Drug Resistance

C h a p t e r

15 Drug Resistance Lori Hazlehurst

OUTLINE 15.1 Introduction 371 15.2 Cancer Cell Drug Resistance 371 15.2.1 Drug Transporters and Drug Resistance 371 15.2.2 Glutathione and Drug Resistance 375 15.2.3 Alterations in Drug Target 376 15.2.4 DNA Repair and Drug Resistance 378 15.3 Drug Resistance in Infectious Diseases 379 15.3.1 Resistance to Penicillin: A Story of Two Mechanisms 379 15.3.2 Malaria and Drug Resistance 381 15.3.3 Tuberculosis Drug Resistance through Decreased Drug Activation 383 15.3.4 Drug Resistance through Drug Transport 384 15.4 Summary 385

15.1 INTRODUCTION The need to increase drug dosage to maintain the desired pharmacological effects can result from the phenomenon known as drug tolerance (discussed elsewhere in the textbook) or as a result of drug resistance. The former is considered to result from changes in the host whereas the latter arises from changes in the target organism—most frequently cancer or infectious diseases. The purpose of this chapter is to define and describe mechanisms of drug resistance encountered in the treatment of infectious diseases or cancers. Drug resistance can lead to a complete loss of drug efficacy because the target organism no longer responds to pharmacologically relevant doses of the drugs. When drug resistance develops in cancer cells, the drug is often rendered useless because increasing the dose of the drug even slightly can lead to unacceptable host toxicity.

15.2 CANCER CELL DRUG RESISTANCE Most cancers will initially respond to standard chemotherapy. However, failure to eliminate the entire tumor population (often referred to as minimal residual

n

Miles Hacker

disease) and the subsequent emergence of drug resistance currently limits the cure rate of many malignancies. It is thought that genetic instability characteristic of tumors as well as the tumor microenvironment can contribute to the emergence of clinical drug resistance. Investigators have developed drug resistant tumor cell lines to aid in the identification of drug resistant mechanism and targets. These resistant cell lines have revealed that cancer cells can employ multiple mechanisms of drug resistance including (Figure 15.1): n n n n n

Altered drug transport Altered drug metabolism Altered drug target Enhanced repair of drug induced damage Altered apoptotic pathway

In this chapter we will discuss each of these mechanisms, and possible ways to overcome these forms of resistance, where available.

15.2.1 Drug Transporters and Drug Resistance 15.2.1.1 Drug Efflux Proteins Exposing a parental tumor cell line to increasing amounts of a chemotherapeutic agent, until a drug resistant population emerges is a classic way to induce drug resistance. Once a resistant cell line has been created, investigators can use biochemical, pharmacological, and molecular techniques within an isogenic system to identify drug resistant mechanisms (Figure 15.2). One of the most frequent phenotypic changes observed in drug resistant cell line models is a decrease in the intracellular concentration of the drug. Most anticancer drugs are designed to diffuse passively through the cell membrane. However, it must be noted that some anticancer agents use existing transport systems to enter the cell. For example, melphalan, an agent that covalently cross-links DNA, contains a phenylalanine group and is reported to use the large neutral amino acid transporter, which is 371

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Drug Resistance

ABC transporters

Drug metabolism Altered drug target

Drug sequestration

Repair DNA damage Altered cell cycle checkpoints Decreased DNA damage response

Balance of pro and anti- apoptotic mediators

Cell death C C APAF-1

Drug Caspase-9

Figure 15.1 A cancer cell can evade drug-induced cell death by several mechanisms, making therapeutic intervention inherently challenging.

Acquired in vitro drug resistance models Parental cell line-Chronic Drug Selection start at the IC20 Dead cells are discarded Surviving cells re-exposed to drug Increase concentration of selection Surviving cells re-exposed to drug

Stable Drug Resistant Phenotype (3-6 months)

Characterize the phenotype (5-1000-fold resistant compared to parental cell line) Often display a multi-drug resistant phenotype

Figure 15.2 Typical method for developing acquired drugresistant cells lines.

372

critical for proper nutrition and cell growth. Other examples of membrane transporters include methotrexate, which uses the folate transport system, and nucleoside analogs, which enter the cell via nucleoside transport proteins. Passive membrane diffusion is usually a slower process than is the carrier-mediated transporter system. Remember, however, that passive diffusion is an unsaturable concentration-driven process whereas carrier-mediated transport is saturable and dependent on the number of transporter proteins present in the cell membrane. Further, the carrier-mediated process can exist in at least two forms—facilitated transport, which can shuttle chemicals across the membrane but can not shuttle against a concentration gradient, or active transport, which is an ATP-dependent transporter capable of moving chemicals against a concentration gradient. Decreased intracellular concentration of a carriermediated drug accumulation can result from mutated carrier protein such that drug recognition is decreased, and there is loss of membrane bound carrier or decreased rate of drug transport across the membrane. Another mechanism of drug resistance resulting from lowered intracellular drug accumulation also relies on a membrane protein, but in this case the pump serves as an efflux mechanism. In this case the drug most often enters the cell by simple passive diffusion, but as the

15.2 Cancer Cell Drug Resistance

drug diffuses across the cell membrane the efflux protein shuttles the drug out of the cell at sufficient rate to prevent drug accumulation to the cytotoxic level. The first phenotypic demonstration of efflux transporters was reported in 1973 when Dano noted that daunorubicin was actively pumped out of daunorubicin-resistant Erhlich ascites tumor cells. Three years later it was reported that a 170 kd protein localized to the cellular membrane of drug resistant cells but not the parental cells; this newly identified protein was called P-glycoprotein or Pgp. Subsequently, laboratories reported that tumor cells that had elevated levels of the Pgp were cross-resistant to a number of chemically divergent anticancer drugs. Overexpression of membrane-bound Pgp has since been named multidrug resistance (MDR). With the advent of powerful molecular tools it has been possible to identify the mRNA sequence of Pgp and show a striking similarity to drug transporters seen in many infectious diseases to be discussed later in this chapter. Pgp is an ATP-binding cassette (ABC) transporter protein composed of two homologous halves, each containing a transmembrane domain (TMD), a total of 12 transmembrane regions, and a nucleotide binding domain (NBD), separated by a flexible linker region. Transport of one drug molecule requires the hydrolysis of two ATP events (H and R). The binding of substrate to the transmembrane regions results in the hydrolysis of one ATP causing a conformational change in the protein and the release of substrate to the outer leaflet of the membrane. Hydrolysis at the second ATP site is required for the transporter to bind another substrate. Thus it appears that ATP hydrolysis occurs sequentially rather than simultaneously to export one molecule of substrate. Upon binding of ATP to the NBDs, Pgp undergoes conformational changes and the TMDs reorganize into three compact domains. This reorganization opens the central pore and allows transport of hydrophobic drugs (transport substrates) directly from the lipid bilayer into the central pore of the transporter. The ability of this protein to efflux drug out of the tumor cell provides the tumor cell a significant means of protection. Further, the lack of chemical specificity of the protein provides the tumor cell protection from a variety of drugs once the membrane protein is expressed. An obvious question to be asked at this point is, If the protein pumps drugs out of the cell could we not inhibit the pump and thereby obviate the resistance factor the protein provides? Yet another approach would be to develop anticancer drugs not affected by the Pgp. In fact there exist a variety of drugs that are not substrates of the membrane pump but unfortunately some of our most important anticancer drugs such as doxorubicin, vincristine, and taxol are affected by this pump. Investigators have attempted to identify drugs capable of just such a pharmacological activity. To date a number of Pgp inhibitors have been identified and are currently classified as first-, second-, or third-generation MDR inhibitors. The first-generation inhibitors

