pET11-a expression vector. The expression vector with insert was used to transform cells of the E. coli strain BL21(DE3). Upon expres-sion the protease was obtained in an insoluble form, inclusion bodies, which were solubilized in 8 M urea, whereupon the protease was refolded by dialy-sis against 1 M urea. The refolded protease was purified by anion-exchange chromatography and subsequent cation-exchange chromatography using elution by a linear NaCl-gradient. The purity and activity of the protease were analysed by SDS-PAGE and activity measurements.

Mutagenesis In order to construct the different variants of HIV-1 protease used in the sub- studies of this thesis, site-directed mutagenesis was adopted. Two different techniques were used to obtain the point-mutated proteases: PCR (paper I & II) and mutagenesis in M13mp18 (paper I & III).

23

Immobilized metal affinity chromatography Immobilized metal affinity chromatography (IMAC) (Porath et al. 1975) utilizes the metal-binding properties of some proteins. IMAC can be used for protein purification and the method is often used today to purify His-tagged proteins, which contain several histidine residues.

A column containing cross-linked agarose beads with attached chelators (e. g., iminodiacetic acid, IDA) is loaded with metal-ions (e. g., Zn2+, Cu2+,Ni2+ or Fe3+). The protein solution is then injected and the metal-binding proteins bind to the immobilized metal-ions. Protein desorption is achieved through change of pH or by injection of a compound that competes for the metal-binding sites, e. g., imidazole. The metal can be removed from the column with a solution of ethylenediamine tetraacetic acid (EDTA).

Spectrophotometric and fluorometric assays Determination of the kinetics of a catalytic reaction requires a measurable change, e. g., in ultraviolet (UV) absorption or fluorescence, as the reaction proceeds. This often requires labeling of the substrate. Kinetic characteriza-tion of an enzyme gives the kinetic parameters, kcat and KM, and the catalytic efficiency (kcat/KM). The inhibition constant (Ki) can be determined by meas-uring the effect of an inhibitor on the catalytic activity of the enzyme.

In the spectrophotometric activity assay a synthetic peptide substrate, based on a natural cleavage site of the HIV protease, was used:

His-Lys-Ala-Arg-Val-Leu-Phe(pNO2)-Glu-Ala-Nle-Ser

(Nashed et al. 1989; Tomaszek et al. 1990). The synthetic substrate is cleaved between leucine (Leu) and p-nitro phenylalanine (Phe(pNO2)) whereby the absorbance is changed for the residue at the carboxy-terminal side of the cleavage site, i. e., Phe(pNO2). The change in absorbance can be followed spectrophotometrically at 300 nm and correlates with product for-mation and thereby enzymatic activity. Inhibition measurements with Cu2+

had to be performed with a substrate lacking the amino-terminal histidine, which would otherwise be able to interact with Cu2+.

In the fluorometric activity assay an internally quenched fluorogenic sub-strate was used:

DABCYL- -Abu-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-EDANS

(Matayoshi et al. 1990), where the peptide sequence was derived from a natural processing site for HIV-1 protease. The substrate contained the fluo-rescent group EDANS and the quenching group DABCYL. Cleavage of the peptide abolished the quenching, resulting in an increase in fluorescence.

24

Incubation of enzyme with the fluorogenic substrate resulted in specific cleavage at the tyrosine (Tyr)-proline (Pro) bond and a time-dependent in-crease in fluorescence intensity that was linearly related to the extent of sub-strate hydrolysis.

Biosensor based interaction analysis Interaction studies were performed using a surface plasmon resonance (SPR) based biosensor system (Johnsson et al. 1995; Cooper 2002). SPR can be used to determine the affinity (KD) and the kinetic rate constants for associa-tion and dissociation (kon and koff) of molecular interactions. The method involves immobilization of a ligand to a sensor surface and subsequent injec-tion of an analyte. Binding of analyte to the immobilized ligand is detected by an increase in refractive index (RI), leading to a resonance signal. The resonance signal resulting from the binding event is proportional to the con-centration of analyte binding to the ligand and measured in resonance units (RU). The result can be followed in real time and is shown in a sensorgram (Fig. 7).

Fig. 7. Schematic sensorgram from a SPR biosensor experiment.

The sensor surface consists of an approximately 100-nm thick carboxy-methylated dextran matrix to which molecules can be immobilized. Immobi-lization of a protein to the sensor surface can be achieved using amine cou-pling, in which amino groups on the protein are covalently linked to the acti-vated dextran matrix. During a binding experiment the surface is subjected to a continuous buffer flow. After injection of analyte and before a new binding event can be studied, the immobilized ligand must be freed from any

25

remaining analyte. This is achieved through regeneration of the surface by injection of a solution that will remove any bound analyte without destroying the immobilized ligand. The surface is then ready for a new injection of ana-lyte.

The interaction is influenced by mass transport, i. e., the capacity of the buffer flow and diffusion to transport analyte to and from the surface. To reduce effects of limitations in mass transport a high flow rate and a low immobilization level should be used. For interactions with association rates faster than the rate of transport the latter will be rate limiting. When evaluat-ing sensorgrams where the association rate is close to or greater than the rate of mass transport, an equation that includes a mass transport rate constant (kt) should be used (Goldstein et al. 1999; Karlsson 1999).

Results and discussion Inhibition of HIV-1 protease by Cu2+

(Paper I)

HIV-1 protease has been shown to be inhibited by metal-ions (Karlström and Levine 1991; Zhang et al. 1991; Woon et al. 1992) and metal-ion com-plexes (Davis et al. 1995; Lebon et al. 1998, 2002) but the mechanism for the inhibition has not yet been completely solved. Two potential binding sites for metals have been proposed; the active site and an allosteric site. In order to resolve this issue, copper binding and copper inhibition of the HIV-1 protease were investigated.

MutantsDifferent variants of HIV-1 protease were constructed to examine which amino acids were responsible for the copper-interacting properties of the enzyme. Metal-binding sites in naturally occurring proteins usually consist of cysteine and histidine residues. Structural modelling identified a putative copper-binding site in the HIV protease and five amino acids in this allos-teric site (Fig. 8) were exchanged. This resulted in the five mutant variants: H69A, C67S, C95S, P1S and F99-. The catalytic efficiencies of the different enzyme variants were in the same range as the wild-type enzyme, except for C95S and F99-. Exchanging Cys95 for serine reduced the catalytic effi-ciency more than 20-fold. Cys95 is important for dimer formation (Davis et al. 2003) and the substitution resulted in a less efficient enzyme. The trun-cated enzyme F99-, in which Phe99 was removed, did not show any catalytic activity after purification and could not be used for further analyses. Phe99 has been shown to be extremely important for formation of the dimer (Toddet al. 1998) and consequently the F99- mutant was assumed to be inactive

26

due to its inability to form a dimer. The copper-binding and copper-inhibiting properties of the remainder of the mutants were compared with each other as well as with the wild-type enzyme.

