Drug Discovery through Enzyme Inhibition and Inactivation

Transcription

1 Drug Discovery through Enzyme Inhibition and Inactivation

2 Learning outcomes The student will be able to: List the types of enzyme inhibitors Describe the mechanism of enzyme catalysis and drug resistance Discuss and apply theory of drug synergism Discuss mechanism of action of reversible and irreversible enzyme inhibitors Describe rational design of enzyme inhibitors

3 Types of enzyme inhibitors Reversible inhibitors involve reversible inhibition of enzyme via non-covalent interactions Irreversible inhibitor (Enzyme inactivator) - inhibits enzyme for an extended period of time via covalent interactions

4 Reversible Enzyme Inhibitors 1. Competitive inhibitors - compete with substrate for active site (structures similar to substrates or products) a) Simple competitive inhibitors b) Alternative substrate inhibitors c) Transition state analogs d) Slow, tight-binding inhibitors 2. Noncompetitive inhibitors - bind to a site other than active site (an allosteric site)

5 Mechanism of reversible inhibition E + I -S +S k on k off E I E S E P E + P E I concentration depends on [I] and [S] and their dissociation constants (K i and K m, respectively). Both S and I compete for E, (so a decrease in [I] or increase in [S], will decrease the [E I] and increases[e S]).

6 a) Simple Competitive Reversible Inhibitors E.g. antihypertensive drugs: ACE inhibitors - Captopril, enalapril, and lisinopril ypertensive Effects of ACE Produces angiotensin II, a vasoconstrictor, also trigger release of a hormone, aldosterone that regulates the electrolyte balance by retention of Na + and water. Angiotensin II is converted to angiotensin III, which also releases aldosterone. ACE also catalyzes hydrolysis of a vasodilator, bradykinin (Arg-Pro-Pro- Gly-Phe-Ser-Pro-Phe-Arg) ypertensive Effects of ACE May be involved in memory retention & neuronal development

7 Lead Discovery mixture of peptides in the venom of a South American pit viper shows inhibition of bradykininase activity and inhibits conversion of angiotensin I to angiotensin II Teprotide (Pyr-Trp-Pro-Arg-Pro Gln-Ile-Pro-Pro) showed highest activity in vivo

8 Lead Modification Testing peptides inhibitors on ACE showed that: Pro is best at C-terminal, Ala at penultimate and an aromatic residue at antepenultimate position. ACE was found to contain Zn 2+ that was believed to assist hydrolysis as a cofactor ACE catalytic site was assumed to resemble carboxypeptidase A

9 Lead Modification With (R)-2-benzylsuccinic acid as a model, a series of peptidomimetic, carboxyalkanoylproline derivatives (e.g. 1) were tested as inhibitors of ACE. All were only weak inhibitors of ACE. To increase potency, C - was changed to S that coordinate better to Zn(II).

10 ypothetical binding of inhibitors to ACE Note: Active site of ACE has two additional binding site than carboxypeptidase Carboxylate analog Thiol analog

11 Effect of Structural Modifications Analog Relative K i Captopril had the best binding properties (K i = 1.7 nm), It was also highly selective for ACE SAR studies showed that every part of captopril is important for binding. Captopril was the first ACE inhibitor on the drug market - hypertension and congestive heart failure. S 3 C S 3 C S S S S 3 C C N N N N N C 3 N N C 2 C 2 C 2 C 2 C 2 C 2 (captopril) , , ,100

12 Captopril showed two reversible side effects (rashes and loss of taste) - Merck hypothesized side effects from S group. Changed S back to C- C 2 replaced by N to increase interaction with enzyme, gave less potent molecule due to high hydrophilicity enalaprilat: R = (S)-PhC 2 C 2, R = was the best candidate A lipophilic group was added to reduce hydrophilicity and also to increase binding to S 1 sub-site on ACE enalaprilat was poorly absorbed orally - remedied by enalapril, an ethyl ester which is hydrolyzed in vivo by esterases to give enalaprilat

13 C 2 C 2 C 2 N N lisinopril 5.29 N 2 (C 2 ) 4 C 2 Does not require prodrug activation.

14 b) Competitive Reversible Inhibitor that Acts as Substrate Examples are Sulfonamide Antibacterial Agents (Sulfa Drugs) Lead discovery Bayer Co. tested azo dyes against streptococci. Prontosil was very effective in mice (1935). No activity in vitro unless a reducing agent is added. In vivo protonsil is metabolized by reduction to the active agent it is a prodrug. the active agent is the bacteriostatic sulfanilamide bacteriostatic - inhibits further growth of bacteria, but does not kill them.