had other unwanted pharmacological features and were not developed as they were not specific MDR inhibitors. Clinical trials revealed that such drugs were limited in activity and had rather significant toxicological potential. Second-generation MDR inhibitors were more effective than their precursors but problems remained. Many of these drugs were substrates for the cytochrome P450 3A4 xenobiotic metabolizing enzyme and as a result caused unpredictable rises in plasma levels of the actual anticancer drug. Given the extraordinarily small therapeutic index for these drugs, any unanticipated elevation of drug plasma levels could have significant effects on host toxicity. Further, these drugs seemed to inhibit all the ABC transporter proteins. Given that this family of proteins has important roles in removal of xenobiotics from the host and limiting chemical access to the CNS, testes, and placenta inhibition of this family of proteins could lead to undesirable consequences as anticancer drugs would now have access to these previously protected tissue compartments. Third-generation MDR inhibitors have been developed. These drugs do not affect cytochrome P450 3A4 nor do they appear to inhibit the normal ABC transporters. To date several such drugs have been tested but none have received FDA approval. Thus, the search for a chemical means to overcome MDR continues. Reports of multidrug resistance in tumor cells that did not express the class Pgp form of MDR led investigators to search for alternate mechanisms of MDR. In 1992, Cole and Deeley identified MRP1 as being overexpressed and sharing homology to MDR1 in a human small cell lung carcinoma cell line selected for resistance to doxorubicin. This protein has three membrane spanning domains, two nucleotide binding domains, and an extracellular N-terminal. Both the structure and drug resistance spectra of MRP1 and Pgp are similar with the exception of the taxanes, which are poor substrates for MRP1. Following this initial observation an additional five MRP-related proteins have been described, and each has its own substrate and protein characteristics. As with the Pgp protein, each of these MRP proteins seems to have important physiological roles other than serving as anticancer drug efflux pumps. Doyle et al. first identified BCRP (breast cancer resistance protein) following exposure of breast cancer cells to an anticancer drug called mitoxantrone, a chemical relative of daunomycin and doxorubicin. This dimeric protein requires either homo- or Homo-dimerization for the protein to function as a membrane-bound pump. Once dimerized the protein can shuttle drug extracellularly from the membrane in a manner similar to that of the other ABC cassette proteins such as Pgp. Finally, a protein has been identified that is unique from the previously discussed ABC proteins. This protein, called lung resistance protein or major vault protein, has a molecular weight of 110 kD and is found not in the cell membrane but rather the nuclear membrane or the cytoplasm. The role of this protein is to prevent the drug from accessing the nucleus by trapping the drug in vesicles called vaults. The vaults then shuttle 373

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the drug to the cell membrane where the drug is then removed from the cell. An alternative pathway for the drug is that the LRP shuttles the drug to the lysosome where the drug is removed by exocytosis. With these discoveries it became apparent that multiple drug transporters were likely to exist and function in vivo. Indeed the subsequent sequencing of the human genome revealed that the ABC transport family consists of 49 members, which are now organized into seven subfamilies (A-G). Most of the 49 transporters recognize and transport a variety of xenobiotics as well as physiological substrates that include bile acids, peptides, steroid ions, and phospholipids. However, of these 49 transporters MRP1, Pgp and BCRP are the best documented for their role in conferring anticancer drug resistance and for this reason will be the focus of our discussion in this portion of the chapter. Pgp, MRP1, and BCRP belong to the ABCB, ABCC, and ABCG family, respectively. Pgp (gene symbol ABCB1) has two nucleotide binding regions (NBD) that are separated by two membrane spanning domains (MSD). Each MSD is comprised of six transmembrane regions (Figure 15.3). MRP1 (gene symbol ABCC1) contains three MSD domains and the NH2 terminus is located on the extracellular surface; in

Out

H In

R

Figure 15.3 Pgp protein in cell membrane.

Table 15.1

contrast, BCRP (ABCG2) has only one MSD region and one NBD and is thought to require homodimerization or possibly even a homotetramer to function as a drug efflux pump. As shown in Table 15.1, some overlap with respect to substrate recognition exists between the identified transporters. Despite the overlap in substrate recognition (Table 15.1), differences between individual transporters include tissue distribution and substrate preference. Thus, these ABC transporters are not functionally redundant molecules. Pgp is considered to prefer large hydrophobic, either uncharged or slightly charged compounds. MRP1 also can extrude large uncharged compounds but it also exports hydrophobic anionic conjugates. MRP1, although ATP-dependent, transports via a cotransport mechanism in which drug molecules are transported with reduced glutathione (GSH), whereas both Pgp and BCRP transport one molecule at a time. Moreover, MRP1 has been shown to export glutathioneconjugated endogenous metabolites including cysteinyl leukotriene and prostaglandin, which might provide an important role in regulating arachodonic acid metabolites. In addition, MRP1 has been shown to export glutathione conjugated anticancer agents such as melphalan, doxorubicin, and cyclophosphamide (see Chapter 9 for an in-depth discussion of drug excretion). Finally, MRP1 plays in important role in transporting glutathione conjugated toxins such as alflatoxin B1, which could provide a protective mechanism against xenobiotic toxins and carcinogens. The substrate preference of BCRP is still under investigation, and literature reports are complicated by the observation that polymorphic variants of BCRP alter the substrate recognition of this transporter. However, recent evidence suggests that, similar to MRP1, BCRP can recognize and export metabolites including sulfated estrogens and methotrexate and folate polyglutamates. As discussed earlier, Pgp, MRP1, and BCRP are expressed in normal tissues and are thought to be important for protecting normal cells from the accumulation of xenobiotics. The tissue distribution of MRP1 is the most ubiquitous and is expressed in most tissues with high

Substrate Recognition

Common Name

Systematic Name

Anticancer Agents

Other Substrates

Pgp

ABCB1

Neutral and cationic organic compounds, many commonly used drugs

MRP1

ABCC1

BCRP

ABCG2

Vincristine Vinblastine Doxorubicin Etoposide Imanitib Vincristine Doxorubicin Etoposide methotrexate Mitoxantrone Topotecan Doxorubicin Imanitib Gefinitib

374

Organic anions Glutathione conjugated-doxorubicin, melphalan, aflatoxin B1 Gluthathione Sulphated oesterogens Methotrexate-polyglutamates Prazosin

15.2 Cancer Cell Drug Resistance

levels found in the lung, testis, kidneys, skeletal muscle, and peripheral blood mononuclear cells but at relatively low levels in the liver. The tissue distribution of Pgp and BCRP expression is more limited compared to MRP1. High expression of Pgp in normal tissue is found in the intestine, liver, kidney, placenta, and the blood–brain barrier. BCRP is found most frequently in the placenta, intestine, breast, and liver. All three transporters are thought to form an intestinal barrier for the absorption of drugs. Pgp plays an integral role in preventing xenobiotics from entering past the blood–brain barrier, whereas MRP1 is localized to the basolateral membrane of the chorid plexus and serves to pump xenobiotics from the blood-cerebrospinal-fluid into the blood. In summary, in addition to conferring drug resistance to tumor cells, ABC transporters are a major determinant of absorption, distribution, and toxicology of drugs. As can be seen from this overview of transportrelated drug resistance, there are myriad ways in which the tumor cell can overcome effects of anticancer drugs via drug efflux. The problem with this is the fact that these transport proteins not only provide resistance for the tumor cell, they also serve important physiological roles for the host. Simple inhibition of such proteins will not likely provide a significant therapeutic advantage for a drug. These and similar problems will be discussed in the section on drug resistance in infectious diseases. Ways to modulate such resistance will be an important focus of pharmacological research for the foreseeable future.