Fig. 8. HIV-1 protease with the putative copper binding-site encircled (A) and enlarged (from a different angle) (B).

Immobilized metal affinity chromatography Immobilized metal affinity chromatography (IMAC) (Porath et al. 1975) was used to distinguish between the different enzyme variants with respect to their copper-binding properties. Copper ion (Cu2+) was immobilized on a chelating column and HIV protease was allowed to bind to the column. Imi-

27

dazole in a linear, 0-300 mM, or a step-wise gradient 20, 60 and 300 mM, was used to elute the protease from the column. The higher the imidazole concentration needed to elute the protease from the IMAC column the higher the affinity for Cu2+. Although a crude method for determination of affinity, this approach was sufficient for comparison of different protease variants having large differences in affinity for the metal-ion. The mutant H69A eluted from the Cu2+-column at a lower concentration of imidazole compared to wild-type (Fig. 9) indicating that His69 was important for the copper-binding properties of the HIV protease. The IMAC chromatogram for C67S and P1S overlapped with that for wild-type, so Cys67 and Pro1 do not seem to be important for copper binding. The C95S mutant did not bind to the column and was found in the flow-through fraction from the Cu2+-IMAC column. Since it has been shown that Cys95 is important for dimer formation (Todd et al. 1998; Davis et al. 2003) and considering the reduced catalytic efficiency of C95S, it was concluded that exchanging Cys95 for serine gave a disturbed enzyme that no longer bound to copper.

Since the protease was eluted from the IMAC column with imidazole the interaction between protease and Cu2+ was reversible. The enzyme was still active after elution from the column, indicating that the enzyme was not subjected to oxidation by Cu2+.

Fig. 9. Elution of wild-type and H69A HIV-1 protease from Cu2+-IDA sepharose. The elution profiles, A280, of IMAC experiments with wild-type and H69A mutant HIV-1 protease have been overlaid. The line represents the stepwise imidazole gra-dient; 20, 60 and 300 mM.

Inhibition measurements HIV-1 protease was tested for inhibition by Cu2+. It was found that H69A exhibited a reduced sensitivity to inhibition by Cu2+ compared to wild-type

28

protease. This was consistent with the reduced affinity of H69A for Cu2+ on the IMAC-column and confirmed the importance of His69 for copper inhibi-tion as well as for copper binding. C95S was more sensitive to Cu2+ inhibi-tion as compared to wild-type enzyme, and the already disturbed C95S mu-tant was thus more easily inhibited. There was no significant effect on the inhibition when Cys67 or Pro1 were replaced by serine, indicating that these two residues were not important for either copper inhibition or for binding to copper, as was shown in the IMAC study. The inhibition of HIV protease did not increase continuously with time, which indicated that the enzyme was not irreversibly inhibited.

Fluorescence spectroscopy The interaction of Cu2+ with the protease was also studied by measuring the intrinsic fluorescence of the interacting molecules. Fluorescence spectros-copy was used to study complex formation and possibly conformational changes upon addition of Cu2+ to the enzyme. The measured signal was be-lieved to contain fluorescence from both the tyrosines and tryptophans of the protease and fluorescence from the active-site inhibitor U-75875.

When copper was added to wild-type or H69A protease, a decrease in fluorescence was observed which was more pronounced for the wild-type enzyme. Subsequent addition of EDTA, after the addition of Cu2+ to enzyme, gave an additional slight decrease in fluorescence. When the active site in-hibitor (U-75875) was added after Cu2+ and EDTA, the fluorescence signal for the wild-type enzyme was almost unaffected, whereas there was an in-crease with H69A. Addition of U-75875 directly to the enzyme resulted in an increase in fluorescence, which did not differ significantly, for both en-zyme variants. Addition of U-75875 without enzyme resulted in a slight increase in fluorescence, although not as high as in the presence of enzyme.

The results from the fluorescence spectroscopic experiments indicate that His69 is likely to be involved in the interaction between Cu2+ and HIV-1 protease and that the interaction results in conformational changes in the enzyme.

Conclusions The conclusions from the binding, inhibition and fluorescence spectroscopy experiments with HIV protease and Cu2+ was that Cu2+ is an allosteric inhibi-tor of the protease resulting in conformational changes and that H69A is important for the interaction.

29

Resistance (Paper II)

In an attempt to circumvent the problem of drug resistance, a series of new classes of inhibitors (Hultén et al. 1997, 1999; Schaal et al. 2001) were ana-lysed. The strategy was to identify efficient inhibitors with significantly dif-ferent structures than those in clinical use. The inhibitors were tested for inhibition of HIV-1 protease variants containing substitutions of amino acid residues known to be associated with clinical resistance (Fig. 10). An addi-tional aim was to use the large set of inhibition data, obtained with different combinations of inhibitors and mutants, to reveal structure-inhibitory rela-tionships.

Resistant mutants HIV-1 protease with single, double, triple and quadruple combinations of G48V, V82A, I84V and L90M substitutions was constructed and used in inhibitor studies in comparison with wild-type enzyme. The catalytic effi-ciency of the mutants was 1-30% of that for the wild-type enzyme, resulting from a reduced kcat for all mutants and an increased KM for all mutants ex-cept G48V. In addition, the mutants generally had a reduced expression level and were less stable than the wild-type enzyme.

Fig. 10. HIV-1 protease with four residues that are involved in resistance towards some of the clinical inhibitors. The residues are only shown in one of the monomers but they are present in both monomers since the protease is a homodimer.

30

Cyclic and linear inhibitors of HIV-1 protease The cyclic inhibitors used in this study were designed to replace not only the substrate, but also a structural water molecule in the active site of HIV-1 protease. In this work, the structure-inhibitory properties of both cyclic sul-famide (Fig. 11) and cyclic urea (Fig. 12) analogues were compared with linear transition-state analogues (Fig. 13), including five clinical inhibitors: amprenavir, indinavir, nelfinavir, ritonavir and saquinavir (Fig. 5).

The inhibition of V82A, I84V and G48V/L90M mutants by the cyclic compounds was less efficient than that of the wild-type enzyme, showing that these mutants were resistant not only towards the clinical inhibitors but also towards the cyclic compounds used in this study. HIV protease variants with other combinations of mutations were analysed but did not show im-proved inhibition profiles for the cyclic compounds. All linear and one cy-clic inhibitor yielded an inhibition of the wild-type enzyme comparable to the clinical protease inhibitors, but all other compounds showed at least an order of magnitude higher Ki values. Both the cyclic and the linear com-pounds were inefficient inhibitors of triple and quadruple mutants.

