BS3013 - Week 8: Enzymes and Drug Targets

What is the main function of an enzyme?

to lower the activation energy — thereby accelerating the reaction rate

black curve = enzyme catalyzed reaction

blue = uncatalyzed: high activation energy (transition state with the highest energy)

What 2 specificities are involved in an enzymatic reaction?

binding and reaction specificity

• binding: recognizes certain substrates only

• reaction: catalyze certain type of reaction only

ex. trypsin = cleaves peptide bone after basic amino acid

Protease = hydrolyzing peptise

What is involved in binding specificity?

certain constituents at active site are involved in binding interactions

• binding to substrate, transition state, intermediates, product

  • max binding interactions with transition state: up to ~ 10^12 times more tightly than with substrate or product

What does reaction specificity include?

rxn: arises from specific acid, base and nucleophilic groups of amino acids

What are the 6 components of enzyme catalysis?

• approximation

  • bring reacting partners to proximity, increasing EM

• Covalent catalysis

  • nucleophilic catalysis: active site amino acid side chain functional groups attach substrate and form covalent bond to it

• general acid-base catalysis

  • active site: acid/base

• electrostatic catalysis

  • ionic charge or dipole interacting with opposite charge developing on the substrate at the transition state of the reaction

• Desolvation:

  • removal of water molecules from charged groups at the active site on substrate binding → ground state destabilization/transition state stabilization

• strain or distortion:

  • binding of substrate to the enzyme inducing a conformational change in the active site - deformation of the enzyme and/or the substrate, leading to strain (destabilization, higher ground energy)

What are the 4 types of proteases?

1. serine protease

2. systeine protease

3. aspartyl protease

4. metalloprotease

What is the mechanism of serine protease-catalyzed hydrolysis?

How does Aspartyl protease work?

What is an enzyme Commission Number system?

depending on the type of enzyme they start with a number

What 2 equations are used relating to enzyme kinetics

michaelis-menten equation

lineweaver-burk equation

What are the 2 main types of enzyme inhibition?

competitive - same site

non-competitive - allosteric site

** both reversible or irreversible

When enzymes are reversible what does this mean?

hat are the 4 types of inhibition of a competitive reversible inhibitor?

simple competitive: designed structure resembles substrate

alternative substrate: binds to enzyme and acts as substrate

• products are useless to the organism

transition state analog: design molecule to resemble substrate at transition state of the reaction (this type would bind much more tightly to the enzyme)

slow, tight-binding: compound with tight binding to the enzyme and equilibrium of binding between it and the enzyme = reached slowly

• inhibition time dependent (reminiscent of the kinetics of irreversible inhibitors)

What is an example of simple competitive inhibition?

inhibitors of angiotension-converting enzyme (ACE)

what are 2 examples of substrate analogs as ACE inhibitors?

What are examples of neuraminidase inhibitor?

Oseltamivir (the free carboxylate form)

• competitive inhibitor of sialic acid on surface proteins of normal host cells

• block viral neuraminidase enzyme, oseltamivir blocks release new viral particles form infected cells

What is an alternative substrate inhibitor?

where the inhibitor is a substrate itself, but the products produced = useless

• preventing the natural substrate from being converted to the product that the organism needs

  • con: product produced could be toxic/side effects

  • drug is “consumed” constantly

ex. sulfonamide antibacterial agents (sulfa drugs)

What is Prontosil? How does it work?

1st sulfonamide antibacterial agent (sulfa drug)

• active principle generated by reductive metabolism reaction

• bacteriostatic agent — inhibits further growth of the bacteria

  • many sulfa drugs = developed based on the p-aminobenzenesulfamide structure

How does sulfa drugs inhibit folic acid biosynthesis?

How do nucleoside analogs work as an alternative substrate inhibitor of reverse transcriptase

How do nucleotide analogs work as inhibitor alternative substrate inhibitors of viral RNA polymerase?