15.2.1.2 Drug Uptake Proteins in Drug Resistance The classic antifolate methotrexate MTX continues to be an important component of the chemotherapeutic armamentarium for a variety of cancers including pediatric ALL, osteogenic sarcoma, lymphoma, and breast cancer. Raltitrexed, another anti-folate, is used throughout much of the world outside of the United States for advanced colorectal cancer. Finally, Pemetrexed was approved in 2004 for the treatment of pleural mesothelioma and shortly thereafter as a second line treatment for nonsmall cell lung cancer. These drugs mimic the natural folate molecule and in so doing utilize the membrane-bound reduced folate carrier (RFC) protein system to gain entry into the cell. The RFC must actively transport sufficient levels of unbound drug to provide intracellular drug concentrations adequate to sustain inhibition of the target enzyme dihydrofolate reductase (DHFR) and for the synthesis of polyglutamated antifolates, the storage form of these drugs. Indeed, antifolate resistance due to decreased RFC expression has been cited in the literature since 1962 and has since emerged as an important mechanism of resistance to classical antifolates. Loss of RFC function can occur by decreased expression of the protein and thus decreased levels of membrane-bound RFC. Once the RFC was cloned it became evident that profound losses of RFC transport and antifolate resistance were associated with

mutations in the RFC. These mutations resulted in decreased transport of the drug or no loss of drug transport and tremendous increase in folate transport. Since the antifolates compete for the binding of DHFR with normal reduced folates, the increased transport of folates provided the cells as much benefit as did the decreased antifolate transport.

15.2.2 Glutathione and Drug Resistance Increased intracellular glutathione levels can contribute to drug resistance by either direct conjugation of the drug and/or by preventing oxidative stress induced by redox cycling drugs such as doxorubicin (refer to Chapters 8 and 9 for an excellent review of glutathione and drugs). Glutathione conjugation to electrophillic anticancer agents are catalyzed by glutathione s-transferases (GSTs) (Figure 15.4). Glutathione conjugation can render compounds biologically inactive as the reactive group is now neutralized and may lead to the inability of the drug to bind the target. For cross-linking agents, such as melphalan glutathione, conjugation inhibits DNA cross-linking, which is considered to be the initiating cytotoxic event of melphalan induced cell death. In addition, glutathione conjugated melphalan is now a good substrate for MRP1 mediated drug efflux. Increased glutathione levels have also implicated resistance platinum analogs as the conjugation of the platinum analog prevents activation of the drug thus preventing drug-DNA interactions.

Pgp

MSD2

MSD1

Out In NH

COOH NBD2

NBD1

A MRP1 NH2 Out

MSD1

MSD3

MSD2

In

COOH NBD1

B

NBD2

MSD1

BCRP Out In NH 2

C

COOH NBD1

Figure 15.4 Predicted secondary structure of Pgp, MRP 1, and BCRP. Pgp contains two membrane spanning domains (MSD) and two nucleotide binding domains (NBD). In contrast, MRP 1 contains MSD and two NBD. Finally BCRP is considered a half-transporter and contains one MSD and one NBD, and requires dimerization to function as transporter.

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In support of the importance of glutathione levels as a mechanism of drug resistance, several investigators have shown that acquired drug resistant cell lines contain elevated levels of glutathione. The rate-limiting step of de novo glutathione synthesis is glutamylcysteine synthase. Depletion of glutathione levels with the pharmacological inhibitor of glutamylcysteine synthase L-buthionine-[S,R]-sulfoximine (BSO) is reported to increase the sensitivity of cells to melphalan in several cell line models. Previous work has demonstrated that the catalytic subunit of glutamylcysteine synthase is increased in acquired melphalan resistant cell lines. The gene expression profiling correlated with our earlier finding showing that melphalan resistant cell lines demonstrated increased glutathione levels. Thus current experimental data suggest that increased glutathione levels and regulation of de novo synthesis of glutathione may be important for mediating resistance to melphalan and other alkylating agents.

15.2.3 Alterations in Drug Target 15.2.3.1 Structural Modifications Assuming the access of the drug to the target is unaltered, the next temporal point along the cell death pathway which can influence cell survival following drug treatment, is the drug target itself. Documented alterations in the target include point mutations, amplifications, alterations in the localization, and activity of the target. Since discussing all anticancer drug targets is beyond the scope of this chapter, for purposes of illustration we will discuss documented alterations in Bcr-Abl. The discovery that Bcr-Abl was the transforming event associated with chronic myelogenous leukemia (CML) provided an ideal target for drug discovery. Indeed Bcr-Abl inhibitors first discovered in 1996, remain the best success story of target-directed drug development in the field of cancer chemotherapy. Typically 90% of CML patients will express Bcr-Abl, which is created by a reciprocal translocation between chromosome 9 and 22, creating a fusion protein consisting of Bcr (breakpoint cluster region) and the proto-oncogene c-abl. The end result

of this is the expression of a chimeric fusion protein referred to as Bcr-Abl. This hybrid protein contains functional domains from both Bcr and Abl. Within the Bcr region there exist two regions, which are causally linked to the oncogenic properties of BcrAbl. Phosphorylation of tyrosine 177 is critical because this event appears necessary for binding of Grb2 and the subsequent activation of the Ras pathway. The second region of Bcr is the coiled-coil domain located in the n-terminus (amino acids 1-63). This region is required for dimerization of Bcr-Abl and is crucial for Abl kinase activity. Abl is classified as a nonreceptor tyrosine kinase and normally the kinase activity of Abl is tightly regulated. In normal cells Abl is expressed largely in the nucleus, but can be found in the cytoplasm as well. Cellular functions associated with Abl are complex and include regulation of cell cycle progression and DNA repair. Similar to other tyrosine kinases, Abl contains an SH3 and SH2 domain at the N-terminal region of the protein. SH3 and SH2 domains allow for docking of proteins containing proline rich domains (SH3 domain) and tyrosine domains that interact with the SH2 domain. Abl also contains nuclear localization signals (NLS) that allow for nuclear import as well as a nuclear export signal, and this is consistent with the observed nuclear and cytoplasmic localization of Abl. The fusion protein Bcr-Abl demonstrates increased constitutive kinase activity compared to Abl and is located predominately in the cytosol. The uncontrolled kinase activity of Bcr-Abl interacts with effector proteins resulting in disregulated cellular proliferation and reduced sensitivity to apoptotic stimuli. Bcr-Abl is known to activate multiple survival pathways including the PI3 kinase/AKT survival pathway and Stat5. Stat5 is a transcriptional regulator of the anti-apoptotic Bcl-2 family members MCL-1 and Bcl-xl. Current data suggest that the transforming capabilities of Bcr-Abl are the culmination of the activation of multiple survival and growth pathways. In summary, Bcr-Abl is a constitutively active tyrosine kinase and is generally accepted as the initiating step in CML. CML typically begins with an indolent chronic phase, which is associated with the gradual expansion of myeloid cells with normal differentiation, and over time will proceed to a

CH3

H3C

O NH

O

C IR

N

N

T N

N G

O

H2N

N

dR

N

NH

?