A set of linear compounds were analysed together with additional mu-tants, but the results revealed a unique profile for each compound and no general structural features could be assigned to a particular inhibition profile. The fact that no clear correlations were identified, indicated the complexity in extracting structure-inhibition and resistance information from this rela-tively extensive data set. To understand the structural and mechanistic fea-tures of inhibition and resistance either larger data sets had to be used or alternative methods and kinetic parameters were required. One way to achieve this was to resolve the affinity of inhibitor interactions into associa-tion and dissociation rate constants (Markgren et al. 2001) (paper III).

SNNR1'

OH OHOO

O O

R1

Fig. 11. Cyclic sulfamide inhibitors

NNR1'

OH OHOO

O

R1

Fig. 12. Cyclic urea inhibitors

31

NH

NH

NH

NH

O

O O

OO

O

OH

OH

R1

R1'

Fig. 13. Linear inhibitors

Interaction kinetics(Paper III)

In drug development it is essential to get as detailed information as possible about the interaction between the drug and its target. Not only the affinity but also the kinetics of an enzyme-inhibitor interaction gives valuable infor-mation. In this study it was possible to determine the association- and disso-ciation rate constants as well as the affinity of the interactions between HIV-1 protease and four clinical inhibitors using optical biosensor interaction analysis.

Stabilized surface In the initial biosensor experiments with immobilized HIV-1 protease there was a baseline drift due to an unstable surface (Markgren et al. 2000). The immobilized HIV protease was bleeding off, either by auto proteolysis, dimer dissociation or a combination of both. To overcome the problem with baseline drift two approaches were applied, one mathematical and one ex-perimental.

In the mathematical approach a new model where the drift was taken into account was used for evaluation of the sensorgrams. This mathematical ap-proach was not optimal in practice since the parameters for drift and binding capacity were time dependent and varied between injection cycles. There-fore, these parameters had to be fitted locally to each curve. This was tedious and time-consuming method.

The experimental approach involved designing an auto proteolysis stabi-lized mutant enzyme (Q7K) and chemical stabilization of the sensor surface by cross-linking. It appeared that a combination of using the stabilized mu-tant and chemical stabilization was the most successful method and gave a completely stable baseline. The stabilized surface was still enzymatically active and the interaction with inhibitors was similar to that of the native enzyme.

32

Kinetic analysis The interaction kinetics between HIV-1 protease and four clinical inhibitors; saquinavir, indinavir, nelfinavir and ritonavir, were investigated. Several of the inhibitors had very high association rates (>~1*106M-1s-1), which made it necessary to determine the kinetics of the interactions using a binding equa-tion which included a mass transport rate constant, kt. The association and dissociation rate constants (kon and koff-values) for the inhibitors were deter-mined using a SPR biosensor. The autoproteolysis-stabilized mutant, Q7K, was immobilized on the sensor surface using amine coupling and subsequent chemical stabilization. Concentration series of the clinical inhibitors were injected over the surface, one concentration and inhibitor at a time, with appropriate regeneration of the sensor surface between every binding event. The sensorgrams obtained were analysed using the BIA evaluation software (Biacore AB) and the interaction kinetic parameters were estimated by global fit of an equation describing 1:1 binding with mass transfer.

Saquinavir had the highest affinity for the protease as a result of the low-est dissociation rate of all the studied clinical inhibitors. The affinity of ri-tonavir was only two-fold lower than that for saquinavir, even though the dissociation rate was six-fold faster. This was explained by the fact that the high dissociation rate was compensated by a three-fold faster association rate. Nelfinavir and indinavir both showed lower affinity for the protease than did the other inhibitors, due to a slower association for nelfinavir and a relatively fast dissociation for indinavir.

This study established that biosensor-based interaction studies can be used to resolve affinity into association and dissociation rates and that these parameters are of importance for an increased understanding of the charac-teristics of enzyme-inhibitor interactions.

Binding models (Paper IV)

The 1:1 binding model used in earlier investigations gave a fit with sufficient accuracy for the conclusions drawn in these studies (Markgren et al. 2001, 2002; Shuman et al. 2003, 2004), but it did not give a satisfactory fit when screening and evaluating a larger data set with multiple variations in experi-mental conditions. To get a better fit to the experimental data from biosensor experiments a new model for enzyme-ligand interaction was developed. The new model, called heterogeneous complex, two conformations, includes the formation of two different conformations of the enzyme-inhibitor complex. This gives a better fit to the experimental data than the 1:1 binding model. A number of other models were also investigated but did not give a better fit than the heterogeneous complex, two conformations model.

33

The models investigated were: 1:1 interaction

A+Bkon

koff

AB

Heterogeneous complex, two conformations

A+Bkon1

koff1

AB

A+Bkon2

koff2

ab

Ligand induced conformational change

A+Bkon

koff

ABk1

k-1

ab

Heterogeneous complex, three conformations

A+Bkon1

koff1

AB

A+Bkon2

koff2

ab

A+Bkon3

koff3

34

Heterogeneous complex, two conformations and pre-equilibrium

A+B A* A*B+Bk1

k-1

kon1

koff1

A+B A* a*b+Bk1

k-1

kon2

koff2

The use of the heterogeneous complex, two conformations model, as a de-scription of the real enzyme mechanism is reasonable considering the high flexibility of the HIV protease. Upon binding of an inhibitor it is assumed that the flaps will have the possibility to embrace the inhibitor in two differ-ent ways, resulting in a heterogeneous complex formation, including two different conformations. Another model, which also gave a better fit to the experimental data, compared to the simple 1:1 binding model, was the ligand induced conformational change model. It is applicable to a mechanism in-volving a conformational change after the inhibitor has bound, which may be interpreted as the closing of the flaps. Using more complex models, i. e., when the heterogeneous model included the formation of three different conformations or a pre-equilibrium step, resulted in a fit equal to the one achieved with the heterogeneous complex, two conformations model.

The heterogeneous complex, two conformations model, was used to ana-lyse sensorgrams from binding experiments with different inhibitors, differ-ent HIV-1 protease variants and at different temperatures. The model worked well and a good fit to the experimental data was achieved in all cases.

ADME (Paper V)

The ability to predict ADME properties of potential drug candidates has a tremendous impact on the drug discovery process, both in terms of cost and the amount of time required to bring a new compound to market. Computa-tional approaches can be used to predict the various physicochemical proper-ties of complex organic molecules from their molecular structures without the need of any experimentally derived parameters, but experimental ap-proaches have to be used later on in the drug development process to con-firm the results from computational methods. However, there is a need for experimental methods that can be used early in the drug development proc-ess to screen a large number of potential drug candidates for their ADME

35

properties. This is of great interest, since advantageous properties of the potential drug candidates may otherwise be lost in the optimization process.

