* 2, 3 & 4 - Cysteine Protease Inhibitors * Flashcards
What’re the 4 major classes of protease enzymes?
Aspartic - HIV
Serine
Cysteine
Metallo
What do protease enzymes do?
They selectively catalyse the hydrolysis of polypeptide bonds and control protein synthesis, turnover and function.
- allowing them to regulate physiological processes
Also crucial for disease propagation
Why’re peptides classically ‘bad drugs’?
Due to their instability in the body, low bioavailability and poor pharmacological profiles
What properties must protease inhibitors possess?
Minimal peptide character
High stability to non-selective proteolytic degradation
Good membrane permeability
Long lifetime in body (blood/cells)
High selectivity for target
Good bioavailability (oral preferred)
Low molecular weight - in line with Lipinski’s rules
How are protease inhibitors traditionally developed?
Truncating polypeptide substrates
Replacing cleavable amide bonds with non-cleavable isostere, optimising potency thru trial and error
Substrate-based drug design
- 3D structural info
Combinatorial chemistry
- HTS
How do a wide range of proteases bind to substrates?
They universally bind in EXTENDED conformations
- peptide backbone, or equivalent, is drawn out in a linear arrangement
Through what means do protease inhibitors bind favourably?
Binding is entropically favourable
Are conformational restricted inhibitors more or less favoured for binding to proteases?
Conformationally restricted inhibitors that adopt receptor-binding conformations are MORE FAVOURED as they are entropically advantaged for binding to a protease
- linear binding arrangement
What does the active site of papain, a group of Cys proteases, contain?
It contains a catalytic triad of Cys25, His159 & Asn175
(Cysteine, histidine and asparagine)
What’s the importance of the catalytic triad in the active site of papain?
Asn175 has been proposed to orientate His159 so that it polarises the thiol on Cys25.
This allows for deprotonation of thiol even at neutral to weakly acidic pH.
This results is the thiolate/imidazolium ion pair becoming highly nucleophilic
Draw a typical mechanism for amide bond cleavage by a Cys thiolate. What stabilises the intermediate?
The intermediate is stabilised by the oxy-anion hole
What structural features do protease inhibitors have?
A peptide segment for recognition
- this is bound to an electrophilic group which can react with the Cys residue of the active site
2 or more amino acids
- to achieve good affinity
Give 3 examples of electrophilic groups used in protease inhibitors
Aldehydes
Nitriles
Halomethyl ketones
Halomethyl ketones have 2 electrophilic sites, how is selectivity achieved at Cys residue of protease?
Attack at either site results in the same product
- though attack at carbonyl is preferred
What’re 3 important Cys protease enzyme targets?
Cathepsin K
Cathepsin B
Caspases
What is the role of Cathepsin K?
Cathepsin K is selectively expressed in osteoclasts (cells that degrade bone to initiate normal bone remodeling) and plays an important role in osteoclasts-mediated bone resorption
What diseases do Cathepsin K inhibitors treat?
Treatment of diseases characterised by excessive bone loss - i.e. osteoporosis
What is the role of Cathepsin B?
Cathepsin B is a lysosomal cysteine protease.
It plays various digestive and processing roles to maintain normal cellular metabolism
What happens when Cathepsin B is over-expressed?
It has been associated with; tumor metastasis, inflammation, bone resorption and myocardial infarction
What are Caspases?
‘Caspase’ is a new term given to ICE-like Cys proteases with absolute specificity for aspartic acid at P1 in their substrates
What does ICE stand for?
Interleukin-1 converting enzyme
What’s the key function of Caspases?
These cytokines are key inflammatory mediators, among many other functions
What are ICE-inhibitors a promising therapeutic candidate for?
For treatment of inflammatory diseases
What is Main protease (Mpro) wrt coronaviruses?
Mpro is a key enzyme in the viral life cycle of coronaviruses as it mediates viral replication and transcription of the virus
What is Mpro’s role wrt Covid?
Mpro’s role is in processing of polyproteins that are translated from (COVID) viral RNA
What amino acid residues are in the Mpro catalytic pocket?
His41 and Cys145
What does this reaction yield?
What does this reaction yield?