O N

N meG

O

H2 N

N

N

dR

dR Nitrosourea DNA alkylation

Figure 15.5 Alkylation of DNA.

376

N

Temolozomide alkylation

15.2 Cancer Cell Drug Resistance

Guanine in DNA Cleth-MGMT

me-MGMT

Methylation (e.g. TMZ)

MGMT

O 6-meG

CH3

H3C

O

dR

N

T

NH

? H2N

MGMT

O 6-ClethG

Replication

O

Spontaneous

N

N meG

O

Chloroethylation (e.g. BCNU)

N

N

O 6-meG : T mismatch

N1-O 6-ethanoG

dR

Futile MMR and replication

MGMT

Spontaneous

Replication

NH

MGMT-DNA crosslink

DNA interstrand crosslink

dR

N

C

N

O N

N G

O

H 2N

N

Replication DNA ds break

Mutation and transformation

Apoptosis

Recombination

Cell death

??

N dR

DNA ds break

Recombination

Survival (resistance, carcinogenesis)

Figure 15.6 MGMT role in cell death and cell survival.

terminal blastic stage. Disease progression is usually associated with additional genetic abnormalities as well as impaired differentiation. Although most patients will respond to Imatinib it is not curative as minimal residual disease can be detected at the molecular level in the bone marrow. Mutations within the kinase domain of Bcr-Abl which can inhibit drug binding is known to contribute to clinical resistance. Additional mechanisms of resistance include the activation of Bcr-Abl independent survival pathways and overexpression of drug transporters. Finally an active area of research is to determine the contribution of the microenvironment of the bone marrow were minimal residual disease is typically found. Determining whether specific soluble factors or extracellular matrixes produced by the bone marrow microenvironment contributes to imanitib resistance may lead to combination therapies targeting minimal residual disease within the bone marrow compartment. Despite the lack of complete molecular understanding of how Bcr-Abl transforms cells into a malignant phenotype, Bcr-Abl provided an ideal target for drug development. In 1996, STI571 or Imanitib was discovered as an inhibitor of Bcr-Abl kinase activity. Imanitib is a competitive inhibitor of the ATP binding domain located in the tyrosine kinase domain of Bcr-Abl. The result of Imanitib binding to Bcr-Abl is the inhibition of the kinase activity and eventual cell death in BcrAbl positive cells. Clinically Imanitib rapidly became the first-line treatment for CML.

15.2.3.2 Altered Levels of Drug Target Methotrexate binds to DHFR and inhibits the cell’s ability to maintain the stores of tetrahydrofolates (THF) necessary for cell survival and division. More than 30 years ago studies were performed that clearly demonstrated that acquired methotrexate resistance resulted from DHFR gene amplification and DHFR enzyme overexpression. Gene amplification can result from a stable expanded chromosome region known as a homogeneous staining region (HSR). This type of gene amplification is stable and replicated with cell division. The other type of gene amplification is an unstable formation of double minutes (DM) in which the DMs are acentromeric and cannot participate in equal chromosome segregation at mitosis. The end result is the potential loss of the gene amplification in daughter cells and the loss of methotrexate resistance. Whether the stable mutation HSR or the unstable DM, cells that overexpress DHFR have a consistent level of resistance depending upon the amount of DHFR present. As the level of DHFR increases, the fraction of active DHFR needed to maintain cellular stores of THF decreases. For example, if 5% of the catalytically active DHFR is needed to maintain THF and DHFR, amplification results in a 100-fold increase in DHFR, then only 0.05% of the total enzyme is necessary to maintain THF. In this case methotrexate must now inhibit greater than 99.95% of the available DHFR to negatively impact THF pools. 377

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Given that the antifolates enter cells by a nonconcentrating RFC transport system, attaining 99.95% inhibition of DHFR would be virtually impossible without also attaining highly toxic or even lethal blood levels of the antifolate. Newer antifolates have been developed that are THF antagonists, which potently inhibit the enzyme thymidylate synthase (TS). Again, cells exposed to increasing levels of the antifolates resulted in an increased resistance to these antifolates. In this circumstance the increased resistance was related to increased expression of TS and not DHFR. Similarly cells made resistant to classical TS inhibitors such as 5-fluorouracil were cross-resistant with the TS inhibitor antifolates.

Many anticancer drugs kill tumor cells by forming covalent bonds with DNA. This class of compounds is known as the alkylating agents and can form interstrand crosslinks, intrastrand crosslinks, or can modify the basic structure of nucleotides making up DNA. Such drug DNA interactions stop DNA synthesis, and tumor cells die when the number of DNA lesions exceeds the capacity of the cell to correct these lesions. Hence an obvious mechanism of drug resistance would be to increase DNA repair processes in the target cell. Previously we discussed the role of glutathione in drug resistance. In that case the glutathione prevented formation of DNA lesions. The present mechanism of resistance relies on the ability of the cell to correct the actual DNA damage caused by the alkylating agents. Alkylation of guanines at the O6 position is common for nitrosoureas and temozolomide. However, the actual product formed is quite different depending on the drug in question. Whereas temozolomide forms a methylated O6 product, the nitrosoureas form a chloroethylated O6 product, which then undergoes a spontaneous rearrangement for an interstrand crosslink. An enzyme, O methyl guanine DNA-methyltransferase (MGMT), was first identified in bacteria, but subsequently a human equivalent was shown to provide cancer cells protection from the lethal effects of the drugs. MGMT can remove the alkylation product before DNA damage can occur. For the drugs such as temolozomide, lethality occurs when the mismatch repair system (MMR) causes a futile replication and double-stranded DNA breaks occur. For the nitrosourea alkylation products, MGMT removes the chloroethyl group prior to the spontaneous formation of the interstrand crosslinks. Interestingly, it appears as though the drug itself induces tumor cell drug resistance as these drugs induce the production of MGMT (thus increasing DNA repair) and at the same time suppress the production of MMR (thus decreasing the formation of lethal ds DNA breaks). One way to overcome this form of resistance is to decrease the effectiveness of MGMT. Pseudosubstrates

378

H3C

O N N

N

NH2

N

O

N

NH

N NH2

N

dR

dR

A

B O 6-Benzylguanine

O 6-(4-Bromothenyl)guanine Br S O

O N

15.2.4 DNA Repair and Drug Resistance

O 6-Methylguanine in DNA

Guanine in DNA

NH

C

N

N N

NH

NH2

N N

NH2

D

Figure 15.7 Alkylation of guanine at the O6 position.