Nucleotide analog reverse transcriptase inhibitors

What is Remdesivir used for?

treatment of Ebola virus & Marburg virus infections

• a prodrug that metabolizes into active form

• acts as a nucleotide analog inhibitor of RNA polymerase

developed by Gilease Sciences

• subsequently found to show antiviral activity against other single stranded RNA viruses

  • respiratory syncytial virus

  • junin virus

  • lassa fever

  • nipah virus

  • hendra virus

  • coronaviruses

What is Favipiravir used for? What kind of inhibitor it is?

approved to treat influenza in Japan

• activity against many RNA viruses: west nile virus, yellow fever, foot-and-mouth disease

• flaviviruses arenaviruses, bunyaviruses and alphaviruses (possible covid-19)

• doesn’t inhibit RNA or DNA synthesis in mammalian cells (nontoxic to humans)

Favipiravir = prodrug (metabolized into active form — favipiravir-ribofuranosyl-5’-triphosphate

What is the strongest interaction between the substrate and enzyme?

What is Pentostatin a potent inhibitor of?

it is a transition state analog — inhibitor of adenosine deaminase

Ki = 2.5 × 10^-12 M, Km of adenosine ~ 10^-5 M

What kind of analogs are slow, tight-binding analogs?

• slow to reach equilibrium

• off rate v low, very tight binding

• functionally equivalent to irreversible inhibitors

What are irreversible enzyme inhibitors?

reversible enzyme inhibitor = need to maintain a high enough conc of inhibitor to sustain E•I complex

competitive irreversible enzyme inhibitor are active-site directed irreversible inhibitor or enzyme inactivator

• compound structure is similar to the substrate, generally forms a covalent bond to an active site residue

not necessary to sustain the inhibitor concentration to retain the enzyme-inhibitor interaction (since enzyme has reacted with irreversible inhibitor)

• complex can’t dissociate and enzyme remains inactive (even in absence of additional inhibitor)

What are the 2 types of enzyme inactivators?

affinity labeling agents

• reactive compounds

mechanism-based enzyme inactivators

• unreactive compounds that are substrates (suicide substrates) of the target enzyme and are activated by the target enzyme

What are affinity labeling agents

• reactive compound

• similar structure to substrate of target enzyme

most cases: equilibrium for reversible EI complex formation = rapid and rate of dissociation is fast

k inact = rate-determining step

• time-dependent loss of enzyme activity

• rate of inactivation = proportional to low conc of inhibitor (becomes independent at high concs due to enzyme saturation

What are some concerns with affinity labeling agents?

potentially toxic — can react with nucleophiles from other biomolecules

• key to effective design = specificity of binding

• enhance selective reactivity of the subsequent enzyme inactivation step

• lower reactivity of the reactive group = can also increase selectivity

What are examples of B-lactam antibiotics?

penicillin & cephalosporins = bacteriocidal

• inactivate an enzyme needed for bacterial growth

• this doesn’t exist in animals (making it an ideal drug)

How does bacterial resistance develop for penicillins and cephalosporins?

penicillins and cephalosporins = not exceeedingly reactive as acylating agents

• few nonspecific acylation reactions occur — non toxicity, once considered “wonder drugs”

resistance:

• half of the strains of staphylococcus aureus = resistant w/n 10 yrs of introduction of penicillin

• 1990, 90% of strains = resistant

Principal cause = resistant strains synthesize and excrete enzyme B-lactamase which catalyze hydrolysis of B-lactam

How do we overcome resistance?

1) pair pencillin with B-lactamase inhibitor → killing resistant strains

** ex of drug synergism

2) new analogs of B-lactam antibiotics can be made

• analogs are poorly recognized by B-lactamase, but active against peptidoglycan transpeptidase

3) switch to other antibiotics (vancomycin) with diff mech of action

4) prevent antibiotic abuse

How does Nirmatrelvir work as an irreversible inhibitor of 3C-like protease of SARS-CoV-2?

3C-like protease process viral polyprotein at multiple sites (usually after glutamine residues

• cysteine proteases

drug forms covalent bond with thiol group of cys residue in the enzyme

What is a mechanism-base enzyme inhibitor?

inactive compound, structurally similar to the substrate or product of an enzyme

• initially serves as a substrate for the enzyme and converted to a product (reactive)

• inactivates the enzyme prior to escape from the active site

Affinity labeling agents vs mechanism based enzyme inhibitors (2 key features)