Oral bioavailability is defined as the fraction of the ingested dose of a drug that is available in the systemic circulation following oral administra-tion. The oral bioavailability of a drug is dependent on solubility, permeabil-ity and transport characteristics across biological barriers, chemical stability in the gastrointestinal tract and various clearance mechanisms.

Analyses of drug interactions with lipid membranes and HSA have been achieved through different experimental strategies (Danelian et al. 2000; Frostell-Karlsson et al. 2000; Rich et al. 2001; Baird et al. 2002; Ahmad et al. 2003; Day and Myszka 2003; Abdiche and Myszka 2004). An in vitroADME study, where absorption and distribution of drugs were investigated, was performed with the purpose of developing efficient methods that could be used early in the drug discovery process to increase our understanding of the bioavailability of the different drugs. A surface plasmon resonance (SPR)-based optical biosensor was used to study drug interactions with lipid membranes and plasma proteins.

HIV-1 protease inhibitorsSeven clinical inhibitors of the HIV-1 protease were used in the ADME-study: amprenavir, atazanavir, indinavir, nelfinavir, ritonavir, saquinavir and lopinavir. Eight taxol-like compounds, with a clinical application in anti-cancer chemotherapy, were also included in the study and the results show that the method can be used with different classes of compounds.

Plasma proteins The distribution of drugs depends to some extent on their binding to plasma proteins, of which human serum albumin (HSA) and 1-acid glycoprotein (AGP) appear to be the most important ones. The drugs bind to the plasma proteins and are transported together in the blood stream to become distrib-uted throughout the body. The drug then has to dissociate from the plasma protein to be able to be absorbed and perform its task inside the cells. In order to be well distributed to all of the cells of the body the drug has to bind well to the plasma protein but must also be able to let go of it when it reaches the target cell.

HSA and AGP were immobilized on the sensor surface at two different detection spots, which made it possible to inject compounds to both proteins simultaneously. Compounds were injected over both plasma protein surfaces at a fixed concentration of 30 µM. A graph displaying the binding levels of the tested compounds showed that there was a correlation between their in-teraction to HSA and AGP. It was possible to determine the kinetics for the interactions between the compounds and HSA. The binding of inhibitors to HSA could be divided into three subgroups depending on their association, kon, and dissociation, koff, rates: one rapid and one slow group and one group

36

with super-slow binding (Fig. 14). The affinities, KD, of the rapid and slow groups are approximately the same, but the kon and koff-values differ, empha-sizing the importance of resolving the affinity into the association and disso-ciation rates.

Fig. 14. HSA interaction kinetic plot of HIV-1 protease inhibitors and taxane ana-logues.

Lipid membranes Absorption to lipid bilayers of the cell membranes is important for the drug to be absorbed by the cells and thus be able to act on its target. In this study two different types of lipid bilayers were used as models for cell membranes. The lipid membranes were used to coat two different spots on the sensor surface, which made it possible to register binding of an inhibitor to one type of lipid bilayer at a time. Screening of the inhibitors at 20, 100 and 150 µM and a ranking of the compounds were performed. Since a simple binding model could not describe the sensorgrams obtained, the maximum binding response after injection of the compound for 60 s was determined for each compound and values were expressed as the fractional binding relative to a reference compound (propanolol).

37

Conclusions It was possible to obtain an estimate of bioavailability of a drug from the absorption of the drug to lipid membranes and the interaction with two im-portant drug-binding proteins in human plasma, HSA and AGP. Structural features of the compounds could be related to their interactions with the lipid bilayer and plasma proteins. This biosensor based method was thus an effi-cient approach for early in vitro ADME characterization and optimization of lead compounds.

Conclusions In the present thesis inhibitors of HIV-1 protease were studied with respect to their ability to inhibit the protease as well as their suitability as drugs against AIDS.

It was shown that it is possible to inhibit the protease allosterically with Cu2+. This is an important finding, since development of resistance makes it essential to strike at the virus in different ways. It was concluded from bind-ing and inhibition studies with copper in combination with different variants of HIV protease that His69, a member of a putative allosteric copper-binding site of the protease, is important for the enzyme-Cu2+ interaction.

Several of the present studies give insight into the flexibility of the prote-ase. A model involving two different conformations between enzyme and inhibitor gave a better fit, than the simple 1:1 binding model, to the experi-mental results in a SPR biosensor experiment. The highly flexible properties of the enzyme are also reflected by the possibility to inhibit the protease allosterically with Cu2+, which shows that regions far away from the active site affect the catalytic ability of the enzyme.

It was clear from the resistance study that it is not easy to predict struc-ture-inhibitory and resistance relationships even from a relatively extensive data set. To better understand the structural and mechanistic features of inhi-bition, the affinity of inhibitor interactions was resolved into association and dissociation rate constants, in a biosensor based interaction study.The problem of predicting bioavailability of compounds was addressed in an ADME study. A biosensor based method that could be used early in the drug development process was employed. It was shown that the method could be used for prediction of both absorption and distribution of different classes of drugs.Altogether, the studies included in this thesis have revealed important char-acteristics of drugs that can potentially be modeled into new compounds with improved efficacy of both wild-type and resistant mutants of HIV-1 protease.

38

Acknowledgements

I would like to express my sincere gratitude to all present and former col-leagues at the Department of Biochemistry at Uppsala University, where I have spent the last years working with this thesis, for a friendly and pleasant working climate.

Special thanks to:

Helena, for always encouraging and supporting me when I need it the most. Your open mind and positive attitude made it possible for me to combine having a family with being a Ph. D. student. You make everything possible! Cynthia, for being a good companion and friend, in good times and bad times, and for accompanying me to movies and restaurants (sushi!) when I needed to get away from “everything”. Peo, for endless discussions about life and research during your time in He-lena’s group and for struggling together with me with the “copper project”. Ulrika, for taking good care of me when I first came to the group and for all the nice times we had. Göran A., for being the handy man in the lab. You know everything worth knowing about the HPLC instrument. Samanta, for a nice collaboration and for being a good friend. I am so happy that you were working together with me that hot summer at Biacore AB. Dan, Göran D., Thomas, Omar, Matthis and Anton for making our lab an excellent place to work. The members of Bengt Mannervik’s and Birgitta Tomkinson’s groups for fruitful discussions and friendship. Bengt, Birgitta T., Gun, Ann-Christin, Birgit, Birgitta E., Ylva, Maryam, Malena, Arna, Lars, Natalia, Usama, Abeer, Inger, Sanela, Carolina, Anna-Karin, Nisse, Lisa, Micke and Franço-ise. Thank you all of you! David Eaker, for the linguistic revision of this summary. The occurring er-rors are due to my last minute changes. Part of my work was done at Biacore AB in Uppsala and I am grateful for having had the opportunity to spend some time in your laboratories and for interesting and fruitful collaboration. Special thanks to Markku, Pär S., Hen-rik J., Annie, Helena N., Robert K., Åsa F. -K. and many others for always being friendly and helpful.