What reagent is used to remove the t-Butyl protecting group?
TFA
- to give -COOH and t-Bu
Identify the electrophilic warhead and the binding pocket of the LHS of the molecule given.
Why is diazomethane (shown) used in the first step of ICE inhibitor synthesis?
It is used as it releases N2
- a Very good leaving group
What is the mechanism for removal of t-butyl using TFA?
t-Butyl is removed in E1 fashion as it forms a stable carbocation
What’s the benefit of the isosteric replacement of the amide group shown?
It increases oral bioavailability from 5% to 30% - in the latter molecule
Suggest the starting materials used to form the given structure
Why is BOP and t-BuOH used in the synthesis of amide bonds?
Because it produces an activated ester in situ
(This is additional content, I’d only focus on the fact that the activated ester is made in situ)
What is the malaria pathogen and how is it transmitted?
The malaria pathogen is a unicellular parasite
It is transmitted from the bite of a female mosquito - Anopheles
Describe the cycle of malaria from infection to transmission
What is the site of action of Falcipain 2 inhibitors?
Haemoglobin metabolism & generation
What is the function of Falcipain 2?
Falcipain 2 (& 3) are localised to the food vacuole and are involved in the breakdown of host proteins - including Haemoglobin
What type of proteases are Falcipain?
Cysteine proteases
What’s significant about Falcipain 2 vs Falcipain 3?
FP3 cleaves native Haemoglobin at double the rate of FP2
Though FP2 is present in higher concentrations
What functional group is cleaved by Falcipain?
Amide - peptide - bonds
What can be added to the amide warhead for Falcipains to display efficacy?
A fluorescent group can be added to the amide bond that will be cleaved
Fluorescent group is now the LG, fluorescing when kicked off through resonance of highly conjugated molecule
What functional groups can be used for attack by Falcipain 2 thiol?
Aldehydes
Vinyl sulphones
Nitriles
What’s the use of inclusion of a morpholine ring in a drug molecule?
Improved water solubility
What sites do Falcipain 2 inhibitors bind to?
S1 S2 S3 & S1’ S2’ S3’
At the P3 site of Falcipain 2, what is the preferred functional group over a Z-protecting group?
At P3, morpholine urea is preferred over Z protecting group as it increases solubility
At the P2 site of Falcipains, what is the preferred functional group?
At P2, Leucine is preferred
At the P1 site of Falcipains, what is the preferred functional group?
At P1, homo-phenylanaline is optimal as it helps to offset host amidase-mediated hydrolysis of drug molecule
What is a scissile bond?
Its a bond that can be broken by an enzyme
What similarities do aldehyde warheads and vinyl sulphone warheads have as FP2 inhibitors?
They both have very similar SAR to each other
P3 = Morpholine urea
P2 = Leucine
P1 = Homo-Phenylalanine
Draw the mechanism for the formation of the active electrophile in the Swern oxidation
Give the mechanism for hydrolysis of amide bonds, the 1st step is given.
CO2 is evolved
Why is EDC used in peptide coupling?
EDC is used as it activates the carboxylic acid
Why is 1-hydroxy benzotriazole used in peptide couplings?
Because it is a good leaving group
What’re the advantages of incorporating a nonpeptidic scaffold in peptidomimetics?
Increased potency
Increased membrane permeability
Improve selectivity by stabilising a biologically active conformation
Enhanced oral bioavailability & stability to degradation by enzymes
Why are aldehydes preferred over vinyl sulphones in peptidomimetics?
Because vinylsulphones show potential toxicity concerns as they are irreversible inhibitors
Aldehydes bind reversibly and hence are more desirable
What’s significant about where Falcipain 2 resides?
It resides in the food vacuole of red blood cells
The pH within the food vacuole is slightly acidic at ~5.5
Here, masked aldehydes can be converted to aldehydes in situ due to low pH
What do more negative docking scores mean for binding of the ligand?
More negative scores = better binding
What is the nature of the S2 pocket in FP2?
the S2 pocket of the enzyme is fairly hydrophobic in nature
What’s the use of adding N-Me piperazine to a drug molecule?
It aids aqueous solubility
- can also be converted to salt forms too