15 Lead Modification Prontosil - beginning of modern chemotherapy Thousands of compounds synthesized and tested - the first SAR studies 1 st example where new leads for other diseases discovered from side effects in clinical studies - antidiabetic and diuretic agents ther developments from these studies: a simple assay for these compounds in body fluids showed antibacterial effect is proportional to the concentration in blood, which varied from patient to patient at a given dose - beginning of monitoring blood drug levels during chemotherapy

16 Mechanism of Action In 1939 Stamp showed microorganisms contained a substance that blocked the antibacterial action of sulfonamides In 1940 Woods hypothesized that this substance has structure similar to the enzyme substrate e also showed that it is p-aminobenzoic acid (PABA) and it compete with sulfanilamide for microbial growth Mid 1940s - Miller shows sulfanilamide inhibits folic acid biosynthesis Richey and Brown purified two enzymes involved in biosynthesis of dihydrofolate Sulfonamides were shown to be substrates for Dihydropteroate synthase (inhibiting formation of dihydrofolate needed for purines and DNA biosynthesis in bacteria)

17 Criteria for a good antimicrobial drug target 1. Target is essential to survival of microorganism 2. Target is unique to the microbe so humans are not harmed 3. Structure and function of the target is highly conserved across a variety of species of that microbe for broad spectrum 4. Resistance to inhibitors of target not easily acquired Dihydropteroate synthase satisfies the first 3 criteria It generally takes 1-4 years for resistance to emerge; resistance to sulfonamides took almost seven years.

18 Sulfonamides- Drug Resistance verproduction of PABA Formation of a dihydropteroate synthase that binds PABA normally, but binds sulfonamides thousands of times less tightly Altered permeability of sulfonamides

19 c) Transition State Analogs Bernhard and rgel theorized that: inhibitor molecules resembling transition state structure are more tightly bound to enzyme than substrate

20 Pentostatin an example of transition state analogs N N N N pentostatin 5.58 Antineoplastic agent isolated from bacterium Streptomyces antibioticus A potent inhibitor of adenosine deaminase K i = 2.5 x M (2.5 pm)(10 7 times lower than K m for adenosine!) 2 -deoxyinosine 2 -deoxyadenosine pentostatin mimics this

21 Multisubstrate transition state analog When more than one substrate is involved, a single stable compound is made with a structure similar to the two or more substrates at the transition state - multisubstrate analog inhibitor.

22 N-Phosphonoacetyl-L-Asp (PALA) an example of Multisubstrate Analog Aspartate transcarbamylase catalyze de novo biosynthesis of pyrimidines from carbamoyl phosphate and L- Asp PALA resembles the transition state P 3 = 5.65 N 2 carbamoyl phosphate P 3 = C - C.. - N 2 C - N 2 N 2 C - isostere - no longer a leaving group, mimics phosphate P 3 = C 2 N 5.67 C - C - PALA N N C - C - N-carbamoyl-L-Asp PALA was inffetctive due to tumor resistance: tumor cells acquired ability to utilize preformed circulating pyrimidine nucleosides increased carbamoyl phosphate increased aspartate transcarbamylase

23 Slow, Tight-Binding Inhibitors Slow-binding inhibitors - equilibrium between enzyme and inhibitor is reached slowly (k on small); inhibition is therefore time-dependent Tight-binding inhibitors - substantial inhibition when [E] and [I] are comparable (k off small) Slow, tight-binding inhibitors have both properties t 1/2 for E I complex can be up to months

24 Slow, Tight-Binding Inhibitors Examples Examples of Slow, Tight-Binding Inhibitors the Antihypercholesterolemic drugs Lovastatin and Simvastatin About 1/2 of body cholesterol is biosynthesized; the rest needs to be eaten. Some people biosynthesize much more than half of what they need. Rate-determining step in cholesterol biosynthesis is conversion of 3- hydroxy-3-methylglutaryl coenzyme A (MG-CoA) to mevalonic acid. Inhibition of MG-CoA reductase should lower plasma cholesterol levels. 2 NADP 2 NADP + 3 C MG-CoA C SCoA 5.68 MG-CoA reductase 3 C C Mevalonic acid 5.69 NADP + NADP NADP + 3 C C SCoA :B -CoAS 3 C C NADP 5.70