(as shown in Figure 15.7) that have been synthesized inhibit the enzyme and thereby prevent the repair process. The problem with this approach is that the MGMT inhibitors provide an unacceptable collateral toxicity. The alkylating agent Cisplatin forms intrastrand crosslink DNA adducts that trigger a series of intracellular events that ultimately results in cell death. If the cell can process and repair these lesions before the apoptotic pathway is initiated, the cell can survive the effects of cisplatin. One of the repair processes thought to have a major impact on resistance to alkylating cytotoxicty is nucleotide excision repair (NER). NER is a highly conserved DNA repair pathway that can deal with a wide variety of DNA damaging agents; most commonly studied are UV T-T dimmers and agents such as the nitrogen mustards, cisplatin, and aryl mutagens. The basic mechanism is shown in Figure 15.8, using the UV dimerization as the model. This process, although simple in description, is a complex process using a number of proteins to complete the process. Initially, proteins must recognize the damaged DNA and bind to the lesion, which then complexes with the damaged DNA binding factor. The damaged area is then demarcated and verified by a complex of proteins referred to as TFIIH. Yet another complex of proteins then binds to the site, readying the lesion for incision. The lesion is then cut, forming a 24-32 nucleotide oligomer, which is then released and the gap filled by DNA polymerases. Finally the DNA ligase enzyme reanneals the repaired sequence with the undamaged DNA sequence. By increasing the amount and/or activity of the DNA repair process the cell can withstand increasing amounts of DNA damage and repair these lesions before the cell process is begun. Given the number of proteins involved in this process it would appear

15.3 Drug Resistance in Infectious Diseases

Damage recognitior

XPA RPA

UV

5⬘ 3⬘

A XPB

D

XP

B

TFIIH

Lesion demarcation and unwinding

RP A TFIIH

C Dual incision

5⬘ 3⬘

ERCC-1 XPF

Incision

Incision XPG

D 27-29 mer Relase of damaged nucleotide

E Gap filling DNA synthesis

DNA polγ/ε PCNA

DNA ligase

5⬘ 3⬘

F Figure 15.8 Nucleotide excision repair (NER).

likely that there exists a number of potential targets for drug discovery and overcoming this form of drug resistance.

15.3 DRUG RESISTANCE IN INFECTIOUS DISEASES With the advent of drugs that could effectively treat previously lethal or severe infectious diseases, the era of infectious diseases was naively declared over in the 1970s. Unfortunately, the inaccuracy of this statement was demonstrated shortly thereafter as more and more drug resistance was noted in an ever-increasing number of infectious diseases. The halcyon days of living in an infectious disease-free world never developed, and now we are faced with counter-attack by the drug resistant infectious agents. In the remainder of this chapter we will discuss ways in which these organisms have been able to avoid the toxic effects of drugs used today.

15.3.1 Resistance to Penicillin: A Story of Two Mechanisms Almost immediately after the introduction of penicillin to the clinic the value of the drug was evident. Different penicillins were developed with differing pharmacological properties, routes of administration, and spectrum of activities, but all penicillins worked in the same manner and required one particular chemical moiety, that being an intact beta-lactam ring (Figure 15.9). Opening of the beta-lactam ring results in the complete loss of pharmacological activity of the drugs. Basically, the bacterial cell wall has layers of peptidogylcans that are then linked together by crosslinking the peptide chains (transpeptidation) of the peptidogylcan building blocks. The crosslinking of these building blocks strengthens and stabilizes the bacterial cell wall. The penicillins inhibit the peptide crosslinking process and thus do not allow the building blocks to polymerize, thus destabilizing and weakening the cell wall. The bacterial membrane being highly permeable now

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Drug Resistance R C HN

O

HC

H C

C

N

S

C CH

CH3 CH3 COO−

O Penicillin

Figure 15.9 Beta lactam ring of penicillin.

expands rapidly, without the support of cell wall lyse. The pharmacologic target for the penicillins is a family of proteins called the penicillin binding proteins (PBP), which play a critical role in transpeptidation. Penicillins bind to these proteins, block transpeptidation, and prevent cell wall synthesis. From this brief description of penicillin mechanism of action and structural requirements, two likely sites of drug resistance stand out. First, if the bacteria were able to break open the beta-lactam ring then the drug would lose its pharmacological activity. Second, if mutations of PBP were to occur, preventing penicillins from binding to the PBP, the penicillins would lose their pharmacological activity. Both have been described in clinical isolates and will be discussed next.

15.3.1.1 Drug Inactivation by Beta-Lactamases The first described mechanism of a bacterial resistance was the production of a beta-lactamase enzyme capable of opening the beta-lactam ring and rendering the penicillins inactive. There exist hundreds of beta-lactamases, but there are only two primary ways in which these enzymes attack the drug. One class of beta-lactamases, the serine-beta-lactamases, is divided further into four categories: group 1 is poorly inhibited by clavulanic acid (a drug designed to prevent or lessen penicillin inactivation by the beta-lactamases); group 2 beta-lactamases are clavulanic acid sensitive and include a family of enzymes called the extended spectrum beta-lactamases; group 3 beta-lactamases are thought to be metallo beta-lactamases that work through activation of water via a Zn center; and group 4 includes clavulanic resistant beta-lactamases, which do not fit into the other categories. The serine beta-lactamases work in a mechanism similar to serine proteases and esterases, where the Ser center performs a ring opening electrophilic attack on the beta-lactam ring. Water then enters the site and enables the hydrolysis of the enzyme drug intermediate. The inactive penicillin is released and the enzyme is ready to attack another ring. This group of beta-lactamases is by far the more clinically important form of drug inactivation. Most gram-positive bacteria are capable of producing and secreting beta-lactamase into the medium surrounding the organism. Thus, if the antibiotic is sensitive to the enzyme, drug inactivation will occur 380

primarily extracellularly. On the other hand, gramnegative bacteria secrete less enzyme into the medium and as a result less extracellular drug inactivation is observed with these organisms. However, many grampositive bacteria do produce the enzyme but retain the enzyme in the periplasmic space. In this way the drug is inactivated after it has diffused across the cell wall but before it binds to its target PBP. It should be noted that another class of beta-lactam antibiotics that also inhibit PBPs has been developed. These drugs, the cephalosporins (Figure 15.10) also require an intact beta-lactam ring to be active and are therefore susceptible to beta-lactamase inactivation. However, given the structural differences from penicillin, the sensitivity of the cephalosporins to the beta-lactamases differs from that of the penicillins. Note that the cephalosporins six-member dihydrothiazine ring is attached to the beta-lactam ring. Note also that whereas the penicillin backbone has but one site for chemical modification, there are two sites for the cephalosporins. As with the penicillins there is a wide array of clinically available cephalosporins with varying sensitivities to the beta-lactamases. There are a number of excellent reviews for in-depth discussions regarding antibiotic sensitivity to the betalactamases. A number of approaches have been taken to overcome beta-lactamase-driven drug resistance, but the two that have been most successful have been developing drugs less sensitive to enzymatic degradation and the coadministration of an enzyme inhibitor such as clavulanic acid with the beta-lactam drug. Given the fact the more than 200 different beta-lactamases have been reported it is not surprising that neither approach has been totally successful. Further, as new beta-lactam drugs are developed the bacteria respond by producing enzymes capable of overcoming the advantages of the new drugs. Remember that the developing resistance is a result of a selection process placed on the bacteria in which the drug selects for bacteria that have the ability to survive the chemical effects of the drug. There is no true adaptive response by the bacteria; rather it is simply a mutational process that enables the organism to live in an environment otherwise toxic to the organism.