39

På ett mer personligt plan vill jag tacka alla er som gör mitt liv, vid sidan av karriären, värt att leva. Speciellt vill jag tacka:

Mikla, för att du är en speciell vän, som jag kan prata med om allt. Petra, för att du är som en syster för mig, även om vi ibland bara har pratat med varandra i telefon någon gång om året. Birgitta O., för tiden då vi gick biokemikurserna tillsammans, för alla påhitt som du har haft och allt kul vi har gjort sedan dess. Lisa N., för allt trevligt vi har haft tillsammans; segling, tedrickning, japans-ka middagar och mycket mer. Vinklubben: Erik, Kattis, Mikla och Per, för allt kul vi har haft tillsammans med vår växande skara av barn. Jocke och Karolina, för att ni gör mig och mina barn glada! Mamma, för all hjälp med barnen och för allt stöd och all omtanke du givit mig.Pappa och Doris, för all trevlig samvaro och goda middagar. Martin, jag är så glad över att just du är min bror. Du finns alltid där när jag behöver dig. Kram, Guldbrorsan !Familjen Lindgren: Göran, Christina, Florie, Anna, Maria, Janne, Astrid, August, och Johan, för att jag får vara en del av er stora och varma familj. Amanda, Klara och Axel, mina små solar , för att ni har lärt mig vad som är viktigast i livet. Jag älskar er !Fredrik, för din positiva livssyn och det liv vi har byggt upp tillsammans. Jag älskar dig !

40

Summary in Swedish

AIDS och HIV När HIV (humant immunbristvirus) började spridas över världen under det tidiga 1980-talet var det början till en av de värsta epidemierna i modern tid. HIV är ett virus som infekterar immunförsvarets celler och förstör dessa. Detta leder till ett försvagat försvar mot sjukdomsalstrande bakterier och virus i vår omgivning och till slut ett tillstånd som kallas AIDS. Om viruset tillåts härja fritt i kroppen kommer olika infektioner och sjukdomar att så småningom leda till döden. Idag finns dock ett antal läkemedel som bromsar virusets framfart och förlänger livet för de flesta HIV infekterade människor som har tillgång till dessa. Ett stort problem är dock den dåliga tillgången på mediciner mot HIV i många av världens utvecklingsländer, där den största delen av världens HIV infekterade befolkning finns. Ett annat problem är virusets förmåga att förändras, mutera, och därmed utveckla resistens, vilket gör medicinerna verkningslösa. Därför behövs hela tiden en utveckling av nya läkemedel som kan slå även mot dessa resistenta virusvarianter.

HIV-1 proteas I HIV finns ett antal enzymer som är nödvändiga för att viruset ska kunna föröka sig och därmed sprida sig och infektera nya celler. Enzymerna är utmärkta måltavlor för läkemedel mot HIV och de mediciner som finns idag är framför allt inriktade på att förstöra funktionen hos dessa enzymer. Ett av enzymerna är HIV proteas, som behövs i virusets mognadsprocess, där det klyver vissa proteiner i dess funktionella delar. Om HIV proteaset hämmas så bildas ett virus men det saknar förmågan att infektera nya celler och där-med stoppas HIV infektionen.

MålI den här avhandlingen utforskas olika hämmare av HIV-1 proteaset ur olika synvinklar, för att hitta nya och förbättrade mediciner mot AIDS, samt för att förbättra och utveckla metoder för att undersöka deras egenskaper.

41

Studier

Hämning med kopparjoner Kopparjoner (Cu2+) har visat sig kunna hämma HIV-1 proteaset. För att ta reda på hur enzymet växelverkar med koppar gjordes studier med förändrade varianter av proteaset, s k mutanter. I mutanterna hade vissa aminosyror bytts ut, för att utvärdera deras roll i hämning och inbindning till kopparjo-ner. Resultaten från experiment med kopparjoner och de olika mutanterna, där hämning, inbindning och strukturella förändringar undersöktes, visade att aminosyran histidin nummer 69 (His69) var viktig för interaktionen. His69 är en av beståndsdelarna i ett område av proteaset som antagits vara kopparbindande. Detta område som kallas för den kopparbindande ytan finns i en region av proteaset långt från enzymets aktiva yta och kopparjonen kal-las därför en allosterisk hämmare.

ResistensOlika läkemedelsresistenta varianter av HIV proteaset har visat sig upp-komma i samband med behandling mot HIV. Nya mediciner utvecklas där-för fortlöpande för att försöka överlista dessa resistenta mutanter. I en studie testades en ny grupp av cykliska hämmare tillsammans med några av de kliniska, för att se om de cykliska fungerade även mot de resistenta mutan-terna. Tanken var att den cykliska strukturen i den nya hämmargruppen skul-le binda på ett nytt sätt till enzymet och därmed hämma även de resistenta mutanterna. Tyvärr kunde inga slutsatser dras om att de cykliska hämmarna skulle vara mer effektiva än de kliniska hämmarna mot de resistenta mutan-terna.

Interaktionskinetik och bindningsmodeller För att undersöka olika hämmares egenskaper vill man få fram så mycket information som möjligt om dem. Man kan t ex ta reda på hur de binder in till enzymet, med vilken hastighet de binder (associerar) till respektive släp-per (dissocierar) från enzymet och hur hårt de binder (affinitet). Utifrån des-sa uppgifter kan man bestämma om molekylen är bra eller dålig på att häm-ma enzymet. Man kan jämföra olika hämmares strukturer och se om en viss struktur hör ihop med en viss egenskap. På så sätt kan man optimera struktu-rerna för att få fram en så bra hämmare som möjligt.

I en studie visades att det går att få fram associations- och dissociations-hastigheter med hjälp av optisk biosensorteknik. Dessa hastighetskonstanter bestämdes för fyra kliniska hämmare av HIV proteaset och det visade sig att konstanterna var karakteristiska för växelverkan mellan enzymet och häm-

42

marna. Den modell, för växelverkan mellan enzym och hämmare, som an-vändes vid utvärdering av biosensorexperimenten (1:1 bindningsmodellen), gav inte en perfekt anpassning till resultaten. En ny modell utvecklades där-för i en studie, där man antog att växelverkan var mer komplicerad än en vanlig 1:1 bindning. I den nya modellen ingick att enzymet och hämmaren kunde bilda inte bara ett slags komplex (som i en 1:1 bindning) utan två oli-ka komplex. Utvärdering med den nya modellen, kallad heterogeneous complex, two conformations, gav en mycket bättre anpassning till de expe-rimentella resultaten. Modellen kunde användas för utvärdering av försök med olika enzymvarianter i kombination med olika hämmare och vid olika temperaturer och gav en bra anpassning i samtliga fall.