25 MG-CoA 3 C C C 3 C 2 C C C P - C 3 P C 2 - N N = 3 P NC 2 C 2 CNC 2 C 2 S N 2 N N CoA

26 Endo and coworkers (Sankyo Co. in Japan) found three compounds from fungus Penicillium citrinum that inhibited sterol biosynthesis mevastatin was most potent. A more potent compound was isolated by Endo from a different fungus - called monacolin K. Lead Discovery Mechanism of Action: Potent competitive reversible inhibitors of MG-CoA reductase (K i for lovastatin 6.4 x M) 3 C R C 3 mevastatin or compactin (R = ) monacolin K, mevinolin, or lovastatin (R = C 3 ) 5.71

27 The active form of lovastatin is the hydrolysis product. This mimics intermediate 5.70 in Scheme C 5.70 C SCoA :B when is replaced by C 3, it is simvastatin (2.5 times more potent than lovastatin) 3 C C C C 3 Active form of lovastatin

28 Irreversible Enzyme Inhibitors Two general types of irreversible inhibitors: Affinity labeling agents (reactive compounds) Mechanism-based enzyme inactivators (unreactive compounds) Note: Irreversible inhibition does not mean permanent loss of the enzyme - gene encodes for other copies (may take hours or days).

29 Affinity Labeling Agents These are reactive compounds that have structure similar to that of substrate of the target enzyme reversible complex S N 2 alkylation or acylation covalent complex Effective Affinity Labeling Agents have: low K i for target enzyme for selectivity reactive group close to a nucleophile in active site of target enzyme

30 -lactam antibiotics Examples affinity labeling agents

31 -lactam antibiotics inactivate Peptidoglycan transpeptidase which catalyze the cross-linking of peptidoglycan to form bacteria cell wall GlcNAc MurNAc GlcNAc NAc 3 C B: + B L-Ala D- -Glu N (D) CC 3 - C NAc L Lys-N 2 N (D) CC 3 C NAc peptidoglycan transpeptidase D-Ala D-Ala D-Ala C 3 GlcNAc MurNAc GlcNAc GlcNAc MurNAc GlcNAc 3 C Ser L-Ala D- -Glu L Lys-N 2 N (D) CC 3 C L Lys N 2 D- -Glu L-Ala 5.10 D-Ala D-Ala L Lys D- -Glu L-Ala C 3 GlcNAc MurNAc GlcNAc GlcNAc MurNAc GlcNAc 3 C L-Ala D- -Glu L Lys-N 2 N (D) CC 3 C N

32 R N a Me N a N b S N b C 2 Me Me C 2 Me The structure of penicillin is comparable with D-Ala-D- Ala which is hydrolyzed by the enzyme Peptidoglycan Penicillin inactivate the enzyme by acylating serine at active site

33 Penicillin Resistance: ccurs via 1. -Lactamase production - hydrolyzes -lactam ring 2. Also, transpeptidase becomes less susceptible to acylation. 3. Membrane permeability is modified. Drug Synergism: combination therapy (penicillin + - lactamase inhibitor) e.g. Augmentin (amoxicillin + clavulanate) Unasyn (ampicillin + sulbactam) S N C 2 - K + clavulanate potassium 5.92 N sulbactam 5.93 C 2

34 Mechanism-Based Enzyme Inactivators is an unreactive molecule with structure similar to substrate or product once bound the target enzyme converts it into a species that inactivates the enzyme Their differences from affinity labeling agents are initially unreactive target enzyme activates them by catalysis

35 Mechanism-Based Inactivation Kinetics k 1 k 2 k E + I E I E I ' 4 k -1 E - I '' k 3 E + P partition ratio = k 3 /k 4 ideally, partition ratio = 0 Note: Inactivation occurs prior to release of the activated species

36 Vigabatrin Examples of Mechanism-Based Inactivation Vigabatrin is an antiepileptic drug Epilepsy - Can arise from imbalance in two neurotransmitters (L-Glu and GABA) Thus an alternative to treat epilepsy could be introduce GABA into brain owever, GABA does not cross blood-brain barrier Vigabatrin crosses blood-brain barrier, and inactivates GABA aminotransferase via mechanism-based inactivation. - C GABA-AT - 3 N C 2 C + 3 N C 2 GAD PLP -C 2 PLP PMP - C 2 NADP + L-Glu -KG SSAD - 2 C NADP - C 2

37 Vigabatrin can cross the BBB because vinyl group: increases lipophilicity of the compound is electron withdrawing it will lower pka of the amino group thus increasing the nonzwitterionic form

38 Mechanism of action of Vigabatrin