O R1

C

S H N

H C

H C

H C H

Acyl side chain

Beta-lactam ring

Dihydrothiazine ring H

C O

C

N C General structure of cephalosporins

Figure 15.10 General structure of cephalosporins.

R2

15.3 Drug Resistance in Infectious Diseases

15.3.1.2 Altered Drug Target As stated earlier, the target for the beta-lactams is the transpeptidase family of enzymes, commonly referred to as the PBP. It should be noted, however, that PBP is at times loosely employed in the literature. Some PBP in fact do bind to the beta-lactams but have yet to be shown to play a role in cell wall synthesis. Further, a bacteria more than likely will contain several types of PBP and only recently has their physiological roles in cell division begun to be understood. PBPs are serine acyltransferases. Once the beta-lactam has bound to the PBP it interacts with the active site serine. The resulting acyltransferase-drug complex can only be hydrolyzed at a very slow rate, thus effectively lowering the enzyme available for cell wall synthesis. Interestingly, the PBP also inactivates the drug by cleaving the beta-lactam ring, but the turnover is so slow that the drug effectively inhibits transpeptidation in a suicide-like mechanism. In contrast, the beta-lactamases also bind the beta-lactams through a serine in the active site of the enzyme but in this case there is an extremely high turnover rate and the enzyme inactivates the drug rather than the drug inactivating the enzyme. There have been approximately 800 PBPs identified thus far. These proteins have been divided into class A and class B proteins, depending primarily on the number of reactions the protein is capable of catalyzing. Class A PBPs are bifunctional and catalyze both the polymerization of the glycan subunits (GT) and the transpepditation (TP) of the peptide chains, whereas the Class B PBPs are monofunctional and serve as transpeptidase enzymes only. Both classes of PBPs are susceptible to beta-lactam attack and inhibition. Once the PBP is acylated by the beta-lactam it is incapable of further hydrolysis of the covalent acyl-enzyme intermediate and transpeptidation is inhibited. Resistance to the beta-lactams can occur through mutation of the PBPs. Although this mechanism is far less studied than the beta-lactamase mechanism, interesting observations have been made. Thus far, there have been reported mutations in PBPs that cause pneumococcal resistance to the beta-lactams. One mechanism has been the mutation in which the serine binding site for the beta-lactams is distant from the active site of the enzyme. Thus, the drug binds to the Ser site, but this site is no longer associated with the active site of the enzyme. The other mechanism has been reported to be a charge modification of the active site, thus preventing the drug access to the active site. Yet another mechanism has been the increased expression of one or more PBP in Enterococcus faecium. This overproduction of the PBP enables the overly produced PBP to take over the functions of the beta-lactam inhibited PBPs. A similar mechanism has been reported for methicillin-resistant Staphylococcus aureus. However where the E. faecium is a naturally occurring type of PBP related resistance, the MRSA results from a horizontal transfer of genetic material. Once transferred, the PBP production can be induced to overproduction following exposure to a beta-lactam. Again,

this overly produced PBP can take over the function of the inhibited PBPs. The future of PBP-targeted antibiotics remains active. A new approach is to develop transpeptidase inhibitors that lack the beta-lactam ring, such as the carbapenems, which are resistant to Ser-based transpeptidases but can be inactivated by the metallo-transpeptidases. Still others are searching for transpepditase inhibitors that do not resemble the beta-lactams. At present a limited number of nonbeta-lactam inhibitors have been identified, but the arylalkylidene-related molecules appear to be excellent precursors for active drug discovery. Another approach has been to not target the transpepditase function of the PBPs, but rather to develop inhibitors of the GT function of the PBPs. GT is essential for bacterial survival and recent molecular studies have clearly demonstrated that GT domains are distinct from the TP domains.

15.3.2 Malaria and Drug Resistance Probably no disease has killed more humans throughout history than malaria. Malaria is transmitted by the bite of an Anopheles mosquito. There are four different parasites that can infect humans, but Plasmodium falciparum is the most virulent. The parasite first migrates to the liver where after several replications the parasites are released into the bloodstream. The parasite then invades the red blood cells and feeds off the hemoglobin. Survival of the parasite relies on the ability of the parasite to package the toxic heme metabolites formed during hemoglobin metabolism in nontoxic hemazoin particles. The parasite causes anemia, fevers, chills, veno-occlusive disorders, liver damage, and death. With the discovery of effective antimalarial drugs and DDT, an amazingly effective mosquitocide, it was felt that malaria could be a disease of the past. Unfortunately, due to environmental concerns, the use of DDT was dramatically curtailed and at about the same time the development of new antimalarial drugs was basically stopped. As a result the mosquito vector of this disease came back strong and as we relied on the same drugs for a prolonged period of time, drug resistance developed in the parasites. In fact, chloroquine, the gold standard for malaria treatment, is rapidly becoming useless as resistance is flourishing worldwide. Similarly, resistance to other drugs such as the antifolates and even the newly introduced arteminisins is becoming problematic.

15.3.2.1 Resistance to Chloroquine: Another Membrane-bound Transporter Protein Chloroquine is a semisynthetic derivative of quinine, the first drug used effectively to treat malaria. Chloroquine was used for decades as the drug of choice to treat malaria because it was a safe, highly effective, and relatively inexpensive drug. The effectiveness of chloroquine was first clearly demonstrated during World War II when in the Pacific Theater more GIs 381

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were hospitalized by malaria than by the Axis forces. By 1957, the first reports of chloroquine resistance were coming from Thailand, followed shortly thereafter in South America. By 1988, chloroquine resistance was encountered worldwide, and today the drug is essentially useless in all but a few areas of the world. Chloroquine kills by concentrating in the food vacuole of the parasite and preventing the formation of the nontoxic heme metabolite hemazoin by the parasite. The parasite then dies from the toxic by-products of its own metabolism of hemoglobin. In the resistant parasite far less chloroquine is maintained in the acidic food vacuole and the parasite is able to detoxify the heme moieties. The exact mechanism of chloroquine action in the food vacuole is not understood, but there are experimental data supporting the idea that chloroquine binds to a heme moiety that both enables high concentrations of chloroquine to develop within the food vacuole and interferes with normal hemoglobin metabolism. The once-touted hypothesis of proton trapping of chloroquine in the acidic food vacuole, which then alters the pH of the organelle, appears inaccurate. How the parasite removes chloroquine from the food vacuole appears to be related to mutations of a specific gene, named pfcrt for “plasmodium falciparum chloroquine resistance transporter.” The protein product of this gene (CRT) is a 49 kD transmembrane protein that has a predicted 10 transmembrane domains. The key mutation seems to be K76T since no chloroquine resistant isolate carries the wild type lysine at position 76. It should be noted that usually a number of other mutations are noted in chloroquine resistant malaria, but only the K76T amino acid switch is seen consistently in the chloroquine resistant malaria. What is the function of the CRT and how does the K76T affect the protein? Bioinformatics place this CRT in a super-family of drug and metabolite membrane transports. Currently it is thought that in the wild type parasite the chloroquine enters the food vacuole as nonprotonated, but is converted to the diprotonated form within the acidic environment and thus prevents the efflux of the drug by the normally positively charged CRT at the K76 site. However, the K76T mutation results in a CRT that can now transport the chloroquine out of the food vacuole. Recent results further suggest that the CRT protein is not an active transporter but rather a gated channel or pore that allows the chloroquine to escape from the vacuole in an energy-independent process. The membrane of the food vacuole has other transporter proteins associated with it. One such protein, encoded by the pfmdr1 gene, shows structural similarity to the human PGP that was discussed earlier in this chapter. This malaria protein has a molecular weight of 162 kD, and is an ABC transporter with 12 transmembrane domains and two ATP binding folds. The mammalian PGP pumps drug out of the cell but the malarial MDR1 actually pumps drugs into the food vacuole. Although this protein can add to the resistance associated with K76T mutation of the CRT protein, by itself it does not provide a chloroquine resistant 382