1) initial inactivity

2) requirement for the enzyme to catalyze the conversion

Advantages of mechanism-based enzyme inactivators

• initially inactive

• no problem of nonspecific alkylation or acylation of other proteins

• converted reactive product = already in active site of the enzyme and will react within

not many ex: most determined ex post facto rather than designed that way

(not easy to design)

What is Selegiline (L-Deprenyl)?

antiparkinsonian drug

• parkinson’s disease = reduction in dopamine conc in the brain

• Selegiline deactivates monoamine oxidase B (MAO B) = involved in metabolism of the inhibitory neurotransmitter dopamine

What are new enzyme targets for epigenetic therapy

enzymes responsible for modulating epigenetic makeup of cells

• DNA methyltransferase

• HATS - histone acetyltransferases

• HDACS - histone deacetylases

  • 2 approved drugs as inhibitors of HDAC for the treatment of cutaneous T-cell lymphoma.

• histone lysine methyltransferase and lysine demethylase

• RNA -modifying enzymes

What is drug resistance mean? how does it happen

formerly effective drug dose no longed effective

• related to microorganisms or cancer cell growth

resistance arises mainly by natural selection

• evolution and adaptations: 1 in 10^7 microorgs (or cancer cells) in a colony have 1+ mutations making it resistant to that drug

• susceptible cells in population get killed → few resistance ones replicate → become predominant population

• resistance = seldom caused by drug induced mutation

What is drug tolerance the cause of?

pt’s body adapts to a particular drug in chronic use → more drug needed to attain initial effect

• lead to decrease in the TI

What is the mechanism of drug resistance? (10)

1) altered drug uptake

2) overproduction of target enzyme

3) altered target enzyme

4) production of drug-destroying enzyme

5) deletion of a prodrug-activating enzyme

6) overproduction of substrate for the target enzyme

7) new pathways for formation of the product of the target enzyme

8) efflux pumps

What is altered drug uptake in drug resistance?

ability of the organism to exclude the drug from the site of action by preventing the uptake of the drug

how does overproduction of the target enzyme lead to drug resistance?

increases target enzyme production though induction of extra copies of the gene encoding the enzyme

How does altering the target enzyme work for drug resistance

mutations of aa residues in the active site can result in poor binding of the drug to active site

• important strat = minimize effect of target enzyme mutation in drug resistance → design a drug similar structure to that of substrate

• mutation would also cause auto-inhibition

production of similar enzyme that can do the same fx

• does the same fx as og enzyme, but drug can’t bind

• ex. methicillin-resistant Staphylococcus aureus (MRSA) - responsible for several difficult to treat infections in humans

Altered active site of action — vancomycin resistance is an ex

• vancomycin = last defence against streptococcal or staphylococcal bacteria

• forms complex with terminal D-alanyl-D-alanine of peptidoglycan, blocking transpeptidation

Vancomycin resistance example (inhibits bacterial wall synthesis — binds to the substrate of the enzyme

last residue = becomes D-lactate → ester bond formed instead of amide bond

• amide can form H-bond with vancomycin

How does drug resistance occur through production of drug-destroying enzyme?

• induction of genes that produce new enzymes to degrade drug

• B-lactamase

How does deletion of a prodrug-activating enzyme work for drug resistance

no active form = no working drug

NH → ribose (so it’s like a nucleoside)

• in some tumor drugs, the enzyme is deleted

How does overproduction of the substrate for the enzyme work for drug resistance?

overproduction of natural substrate = competitively block ability of the drug to bind at the active site of the target enzyme

how do new pathways form for product of target enzyme?

• drug is used to block production of a metabolite using enzyme inhibition

• organism can bypass this by creating a new pathway to produce that same metabolite

how do efflux pumps work against drugs → resistance?

tumor cells and microorganisms can develop protein transporters that bind to drug that carry the drugs out of the cell

How can we fight against drug resistance? what is the main strat?

drug synergism

• therapeutic effect of 2+ drugs used in combo

• combo is greater than the sum of the effects of the drugs administered individually

improve use of antibiotics

• avoid overuse and abuse

What are the approaches to drug synergism?

inhibition of drug-destroying enzyme

sequential blocking: inhibition of 2+ consecutive steps in metabolic pathway

inhibition of enzymes in different metabolic pathways

• if the cause of drug resistance = production of a new metabolic pathway

efflux pump inhibitors

use of multiple drugs for same target