ADME För att ett läkemedel ska kunna nå sitt mål och utföra sin uppgift i kroppen måste det först transporteras dit och sedan tas upp av cellerna där. Ett läke-medels egenskaper när det gäller transport (distribution) och upptag (absorp-tion) undersöks noga i de kliniska prövningarna, innan det släpps ut på marknaden för försäljning. Andra viktiga egenskaper som undersöks är hur det tas om hand av kroppens system för ämnesomsättning (metabolism) och hur det utsöndras (excretion) från kroppen. Dessa studier kallas följaktligen för ADME (absorption, distribution, metabolism, excretion). För att kunna ta reda på ADME-egenskaper så tidigt som möjligt i processen för läkemedels-utveckling är det viktigt att utveckla metoder för detta. I en studie undersök-tes absorptions- och distributionsegenskaperna hos ett antal läkemedel mot AIDS och cancer med hjälp av modellsystem och optisk biosensorteknik. Modellsystemet för undersökning av absorption bestod av en yta av lipid-membran, liknande en cellvägg. Modellsystemet för undersökning av distri-bution bestod av ytor belagda med olika plasmaproteiner som har visat sig vara viktiga för transport av läkemedel i blodet. Läkemedel fick sedan kom-ma i kontakt med dessa ytor och inbindningen kunde registreras. Det visade sig att preliminära absorptions- och distributionsegenskaper var möjliga att få fram för dessa läkemedel och gick att koppla till andra egenskaper och strukturerna hos läkemedlen.

SlutsatserDe studier som presenteras i denna avhandling avslöjar viktiga egenskaper hos de undersökta HIV proteashämmarna samt metodutveckling för att un-dersöka dessa. I förlängningen skulle denna kunskap kunna användas för att utveckla nya läkemedel med förbättrad effektivitet mot HIV och dess resis-tenta varianter.

43

References

Abdiche YN, Myszka DG. Probing the mechanism of drug/lipid membrane interactions using Biacore. Anal Biochem 2004;328(2):233-43.

Ahmad A, Ramakrishnan A, McLean MA, Breau AP. Use of surface plasmon resonance biosensor technology as a possible alternative to detect differences in binding of enantiomeric drug compounds to immobilized albumins. Biosens Bioelectron 2003;18(4):399-404.

Baca M, Kent SB. Catalytic contribution of flap-substrate hydrogen bonds in "HIV-1 protease" explored by chemical synthesis. Proc Natl Acad Sci U S A1993;90(24):11638-42.

Baird CL, Courtenay ES, Myszka DG. Surface plasmon resonance characterization of drug/liposome interactions. Anal Biochem 2002;310(1):93-9.

Barré-Sinoussi F, Chermann JC, Rey F, Nugeyre MT, Chamaret S, Gruest J et al. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 1983;220(4599):868-71.

Boden D, Markowitz M. Resistance to human immunodeficiency virus type 1 protease inhibitors. Antimicrob Agents Chemother 1998;42(11):2775-83.

Boggetto N, Reboud-Ravaux M. Dimerization inhibitors of HIV-1 protease. Biol Chem 2002;383(9):1321-4.

Clavel F, Guétard D, Brun-Vézinet F, Chamaret S et al. Isolation of a new human retrovirus from West African patients with AIDS. Science1986;233(4761):343-6.

Cooper MA. Optical biosensors in drug discovery. Nat Rev Drug Discov2002;1(7):515-28.

Danelian E, Karlén A, Karlsson R, Winiwarter S, Hansson A et al. SPR biosensor studies of the direct interaction between 27 drugs and a liposome surface: correlation with fraction absorbed in humans. J Med Chem 2000;43(11):2083-6.

Darke PL, Nutt RF, Brady SF, Garsky VM, Ciccarone TM, Leu CT et al. HIV-1 protease specificity of peptide cleavage is sufficient for processing of gag and pol polyproteins. Biochem Biophys Res Commun 1988;156(1):297-303.

Davis DA, Branca AA, Pallenberg AJ, Marschner TM, Patt LM, Chatlynne LG et al. Inhibition of the human immunodeficiency virus-1 protease and human immunodeficiency virus-1 replication by bathocuproine disulfonic acid Cu1+. Arch Biochem Biophys 1995;322(1):127-34.

Davis DA, Brown CA, Newcomb FM, Boja ES, Fales HM, Kaufman J et al. Reversible oxidative modification as a mechanism for regulating retroviral protease dimerization and activation. J Virol 2003;77(5):3319-25.

Day YS, Myszka DG. Characterizing a drug's primary binding site on albumin. J Pharm Sci 2003;92(2):333-43.

De Clercq E. HIV-chemotherapy and -prophylaxis: new drugs, leads and approaches. Int J Biochem Cell Biol 2004a;36(9):1800-22.

44

De Clercq E. Antiviral drugs in current clinical use. J Clin Virol 2004b;30(2):115-33.

Dorsey BD, Levin RB, McDaniel SL, Vacca JP, Guare JP, Darke PL et al. L-735,524: the design of a potent and orally bioavailable HIV protease inhibitor. J Med Chem 1994;37(21):3443-51.

Farmerie WG, Loeb DD, Casavant NC, Hutchison CA, Edgell MH, Swanstrom R. Expression and processing of the AIDS virus reverse transcriptase in Escherichia coli. Science 1987;236(4799):305-8.

Frostell-Karlsson A, Remaeus A, Roos H, Andersson K, Borg P, Hämäläinen MD et al. Biosensor analysis of the interaction between immobilized human serum albumin and drug compounds for prediction of human serum albumin binding levels. J Med Chem 2000;43(10):1986-92.

Fujiwara T, Craigie R. Integration of mini-retroviral DNA: a cell-free reaction for biochemical analysis of retroviral integration. Proc Natl Acad Sci U S A1989;86(9):3065-9.

Gallo RC, Salahuddin SZ, Popovic M, Shearer GM, Kaplan M, Haynes BF et al. Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science 1984;224(4648):500-3.

Goldstein B, Coombs D, He X, Pineda AR, Wofsy C. The influence of transport on the kinetics of binding to surface receptors: application to cells and BIAcore. JMol Recognit 1999;12(5):293-9.

Grandgenett DP, Mumm SR. Unraveling retrovirus integration. Cell 1990;60(1):3-4. Griffiths JT, Phylip LH, Konvalinka J, Strop P, Gustchina A, Wlodawer A et al.

Different requirements for productive interaction between the active site of HIV-1 proteinase and substrates containing -hydrophobic*hydrophobic- or -aromatic*pro- cleavage sites. Biochemistry 1992;31(22):5193-200.

Hansen J, Schulze T, Moelling K. RNase H activity associated with bacterially expressed reverse transcriptase of human T-cell lymphotropic virus III/lymphadenopathy-associated virus. J Biol Chem 1987;262(26):12393-6.

