33/34 - Intro + Hydrolases (+Proteases) Flashcards
Type of Inhibitor?
bind to either free enzyme or the E-S complex
minimal effect on Km
&
LOWER Vmax
NON-Competitive Inhibitors
NOncompetitive inibitors are NOt so picky
bind to either free enzyme or E-S complex
Ex.
anti HIV - RT inhibitors
Nevirapine
What type of Protease Mechanism?
Metalloproteasese // Aspartic Acid
Proteases
Uses WATER
no covalent bonds involved
ACID-BASE CATALYSIS
Protease mechanism
Use a non-covalent, acid-base mechanism
EX.
Captopril –> ACE
Ace = zinc protease
Indinavir –> Asp Protease (HIV)
Type of Inhibitor?
Inhibitor once bound NEVER comes off
- *covalent bond formation** between the
- *enzyme + reactive group of the inhibitor**
IRREVERSIBLE Inhibitors
Ex.
ASPIRIN / PENICILLIN
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
SUBSTRATE MIMICS
Mimic the structure of the peptide substrate
CAPTOPRIL
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
COMPETITIVE INHIBITORS
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)
COVALENT CATALYSIS
the tetrahedral intermediate
involves a
stable covalent bond to the enzyme’s catalytic nucleophile
What is the MECHANISM of Aspartic Protease (HIV)?
inhibited by INDINAVIR
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
What Drug targets ACE?
CAPTOPRIL = Capoten
indicated for CV disorders
HT / CHF / renal insuficiency
SUBSTRATE MIMIC
ACE = Zinc Protease
(Acid-Base Catalysis)
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
TRANSITION STATE MIMICS
indinavir
What TYPES of PROTEIN-TARGETS are there?
MOST TARGETS are PROTEINS!
Receptors > Enzymes
Transporters > other
enzyme interacting proteins / structural proteins
Efficacy or Potency?
^^^INCREASE^^^ in % RESPONSE
- *Maximum effect** that a drug can produce
- regardless of DOSE*
EFFICACY
Type of Inhibitor?
bind to the enzyme-substrate complex
but NOT to the free enzyme
both Km & Vmax are REDUCED
UN-Competitive** **Inhibitor
UNcompetitive inhibitors UNiquely bind
the enzyme-substrate complex
- uncommon*
ex. mycophenolic acid = immunosuppressive
Why does Biochemical mechanism MATTERS
in Drug-discovery
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
Common Features
of other drugs that INHIBIT ACE
All are Carboxyl
Enalapril / Lisinopril / Ramipril
except for Captopril = sulfhydryl class
Metal-Chelating Group
that interacts with ZINC
Thiol / Carboxylate / Phosphinate
Some are prodrugs
Enalapril / Ramipril
They vary in onset of action –> dose adjusting
ZINC’s Role
in ACE’s Protease Acid-Base Catalysis
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
Therapeutic Index
TI
TD50 / ED50
Dose that elicits a TOXIC response in 50% of treated individuals
////
Dose that is Therapeutically EFFECTIVE in 50% of treated individuals
Lower the EC50 –>
what affect on Potency/Efficacy?
The lower the EC50 the
HIGHER THE POTENCY
EC50 is the
concentration of a drug that gives half‐maximal response.
What DRUG targets HIV Protease?
& what is the indication?
INDINAVIR = Crixivan
HIV Infection / AIDS
non-petidic
TRANSITION STATE MIMIC
What TYPE OF ENZYME requires the formation of a
TRANSITION STATE=Tetrahedral Intermediate
as a prequisite for peptide-bond scission?
PROTEASE
a type of hydrolase
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.
PHENOTYPIC Screening
for drug discovery
EXAMPLE:
antibody discovery with bacterial growth
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
Target-Based
Drug Discovery
High throughput screening
What is the TARGET of INDINAVIR = Crixivan?
HIV Protease
Aspartic-Acid Protease =
ACID-BASE CATALYSIS
How do
- *HIV Protease Inhibitors = Indinavir** (Asp-Protease)
- *work?**
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
What are the 2 types of
Target-driven drug discovery?
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
Mechanism for ACE-I (Captopril’s)
COUGH side Effect
& Alternative
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
What is the TARGET of CAPTOPRIL = Capoten?
captopril = substrate mimic
- *ACE**
- *Zinc Protease** = Acid-Base Catalysis
Indication / MoA
of ACE-Inhibitors
Hypertension
CV Disorders / CHF / renal insufficiency
PREVENT the formation of Angiotensin II
which is a peptide hromone that leads to:
Vasoconstriction –> Increase in BP
How does CAPTOPRIL inhibit/bind to ACE?
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
What is a PROTEASE?
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
What type of Enzyme?
Catalyze the hydrolysis of a substrate
The chemical breakdown of a molecule due to:
reaction with water
HYDROLASE
Example:
Proteases
ACE / HIV protease / Renin / proteasome / Thrombin
Efficacy or Potency?
<<left><br></br><strong>Amount</strong> of drug that is needed to <strong>produce a given effect</strong> </left>
- *POTENCY**
- Less drug* is needed, the MORE POTENT the drug is
How to BLOCK the action of Proteases?
- *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
What type of inhibitor is
CAPTOPRIL = Capoten
REVERSIBLE** & **COMPETITIVE
it binds for the same enzyme = ACE
it mimics the substrate
Lewis Acid
- *ZINC**
- *ELECTROPHILE**
ACCEPTS a Pair of electrons
ACID = ACCEPT
