Chapter 7

Drug Resistance and Drug Synergism

The Organic Chemistry of Drug Design and Drug

Action

Drug ResistanceWhen a formerly effective drug dose is no longer effective.

Arises mainly from natural selection - replication of a naturally resistant strain after the drug has killed all of the susceptible strains.

On average, 1 in 10 million organisms in a colony has one or more mutations that makes it resistant.

Resistance is different from tolerance - this is when the body adapts to a particular drug and requires more of the drug to attain the same initial effect - lowers the therapeutic index.

It is also possible to develop tolerance to undesirable effects of drugs, such as sedation by phenobarbitol - raises the therapeutic index.

Mechanisms of Drug Resistance1. Altered drug uptake - exclusion of drug from site of action by blocking uptake of drug - altered membrane with more + or - charges

2. Overproduction of the target enzyme - gene expression

3. Altered target enzyme (mutation of amino acid residues at the active site) - drug binds poorly to altered form of the enzyme

4. Production of a drug-destroying enzyme - a new enzyme is formed that destroys the drug

Mechanisms of Drug Resistance (cont’d)

5. Deletion of a prodrug-activating enzyme - the enzyme needed to activate a prodrug is missing

6. Overproduction of the substrate for the target enzyme - blocks inhibitor binding

7. New metabolic pathway for formation of the product of the target enzyme - bypass effect of inhibiting the enzyme

8. Efflux pump - protein that transports molecules out of the cell

Mutation of dihydrofolate reductase results in resistance to trimethoprim

Another Example of a Mutated Target Enzyme

M184V and M184I mutants of reverse transcriptase are produced by HIV when exposed to these drugs

If your drug has a structure similar to the substrate, mutations will lower binding of the substrate as well as the inhibitor.

Vancomycin is the antibiotic of last resort

Vancomycin binds to the D-Ala-D-Ala inpeptidoglycan

FIGURE 7.1 Structure of a peptidoglycan segment prior to cross-linking with another peptidoglycan fragment catalyzed by peptidoglycan transpeptidase. This structure is an alternative depiction of the transpeptidase substrate shown on the left in Scheme 4.13, graphic A.

The complex of vancomycin and peptidoglycan

FIGURE 7.2 Complex between vancomycin and the terminal D-alanyl-D-alanine of the peptidoglycan

Resistant cells make D-Ala-D-lactate

FIGURE 7.4 Biosynthesis of D-alanyl-D-lactate and incorporation into the peptidoglycan of vancomycin-resistant bacteria

Vancomycin binds weakly to the D-Ala-D-lactate

FIGURE 7.5 Complex between vancomycin and the peptidoglycan with terminal D-alanyl-D-lactate instead of D-alanyl-D-alanine in vancomycin-resistant bacteria

HIV develops resistance to Lopinavir

The resistance results from mutations in the HIV protease target enzyme.

Ritonavir inhibits HIV protease, but also inhibits Cyp450

Lopinavir was made from Ritonavir to avoid Cyp450 inhibition, but it ismetabolized very fast. Ritonavir inhibits susceptible HIV and helps reduce metabolism of Lopinavir, which inhibits the mutant HIV strains.

Imatinib inhibits Bcr-Abl kinase

Mutations at many locations in Bcr-Abl result in weak inhibition by Imatinib.H396, E255, and T315 are mutation sites.

Analogues resistant to most mutations

But not T315I!

Design of an inhibitor for T315I Bcr-Abl

FIGURE 7.6 Evolution of 7.11 optimization for inhibition of Bcr-Abl (T315I)

An alternative inhibitor of Bcr-Abl and T315I mutant—DCC-2036.

DCC-2036 bound to T315I Bcr-Abl

FIGURE 7.7 Image based on X-ray crystal structure of DC-2036 complexed to Bcr-Abl (T315I). Note the position of the I315 residue and the hydrogen bonds to Met318; Met318 is analogous to Met793 in Figures 5.2 and 5.3.

Other kinase inhibitors susceptible to mutations of “gatekeepers”

The T790M mutation in EGFR kinase affects gefitinib and Erlotinib binding

L1196M of ALK give resistanceof lung cancer to crizotinib

Mutations of topoisomerase can give resistance to amsacrine

Fluconazole is an antifungal drug

Mutations of lanosterol 14α-demethylasecan cause resistance.