Hultén J, Andersson HO, Schaal W, Danielson HU, Classon B, Kvarnström I et al. Inhibitors of the C(2)-symmetric HIV-1 protease: nonsymmetric binding of a symmetric cyclic sulfamide with ketoxime groups in the P2/P2' side chains. J Med Chem 1999;42(20):4054-61.

Hultén J, Bonham NM, Nillroth U, Hansson T, Zuccarello G, Bouzide A et al. Cyclic HIV-1 protease inhibitors derived from mannitol: synthesis, inhibitory potencies, and computational predictions of binding affinities. J Med Chem1997;40(6):885-97.

Hyland LJ, Tomaszek TA, Roberts GD, Carr SA, Magaard VW, Bryan HL et al. Human immunodeficiency virus-1 protease. 1. Initial velocity studies and kinetic characterization of reaction intermediates by 18O isotope exchange. Biochemistry 1991;30(34):8441-53.

Jacobsen H, Hanggi M, Ott M, Duncan IB, Owen S, Andreoni M et al. In vivo resistance to a human immunodeficiency virus type 1 proteinase inhibitor: mutations, kinetics, and frequencies. J Infect Dis 1996;173(6):1379-87.

Johnsson B, Löfås S, Lindquist G, Edström et al. Comparison of methods for immobilization to carboxymethyl dextran sensor surfaces by analysis of the specific activity of monoclonal antibodies. J Mol Recognit 1995;8(1-2):125-31.

Kaldor SW, Kalish VJ, Davies JF, Shetty BV, Fritz JE, Appelt K et al. Viracept (nelfinavir mesylate, AG1343): a potent, orally bioavailable inhibitor of HIV-1 protease. J Med Chem 1997;40(24):3979-85.

45

Karlsson R. Affinity analysis of non-steady-state data obtained under mass transport limited conditions using BIAcore technology. J Mol Recognit 1999;12(5):285-92.

Karlström AR, Levine RL. Copper inhibits the protease from human immunodeficiency virus 1 by both cysteine-dependent and cysteine-independent mechanisms. Proc Natl Acad Sci U S A 1991;88(13):5552-6.

Kempf DJ, Sham HL, Marsh KC, Flentge CA, Betebenner D, Green BE et al. Discovery of ritonavir, a potent inhibitor of HIV protease with high oral bioavailability and clinical efficacy. J Med Chem 1998;41(4):602-17.

Kim EE, Baker CT, Dwyer MD, Murcko MA, Rao BG, Tung RD et al. Crystal structure of HIV-1 protease in complex with VX-478, a potent and orally bioavailable inhibitor of the enzyme. J Am Chem Soc 1995;117:1181-2.

Kohl NE, Emini EA, Schleif WA, Davis LJ, Heimbach JC, Dixon RA et al. Active human immunodeficiency virus protease is required for viral infectivity. Proc Natl Acad Sci U S A 1988;85(13):4686-90.

Konvalinka J, Strop P, Velek J, Cerna V, Kostka V, Phylip LH et al. Sub-site preferences of the aspartic proteinase from the human immunodeficiency virus, HIV-1. FEBS Lett 1990;268(1):35-8.

Kräusslich HG, Schneider H, Zybarth G, Carter CA, Wimmer E. Processing of in vitro-synthesized gag precursor proteins of human immunodeficiency virus (HIV) type 1 by HIV proteinase generated in Escherichia coli. J Virol1988;62(11):4393-7.

Lapatto R, Blundell T, Hemmings A, Overington J, Wilderspin A, Wood S et al. X-ray analysis of HIV-1 proteinase at 2.7 A resolution confirms structural homology among retroviral enzymes. Nature 1989;342(6247):299-302.

Le Grice SF, Mills J, Mous J. Active site mutagenesis of the AIDS virus protease and its alleviation by trans complementation. EMBO J 1988a;7(8):2547-53.

Le Grice SF, Zehnle R, Mous J. A single 66-kilodalton polypeptide processed from the human immunodeficiency virus type 2 pol polyprotein in Escherichia coli displays reverse transcriptase activity. J Virol 1988b;62(7):2525-9.

Lebon F, Boggetto N, Ledecq M, Durant F, Benatallah Z, Sicsic S et al. Metal-organic compounds: a new approach for drug discovery. N1-(4-methyl-2-pyridyl)-2,3,6-trimethoxybenzamide copper(II) complex as an inhibitor of human immunodeficiency virus 1 protease. Biochem Pharmacol2002;63(10):1863-73.

Lebon F, de Rosny E, Reboud-Ravaux M, Durant F. De novo design of a new copper chelate molecule acting as HIV-1 protease inhibitor. Eur J Med Chem1998;33:733-7.

Lightfoote MM, Coligan JE, Folks TM, Fauci AS, Martin MA, Venkatesan S. Structural characterization of reverse transcriptase and endonuclease polypeptides of the acquired immunodeficiency syndrome retrovirus. J Virol1986;60(2):771-5.

Markgren PO, Hämäläinen M, Danielson UH. Kinetic analysis of the interaction between HIV-1 protease and inhibitors using optical biosensor technology. Anal Biochem 2000;279(1):71-8.

Markgren PO, Lindgren MT, Gertow K, Karlsson R, Hämäläinen M, Danielson UH. Determination of interaction kinetic constants for HIV-1 protease inhibitors using optical biosensor technology. Anal Biochem 2001;291(2):207-18.

Markgren PO, Schaal W, Hämäläinen M, Karlén A, Hallberg A, Samuelsson B et al. Relationships between structure and interaction kinetics for HIV-1 protease inhibitors. J Med Chem 2002;45(25):5430-9.

46

Matayoshi ED, Wang GT, Krafft GA, Erickson J. Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer. Science1990;247(4945):954-8.

McQuade TJ, Tomasselli AG, Liu L, Karacostas V, Moss B, Sawyer TK et al. A synthetic HIV-1 protease inhibitor with antiviral activity arrests HIV-like particle maturation. Science 1990;247(4941):454-6.

Mildner AM, Rothrock DJ, Leone JW, Bannow CA, Lull JM, Reardon IM et al. The HIV-1 protease as enzyme and substrate: mutagenesis of autolysis sites and generation of a stable mutant with retained kinetic properties. Biochemistry1994;33(32):9405-13.

Miller V. International perspectives on antiretroviral resistance. Resistance to protease inhibitors. J Acquir Immune Defic Syndr 2001;26 Suppl 1:S34-50.

Moelling K, Schulze T, Knoop MT, Kay J, Jupp R, Nicolaou G et al. In vitro inhibition of HIV-1 proteinase by cerulenin. FEBS Lett 1990;261(2):373-7.

