33/34 - Intro + Hydrolases (+Proteases)

Question 1

Type of Inhibitor?

bind to either free enzyme or the E-S complex

minimal effect on Km
&
LOWER Vmax

Answer 1

NON-Competitive Inhibitors

NOncompetitive inibitors are NOt so picky
bind to either free enzyme or E-S complex

Ex.
anti HIV - RT inhibitors

Nevirapine

Question 2

What type of Protease Mechanism?

Metalloproteasese // Aspartic Acid
Proteases

Uses WATER
no covalent bonds involved

Answer 2

ACID-BASE CATALYSIS
Protease mechanism

Use a non-covalent, acid-base mechanism

EX.
Captopril –> ACE
Ace = zinc protease
Indinavir –> Asp Protease (HIV)

Question 3

Type of Inhibitor?

Inhibitor once bound NEVER comes off

• *covalent bond formation** between the
• *enzyme + reactive group of the inhibitor**

Answer 3

IRREVERSIBLE Inhibitors

Ex.

ASPIRIN / PENICILLIN

Question 4

1/2 Methods to block the action of proteases

EX. Captopril –> ACE

Inhibitors that target the Active site

A good substrate can be converted to a good inhibitor by
replacement of the part of the substrate that directly reacts with
the active site of the protease

Answer 4

SUBSTRATE MIMICS

Mimic the structure of the peptide substrate

CAPTOPRIL

Question 5

Type of Inhibitor

Act by competing with endogenous substrate for binding to the enzyme

Negative:
their efficacy can be compromised in the presence of
EXCESS / buildup of substrate

Answer 5

COMPETITIVE INHIBITORS

Question 6

What type of Protease Mechanism?

Serine // Cysteine // Threonine
proteases

Difficult to cleave an amide, b/c of delocalized charge

Active site has a proton withdrawing group = HISTINE
that facilitates an NU- attacking group
–> creates an ESTER (easier to hydrolyze vs amide)

Answer 6

COVALENT CATALYSIS

the tetrahedral intermediate
involves a
stable covalent bond to the enzyme’s catalytic nucleophile

Question 7

What is the MECHANISM of Aspartic Protease (HIV)?

inhibited by INDINAVIR

Answer 7

Acid-Base Catalysis

Asp residues polarize water & carbonyl
Asp abstracts a proton from water –> water attacks the carbonyl
VVVVV
forming the tetrahedral intermediate
VVVVV
rearrangement, assisted by 2 ASP residues
leads to the formation of AMINE + COOH products

Question 8

What Drug targets ACE?

Answer 8

CAPTOPRIL = Capoten

indicated for CV disorders
HT / CHF / renal insuficiency

SUBSTRATE MIMIC
ACE = Zinc Protease
(Acid-Base Catalysis)

Question 9

1/2 Methods to BLOCK the action of PROTEASES?

EX. Indinavir –> HIV protease

Compounds that resemble the transition state
but that can NOT go on to complete the reaction

Answer 9

TRANSITION STATE MIMICS

indinavir

Question 10

What TYPES of PROTEIN-TARGETS are there?

Answer 10

MOST TARGETS are PROTEINS!

Receptors > Enzymes

Transporters > other
enzyme interacting proteins / structural proteins

Question 11

Efficacy or Potency?

^^^INCREASE^^^ in % RESPONSE

• *Maximum effect** that a drug can produce
• regardless of DOSE*

Answer 11

EFFICACY

Question 12

Type of Inhibitor?

bind to the enzyme-substrate complex

but NOT to the free enzyme

both Km & Vmax are REDUCED

Answer 12

UN-Competitive** **Inhibitor

UNcompetitive inhibitors UNiquely bind
the enzyme-substrate complex

• uncommon*
  ex. mycophenolic acid = immunosuppressive

Question 13

Why does Biochemical mechanism MATTERS

in Drug-discovery

Answer 13

Plays a role in HIT PRIORITIZATION

Knowing the mechanism aids in
Structure-based drug design –> lead optimization

We prefer to have
Reversible / Competitive Inhibitors
instead of _irreversible inhibition_
due to toxicity

Question 14

Common Features
of other drugs that INHIBIT ACE

All are Carboxyl
Enalapril / Lisinopril / Ramipril
except for Captopril = sulfhydryl class

Answer 14

Metal-Chelating Group
that interacts with ZINC
Thiol / Carboxylate / Phosphinate

Some are prodrugs
Enalapril / Ramipril

They vary in onset of action –> dose adjusting

Question 15

ZINC’s Role

in ACE’s Protease Acid-Base Catalysis

Answer 15

ZINC = ion cofactor that acts as an:
Lewis Acid** or **Electrophile
Lewis Acid / Electrophile = ACCEPTS electron pair

ACTIVATES the CARBONYL GROUP
(making it more electrophilic / highly reactive)
&
STABILIZES the CARBOXYLATE ANION

GLU = base

Question 16

Therapeutic Index
TI

Answer 16

TD₅₀ / ED₅₀

Dose that elicits a TOXIC response in 50% of treated individuals
////
Dose that is Therapeutically EFFECTIVE in 50% of treated individuals

Question 17

Lower the EC₅₀ –>
what affect on Potency/Efficacy?