A proteasome inhibitor used to treat multiple myeloma

Overproduction of proteasome subunits causes resistance to bortezomib

Bortezomib bound to the proteasome

FIGURE 7.8 Image based on X-ray crystal structure of bortezomib complexed to 20S proteasome

Overproduction of substrate

Overproduction of p-aminobenzoate can give resistance to sulfa drugsSCHEME 5.3 Biosynthesis of bacterial dihydrofolic acid

Approaches When a Drug-Destroying Enzyme is Produced

1. Make an analog that binds poorly to this new enzyme

3. Inhibit the new enzyme

2. Alter structure of drug so it is not modified by the new enzyme, such as tobramycin (5.14), which lacks the OH group of kanamycins (5.12) that is phosphorylated by resistant organisms.

resistant organisms phosphorylate here

no OH group

Neomycins are also phosphorylated to inactivate

Overcoming kanamycin resistance

SCHEME 7.1 An approach to avoid resistance to kanamycin A

A bifunctional analogue

These compounds inhibit the phosphorylation as well as still bind to the ribosome

Bleomycin resistance occurs by hydrolysis

SCHEME 7.2 Action of bleomycin hydrolase to promote tumor resistance to bleomycin

Inactivation of nitrogen mustard by glutathione-S-transferase

SCHEME 7.3 Inactivation of a nitrogen mustard by reaction with glutathione (GSH).

Resistance due to loss of prodrug activation

6-Mercaptopurine is activated by hypoxanthine-guanine ribosyltransferase

Cytidine kinase activates antiviral drugs

SCHEME 7.4 Conversion of prodrugs fludarabine and cladrabine to their active form in cells catalyzed by cytidine kinase.

Resistance to PALA arises from cells using preformed pyrimidines

Alternative pathways in drug resistance

FIGURE 7.9 Example of resistance resulting from activation of alternative pathways

Alkylation of DNA can be reversed

SCHEME 7.5 O6-Alkylation of guanine by an alkylating agent and its reversal by O6-alkylguanine-DNA alkyltransferase

Resistance to dactinomycin is caused by efflux pumps

Drug Synergism

Arises when the therapeutic effect of two or more drugs used in combination is greater than the sum of the effect of the drugs individually.

Mechanisms of Drug Synergism

5. Use of multiple drugs for same target - about 1 in 107

bacteria resistant to a drug; if you use two drugs, then only 1 in 1014 is resistant to both

1. Inhibition of a drug-destroying enzyme protects the drug from destruction

2. Sequential blocking - inhibition of two or more consecutive steps in a metabolic pathway - overcoming difficulty of getting 100% enzyme inhibition

3. Inhibition of enzymes in different metabolic pathways- block both biosynthetic routes to the same metabolite

4. Efflux pump inhibitors can be made to prevent efflux of the drug

Resistance to β-lactam antibiotics is due to β-lactamases

Mechanism of β-lactamase inactivation

SCHEME 7.6 Proposed mechanism of inactivation of β-lactamase by clavulanate

Pentostatin prevents metabolism of vidarabine by adenosine deaminase

Sulfa drugs are synergized by DHFR inhibitors

Methotrexate synergizes with thymidylate synthase inhibitors—5-fluorouracil

Combining RAF and MEK inhibitors improves efficacy in treatment of resistant melanoma

RAF inhibitor

MEK inhibitor

V600E RAF is overactive

Inhibition of alternative pathways is synergistic

EGFR kinase inhibitor

MET inhibitor

MET and VEGFR inhibitor

Efflux pump inhibitors

Mechanism of IMP dehydrogenase (IMPDH)

SCHEME 7.7 Mechanistic steps for conversion if inosine 5′-monophosphate (IMP) to xanthosine 5′-monophosphate (XMP) catalyzed by the enzyme IMPDH.

IMPDH inhibitors with different mechanisms

Mechanism of IMPDH inhibition by mizoribine

FIGURE 7.10 Schematic drawing showing how the complex (B) of mizoribine monophosphate to IMPDH is believed to mimic the tetrahedral intermediate (A) for E-XMP hydrolysis (compare Scheme 7.7).

The two IMPDH inhibitors are synergistic