phenotype. However, numerous reports find a reciprocal relationship between chloroquine resistance and resistance to other drugs that act on the food vacuole such as mefloquine and halofantrine. Exactly how the MDR1 protein affects parasite sensitivity to food vacuole poisons remains a mystery.

15.3.2.2 Antifolate Resistance Assessment of resistance to the antifolates has been much more straightforward than it has for chloroquine, since the molecular targets for this class of drugs have been known since the 1930s. The malaria parasite relies on the intracellular synthesis of the folate molecule since the parasite cannot effectively scavenge folates from the surrounding environment. These organisms have an enzyme dihydropteroate synthase (DHPS). They also contain dihydrofolate reductase (DHFR) to insure sufficient levels of tetrahydrofolates needed for many cellular processes. The DHPS is an enzyme unique to the malaria parasite but both the host and the parasite have DHFR. Fortunately drugs that target the parasitic DHFR do so at affinities 100 times greater than that of the mammalian DHFR. Antifolates with clinically important activity include the sulfa drugs, which target DHPS, as well as pyrimethamine and proguanil, which target DHFR. The most commonly used clinical formulation of the antifolates is to use a combination of a sulfa drug such as sulfadoxine and pyrimethamine to inhibit both enzymes involved in folate metabolism. The primary goal of this combination is to inhibit DNA synthesis when the parasite begins DNA synthesis for cell replication. The genetic basis for antifolate resistance relies almost exclusively on mutations found in the protein targets such that the drug no longer binds with sufficient affinity to allow inhibition of the enzyme. There need be but a few point mutations in these enzymes to result in loss of drug affinity. The most common DHFR mutations are S108N, N51I, and C59R. Similarly, point mutation patterns appear common for the DHPS. Further these mutations seem geographically related in that sulfadoxine resistance in Africa is commonly associated with A437G/K540E mutations, whereas sulfadoxine resistance in Southeast Asia is associated with A437G/A581G or A437G/A581G/ K540E mutations. In Southeast Asia and South America a fourth mutation, I164L, in DHFR in combination with the three mutations just cited results in an organism completely resistant to antifolate resistance. Surprisingly, a method of antifolate resistance commonly cited in tumor cells is gene amplification resulting in increased target protein and decreased anti-folate efficacy, which has yet to be identified in any clinically relevant isolate. A further difference in antifolate resistance between mammalian cells and the parasitic cell is altered polyglutamation of the antifolate drugs. In mammalian cells, folates are taken up by a folate transport system and once inside the cell are polyglutamated to trap the folates intracellularly. A reported mechanism of resistance in the cancer cell is the loss of

15.3 Drug Resistance in Infectious Diseases

polyglutamation of the antifolate, and cells can no longer concentrate and store the antifolate. Cytotoxic levels of the drug cannot be attained and the cell becomes resistant to drug. There does not appear to be such a process in the parasite and thus antifolate trapping is not an important process in the parasite.

15.3.2.3 Recent Drugs, Drug Resistance, and Overcoming Resistance Unfortunately, as new drugs are finally entering the clinic for the treatment of malaria, resistance is developing rapidly against these drugs. Two such drugs are atovaquone and artemisinins. One approach that is gaining traction is to end monodrug therapy and incorporate multidrug therapy to treat malaria. The concept behind drug combination is that a simple point mutation may not provide the protective cover that the parasite needs when two drugs are used simultaneously. This approach is being considered in the therapy of all infectious diseases. Whether this approach does in fact slow the development of drug resistance is at present unknown. Obviously the other approach to overcoming drug resistance is to identify new parasitic targets and develop drugs to affect these targets. Here again, unless something such as multidrug therapy is used when these drug are introduced, clinical drug resistance will develop and will develop rapidly.

15.3.3 Tuberculosis Drug Resistance through Decreased Drug Activation Tuberculosis (TB) is an infectious disease that remains a leading killer (>2 million/year) primarily in individuals in poor living conditions. Further, one to two billion people are latently infected worldwide. Fortunately, less than 15% of latenyl infected people will go on to develop clinical TB; this represents a vast storehouse of potential cases of TB. The most important causative agent of this disease is Mycobacterium tuberculosis, a slim, aerobic, acid-resistant bacterium. Since 1945 a number of drugs have been developed and proved effective in TB. Unfortunately, due to inappropriate therapy and poor knowledge of the disease we have not been able to control TB. Of the many drugs used to treat TB, isoniazid (INH: isonicotinic acid hydrazide) has been the most widely used and is most effective. The structure for INH is shown in Figure 15.11. H N

O

NH2

N

Figure 15.11

Structure of isoniazide.

The exact mechanism(s) of action has not been identified for INH, but it has been well established that INH serves as a prodrug. The drug gains access inside the bacterium through small water-filled pores by passive diffusion. Once inside the cell the nontoxic INH is thought to undergo metabolic activation. A multifunctional catalase/peroxidase, KatG, is responsible for the activation of INH. The physiological role for KatG is thought to be protection against host phagocytic NADPH oxidase-derived peroxides and nitroxides. Interaction between INH and KatG could have two deleterious consequences for the bacterium. First, the enzyme converts the inactive prodrug to a variety of metabolites with tuberculocide activities; and second, the binding of INH to the KatG decreases the efficacy of this protective enzyme against peroxides and nitroxides. It is currently believed that in addition to the generation of a number of INH-derived radicals, the activated INH inhibits the enzyme enoyl-ACP reductase, an enzyme critical for the formation of mycolic acid, a precursor essential for mycobacterial cell wall synthesis. The activated INH is capable of acylation or oxidation of a number of amino acids in a variety of proteins. What impact these reactions have on protein function is still being determined. Interesting, INH has little activity in bacteria other than Mycobacterium. Given that INH is a prodrug that requires intracellular activation for anti-TB activity, a logical site for drug resistance would be a mutation of the KatG protein. A correlation between INH activation and peroxidase enzyme dates back to the 1960s, when it was reported that INH resistant M tuberculae also lacked peroxidase/catalase activity. Subsequently molecular techniques have confirmed this observation and have identified mutations in the KatG gene that render the bacterium resistant to INH. Most frequently point mutations have been described such as S315T, but a range of other mutations have been identified. Crystal structure analysis of the wild type KatG and the S315T mutated KatG have provided a possible explanation for the INH resistance. In the wild type protein the heme access pore is 7 angstroms; in the mutated form the pore is 4.7 angstroms, suggesting decreased INH access to the oxidizing site of the mutated protein. This mutation prevents that activation of INH but does not affect the ability of KatG to serve as a peroxidase catalase. It must be noted here that more than 40 additional point mutations in the KatG gene have been correlated with INH resistance, but the S315T is the most commonly identified mutation in clinical isolates. Other sites of mutation associated with INH resistance include the inhA gene, which codes for the enoyl-ACP reductase, the putative target for activated INH. Since this type of resistance is to the activated drug and the activation of a prodrug, this mechanism of resistance will not be discussed in detail here. The combination of KatG and inhA mutations account for approximately 80% of all INH-resistant clinical isolates. The remaining resistance phenotypes have yet to be identified. 383