Nashed NT, Louis JM, Sayer JM, Wondrak EM, Mora PT, Oroszlan S et al. Continuous spectrophotometric assay for retroviral proteases of HIV-1 and AMV. Biochem Biophys Res Commun 1989;163(2):1079-85.

Navia MA, Fitzgerald PM, McKeever BM, Leu CT, Heimbach JC, Herber WK et al. Three-dimensional structure of aspartyl protease from human immunodeficiency virus HIV-1. Nature 1989;337(6208):615-20.

Porath J, Carlsson J, Olsson I, Belfrage G. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 1975;258(5536):598-9.

Potter SJ, Chew CB, Steain M, Dwyer DE, Saksena NK. Obstacles to successful antiretroviral treatment of HIV-1 infection: problems & perspectives. Indian J Med Res 2004;119(6):217-37.

Rezacova P, Brynda J, Fabry M, Horejsi M, Stouracova R, Lescar J et al. Inhibition of HIV protease by monoclonal antibodies. J Mol Recognit 2002;15(5):272-6.

Rich RL, Day YS, Morton TA, Myszka DG. High-resolution and high-throughput protocols for measuring drug/human serum albumin interactions using BIACORE. Anal Biochem 2001;296(2):197-207.

Roberts NA, Martin JA, Kinchington D, Broadhurst AV, Craig JC, Duncan IB et al. Rational design of peptide-based HIV proteinase inhibitors. Science1990;248(4953):358-61.

Robinson BS, Riccardi KA, Gong YF, Guo Q, Stock DA, Blair WS et al. BMS-232632, a highly potent human immunodeficiency virus protease inhibitor that can be used in combination with other available antiretroviral agents. Antimicrob Agents Chemother 2000;44(8):2093-9.

Rodriguez EJ, Angeles TS, Meek TD. Use of nitrogen-15 kinetic isotope effects to elucidate details of the chemical mechanism of human immunodeficiency virus 1 protease. Biochemistry 1993;32(46):12380-5.

Rosé JR, Salto R, Craik CS. Regulation of autoproteolysis of the HIV-1 and HIV-2 proteases with engineered amino acid substitutions. J Biol Chem1993;268(16):11939-45.

Salto R, Babé, LM, Li J, Rosé, JR et al. In vitro characterization of nonpeptide irreversible inhibitors of HIV proteases. J Biol Chem 1994;269(14):10691-8.

Schaal W, Karlsson A, Ahlsén G, Lindberg J, Andersson HO, Danielson UH et al. Synthesis and comparative molecular field analysis (CoMFA) of symmetric and nonsymmetric cyclic sulfamide HIV-1 protease inhibitors. J Med Chem2001;44(2):155-69.

Schechter I, Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 1967;27(2):157-62.

47

Sham HL, Kempf DJ, Molla A, Marsh KC, Kumar GN, Chen CM et al. ABT-378, a highly potent inhibitor of the human immunodeficiency virus protease. Antimicrob Agents Chemother 1998;42(12):3218-24.

Shuman CF, Hämäläinen MD, Danielson UH. Kinetic and thermodynamic characterization of HIV-1 protease inhibitors. J Mol Recognit 2004;17(2):106-19.

Shuman CF, Markgren PO, Hämäläinen M, Danielson UH. Elucidation of HIV-1 protease resistance by characterization of interaction kinetics between inhibitors and enzyme variants. Antiviral Res 2003;58(3):235-42.

Strickler JE, Gorniak J, Dayton B, Meek T, Moore M, Magaard V et al. Characterization and autoprocessing of precursor and mature forms of human immunodeficiency virus type 1 (HIV 1) protease purified from Escherichia coli. Proteins 1989;6(2):139-54.

Todd MJ, Semo N, Freire E. The structural stability of the HIV-1 protease. J Mol Biol 1998;283(2):475-88.

Tomaszek TA, Magaard VW, Bryan HG, Moore ML, Meek TD. Chromophoric peptide substrates for the spectrophotometric assay of HIV-1 protease. Biochem Biophys Res Commun 1990;168(1):274-80.

Tözsér J, Bagossi P, Weber IT, Louis JM, Copeland TD, Oroszlan S. Studies on the symmetry and sequence context dependence of the HIV-1 proteinase specificity. J Biol Chem 1997;272(27):16807-14.

Tözsér J, Gustchina A, Weber IT, Blaha I, Wondrak EM, Oroszlan S. Studies on the role of the S4 substrate binding site of HIV proteinases. FEBS Lett1991;279(2):356-60.

Tözsér J, Weber IT, Gustchina A, Bláha I, Copeland TD, Louis JM et al. Kinetic and modelling studies of S3-S3' subsites of HIV proteinases. Biochemistry1992;31(20):4793-800.

Tözsér J, Zahuczky G, Bagossi P, Louis JM, Copeland TD, Oroszlan S et al. Comparison of the substrate specificity of the human T-cell leukemia virus and human immunodeficiency virus proteinases. Eur J Biochem2000;267(20):6287-95.

Wain-Hobson S, Sonigo P, Danos O, Cole S, Alizon M. Nucleotide sequence of the AIDS virus, LAV. Cell 1985;40(1):9-17.

Wlodawer A, Erickson JW. Structure-based inhibitors of HIV-1 protease. Annu Rev Biochem 1993;62:543-85.

Wlodawer A, Miller M, Jaskólski M, Sathyanarayana BK, Baldwin E, Weber IT et al. Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease. Science 1989;245(4918):616-21.

Wlodawer A, Vondrasek J. Inhibitors of HIV-1 protease: a major success of structure-assisted drug design. Annu Rev Biophys Biomol Struct 1998;27:249-84.

Woon TC, Brinkworth RI, Fairlie DP. Inhibition of HIV-1 proteinase by metal ions. Int J Biochem 1992;24(6):911-4.

Zhang ZY, Reardon IM, Hui JO, O&apos, Connell KL, Poorman RA et al. Zinc inhibition of renin and the protease from human immunodeficiency virus type 1. Biochemistry 1991;30(36):8717-21.

Acta Universitatis UpsaliensisComprehensive Summaries of Uppsala Dissertations

from the Faculty of Science and TechnologyEditor: The Dean of the Faculty of Science and Technology

Distribution:Uppsala University Library

Box 510, SE-751 20 Uppsala, Swedenwww.uu.se, acta@ub.uu.se

ISSN 1104-232XISBN 91-554-6087-9

A doctoral dissertation from the Faculty of Science and Technology, UppsalaUniversity, is usually a summary of a number of papers. A few copies of thecomplete dissertation are kept at major Swedish research libraries, while thesummary alone is distributed internationally through the series ComprehensiveSummaries of Uppsala Dissertations from the Faculty of Science and Technology.(Prior to October, 1993, the series was published under the title “ComprehensiveSummaries of Uppsala Dissertations from the Faculty of Science”.)