Answer 17

The lower the EC50 the
HIGHER THE POTENCY

EC50 is the
concentration of a drug that gives half‐maximal response.

Question 18

What DRUG targets HIV Protease?
& what is the indication?

Answer 18

INDINAVIR = Crixivan

HIV Infection / AIDS

non-petidic

TRANSITION STATE MIMIC

Question 19

What TYPE OF ENZYME requires the formation of a
TRANSITION STATE=Tetrahedral Intermediate
as a prequisite for peptide-bond scission?

Answer 19

PROTEASE

a type of hydrolase

Question 20

Which of the 1 of 2 types of Drug Discovery?

Empirical Approach
You don’t know what you are targetting
but you know what the expected outcome is

Strategy for the ID of molecules with particular biologic effects in
cell-based assays** or **animal models.

Answer 20

PHENOTYPIC Screening
for drug discovery

EXAMPLE:
antibody discovery with bacterial growth

Question 21

Which of the 1 of 2 types of Drug Discovery?

• *Molecular Approach**
• *Structure-guided** drug design

Hypothesis Driven
We know more about the disease
The target has proven function in tha pathophysiology of a disease

Answer 21

Target-Based
Drug Discovery

High throughput screening

Question 22

What is the TARGET of INDINAVIR = Crixivan?

Answer 22

HIV Protease

Aspartic-Acid Protease =
ACID-BASE CATALYSIS

Question 23

How do

• *HIV Protease Inhibitors = Indinavir** (Asp-Protease)
• *work?**

Answer 23

HIV Protease is required for viral cell life
inhibition –> stops ​viral spread

TRANSITION STATE MIMIC
for peptide cleaveage

Non-Hydrolyzable Alcohol –> instead of Amide
unable to be cleaved

Question 24

What are the 2 types of
Target-driven drug discovery?

Answer 24

Target-Based HTS
High throughput screening
Diverse library of compounds –> TARGET of interest
–> HIT Identification

Structure-Guided Drug Design
3-D structure of enzyme –> try and MATCH/FIT in the target

Question 25

Mechanism for ACE-I (Captopril’s)
COUGH side Effect
& Alternative

Answer 25

Instead use –> ARBs = -Sartans

ACE also accepts bradykinin as a substrate
converting it to inactive peptides

Inhibiting ACE –> accumalation of inflammatory kinins
which can cause the COUGH

Question 26

What is the TARGET of CAPTOPRIL = Capoten?
captopril = substrate mimic

Answer 26

• *ACE**
• *Zinc Protease** = Acid-Base Catalysis

Question 27

Indication / MoA
of ACE-Inhibitors

Answer 27

Hypertension
CV Disorders / CHF / renal insufficiency

PREVENT the formation of Angiotensin II
which is a peptide hromone that leads to:
Vasoconstriction –> Increase in BP

Question 28

How does CAPTOPRIL inhibit/bind to ACE?

Answer 28

MIMICS 2 Carboxy-Terminal Residues

Binding of SulfHydryl group –> ZINC
displaces water molecules

Binding of the proline-COOH –> Cationic site on enzyme

Incorporation of PROLINE + Methyl
since Ala-Pro has greatest affinity for ACE

Question 29

What is a PROTEASE?

Answer 29

A type of HYDROLASE
that hydrolyzes Proteins & Peptides
Cleavageof theamino linkagesjoiningAA’s

accelerates the formation of a TRANSITION STATE
(tetrahedral intermediate)​
important process in
blood clotting / protein digestion / peptide+hormone processing

Two CLASSES of Protease mechanisms:
Covalent + Acid Base
Catalysis

Question 30

What type of Enzyme?

Catalyze the hydrolysis of a substrate

The chemical breakdown of a molecule due to:
reaction with water

Answer 30

HYDROLASE

Example:
Proteases

ACE / HIV protease / Renin / proteasome / Thrombin

Question 31

Efficacy or Potency?

<<left><br></br><strong>Amount</strong> of drug that is needed to <strong>produce a given effect</strong> </left>

Answer 31

• *POTENCY**
• Less drug* is needed, the MORE POTENT the drug is

Question 32

How to BLOCK the action of Proteases?

Answer 32

• *SUBSTRATE MIMICS**
• *mimic** the substrate but replace the part that directly reacts with the active site of the protease –> target the catalytic mechanism
• *TRANSITION STATE MIMICS**
• *resemble the TS** but it can NOT go on to complete the rxn

Question 33

What type of inhibitor is
CAPTOPRIL = Capoten

Answer 33

REVERSIBLE** & **COMPETITIVE

it binds for the same enzyme = ACE

it mimics the substrate

Question 34

Lewis Acid

Answer 34

• *ZINC**
• *ELECTROPHILE**

ACCEPTS a Pair of electrons

ACID = ACCEPT