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15.3.4 Drug Resistance through Drug Transport As was discussed with cancer cell drug resistance, infectious diseases also express drug resistance through decreased drug uptake or increased drug efflux. In the late 1970s, tetracycline resistance was first attributed to an efflux mechanism. Since then efflux-based mechanisms of antibiotic resistance has been described for a wide range of antibacterial agents. Based on what we discussed earlier in the chapter it is not surprising that some of these efflux mechanisms are relatively drug specific, but many accommodate an array of drugs. One point to keep in mind is the fact that there are gram-negative and gram-positive bacteria. The former have a complex cell envelope surrounding the organism. This cell envelope limits the access of many drugs and as a consequence drug resistance in gram-negative bacteria in many cases is attributable to a synergy between the membrane-bound drug efflux pumps and the cell envelope mediated poor drug uptake. Bacterial drug efflux transporters are classified into five families: n n

n n

n

The major facilitator family (MFS) The ATP binding cassette superfamily described previously for cancer cells and the malaria parasite The small multidrug resistance family (SMR) Resistance nodulation cell division superfamily (RND) The multidrug and toxic compound extrusion family (MATE)

Of these families the ABC and MFS families are by far the largest. Gram-positive bacteria usually require only a single component efflux mechanism to pump the drug out of the cytoplasmic membrane whereas gram-negative bacteria have a multicomponent efflux mechanism including the membrane efflux pump (MFP) and an outer membrane protein component (OMP) to efflux out of the cytoplasmic membrane and across the cell envelope.

15.3.4.1 Major Facilitator Superfamily The MFS is an ancient and diverse set of proteins numbering more than 1000 sequenced membranes that are involved in transporting sugars, metabolites, anions, and drugs. Most MFSs are single-component systems,

OMP H+

H+

but a few multicomponent systems have been described for gram-negative bacteria. The MFP proteins have 12 to 14 TMD and catalyze drug efflux using at least different subfamilies of drug/Hþ antiporters (DHA). DHA 1 and 2 are found in both prokaryotes and eukaryotes whereas DHA 3 is prokaryote specific. Members of the DHA 1 family export a variety of chemicals including sugars, polyamines, monamines, acetylcholine, paraquat, and methylglyoxal. DHA 2 has a more limited spectrum of substrates, which includes dyes and bile salts. DHA 3 is known to efflux a number of antibiotics including the macrolides and tetracyclines. The tetracycline efflux pumps are the best studied of the MFS family and are present in both gram-negative and gram-positive bacteria.

15.3.4.2 ABC Transporter Superfamily This superfamily of transporters contains both uptake and efflux transporter systems and requires the hydrolysis of ATP to provide the energy to shuttle drug across membranes. The pumps are multiprotein complexes with six TMD, and associate on the cytoplasmic faceoff the inner membrane with two ATPase subunits. Although there are significant similarities between the proteins and the mammalian ABC efflux proteins involved in MDR, drug efflux pumps belonging to the ABC transporter superfamily are rare in prokayotes.

15.3.4.3 Small Drug Resistance Family (SMR) This family of transported proteins consists of proteins having approximately 110 amino acids, four TMD, and are powered by a proton motive force similar to mitochondria. These proteins must form tetramers to function as transporters. These pumps are capable of effluxing drugs, dyes, and cations.

15.3.4.4 Resistance Nodulation Cell Division Superfamily (RND) These proteins are encoded in chromosomal genes, but an RND protein recently was found encoded in a plasmid. These transporters serve as antiporters and have been shown to be important in acquired and intrinsic resistance in gram-negative bacteria. All RND described thus far serve as multidrug efflux transporters.

Outer membrane

Na+ H+ MFP

Plasma membrane ADP + Pi

ATP Drug MFS

Figure 15.12

384

Drug SMR

Drug MATE

Membrane bound drug transporters.

Drug RND

Drug ABC

Review Questions

In gram-negative bacteria, a unique arrangement must be established between the cytoplasmic RND consisting of RND transmembrane protein with 12 TMD, an MFP, and an OMP. A characteristic feature of the RND topology is the presence of two large periplasmic loops between TMD 1 and 2 and TMD 7 and 8. The N-terminus halves of the RND family of proteins are identical to the C-terminus halves. It is thought that these proteins arose from an intragenic duplication process that occurred before the divergence of the RFD family.

15.3.4.5 Multidrug and Toxic Compound Extrusion (MATE) This family of proteins was at one time thought to be a member of the MFS superfamily due to similar membrane topology. However, they show no sequence homology and are now described as a separate family of transporter proteins. Proteins in this family use a sodium gradient as the energy source for effluxing drugs such as the fluoroquinolines and other dyes. The combination of these families of drug transporters has resulted in a variety of resistant phenotypes in both gram-negative and gram-positive bacteria. However, this type of drug resistance is far less common in bacteria than it is in cancer cell drug resistance.

15.4 SUMMARY We have discussed examples of drug resistance reported for both cancer cells and infectious diseases. There are myriad ways in which both populations of cells develop drug resistance depending upon the drug type and the cell type. Although this phenomenon of drug resistance is, on the surface, a roadblock to effective chemotherapy, it provides the pharmacologist newer targets for drug development as well as a better understanding of drug action and cell response to the drugs. Hopefully, this chapter has

provided more insight into drug resistance and encourages you to learn more about this fascinating phenomenon.

REVIEW QUESTIONS 1. What is the difference between inherent (natural) and acquired drug resistance? 2. Compare and contrast the MDR and MRP1 with respect to structure, function, and drug selectivity. 3. Discuss some of the unwanted potential consequences when we try to inhibit MDR to overcome drug resistance. 4. Describe the role of RFC in drug resistance. 5. How are BER and NER used as means to drug resistance? How do they differ and how are they similar? How might these pathways be important in normal cell function? 6. What are PBPs? How are they important in drug action and drug resistance? 7. Membrane-bound drug transporter proteins are important in drug resistance. What types of transporters are used in chloroquine resistance and what do they do? 8. Antifolates are important in the treatment of cancer and infectious diseases. Discuss mechanisms of antifolate drug resistance in cancer and malaria. 9. Discuss the rationale of using multidrug therapy rather than monotherapy if monotherapy is effective at present. 10. How does isoniazide kill M. tuberculae organisms? What is a common mechanism of drug resistance to INH? 11. Cytosine arabinoside (Ara-C) is an anticancer drug, and an example of host effects causing the need for large doses of drug to have a clinical effect far greater than would have been predicted from in vitro studies. What is the reason for this? How is it overcome?

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