Proteases as drug targets

Question 1

Proteases (peptidases)

Answer 1

Cleave and degrade proteins -
To regulate biochemical processes
To removed damaged/modified proteins
To obtain amino acids for other purposes
Can be dangerous to the cells that produce them.

They need to be tightly regulated -
Often activate only after partial proteolytic cleavage of inactive pro-enzymes
Often contained within membrane bound organelles (lysosomes, endosomes, plasmodium food vacuole) or in tightly regulated machines (the proteasome)

Question 2

Lysosomes

Answer 2

Bound by single membranes
Degrade cellular macromolecules, critical for cellular turnover.
Important role in Autophagy, which is an essential part of cellular remodelling (in protozoa).
Possess a number of catabolic enzymes (nucleases,proteases,phosphatases,glycosylases)
Have an acidic pH of 4.2, lysosomal enzymes work best at this pH which is critical so they cannog cause cellular damage if inadvertently released.
Primary lysosomes fuse with endosomes to degrade endocytosed material and autophage.

Question 3

The proteosome

Answer 3

A complex of protiens, including proteases that can degrade cellular proteins specifically targetted to this complex.
A number of proteins (these involved in celk cycle) are targetted to this compartment using ubiquitin as a signal.
Other damaged proteins are also tagged and degraded in the molecular protein shredder.

Question 4

Classes of protease

Answer 4

Serine proteases 
Threonine proteases
Cysteine proteases
Aspartate proteases
Glutamate proteases
Metalloproteases
The pre-fix indicates what the binding catalytic site contains.

Question 5

Retrovirus genes

Answer 5

Gag- processed to matrix and other core proteins that determine retroviral core
Pol - reverse transcriptase, RNase H and integrase functions.
Env - envelope protein, residues in lipid layer, determines viral tropism.

Question 6

HIV viron structure

Answer 6

Glycoproteins gp120 and gp41 form 72 spikes on the surface.
CD4 receptor site is on gp120.
P24 capsid protein forms a cone shaped shell around the ribonucleoprotein.
P17 matrix protein lines the inner surface of the lipid envelope.
P7 + P9 form a ribonucleoprotein complex with the RNA genome.
Bound to diploid ssRNA are 50 molecules of RT alongside integrase and protease enzymes.

Question 7

HIV genome

Answer 7

Single stranded
Two identicle copies, diploid
Each 10kb.
Complex gene organisation
Encodes gag,p ol and env like all retroviruses.
Pol gene Encodes 3 proteins: RT, integrase and protease (retropepsin).
Additional control genes enhance virus replication (rev,tat,vif,nef)
The gag and pol genes are transcribed and translated into large polyproteins that must be processed into individual proteins to reassemble viral partcles using retropepsin.

Question 8

Structure based drug design

Answer 8

3D structure of ligand bound to structure is imaged
A drug is designed to have similar binding and shape to the ligand
Or to bind at another site, inactivating the molecule.

Question 9

HIV protease inhihitors

Answer 9

Saquinavir, ritonavir, indinavir, nelfinavir, amprenavir

All varients on the peptidomimetic theme.

Question 10

Malaria (plasmodium falciparum)

Answer 10

Enzymes involved in haemoglobin digestion by plasmodium falciparum -
Plasmepsins I and II (aspartic proteases)
Falcipains 2, 2’, and 3 (cysteine proteases)
Falcilysin (metalloprotease, to produce small peptides).
Small peptides are then transported to the cytoplasm from the food vacuole and broken down into amino acids by amino peptidase.
Aspartate and cysteine protease inhibitors like pepstatin and E-64 prevent breakdown of Hb and kills plasmodium in RBCs.
Efforts underwag to make more specific inhibitors of plasmodium proteases.

Question 11

Why target proteases in antiparasitic drug discovery?

Answer 11

They are valudated drug targets.
Good exp tools are already available.
There is a large body of data on host and some other enzymes.
Expertise already exists in acedemia and industry.
Potential for piggybacking or multi-target drugs thus minimising resistance problems.
Multidisease drugs are a possibility.

Question 12

American trypanosomiasis (changas disease)

Answer 12

Caused by trypanosomiasis cruzi
Vector in triatomine bug
Natural host is a large resevoir of mammals
Injestion of parasite with blood meal, faeces or parasite entering wound/eyes.
Romanas sign is a typical manifestation in early stages of the disease (purple swollen eyelids).
Chagasic megasyndromes are found in chronic manifestations of the disease. (megacolon, megaesophagus, megacardiapathy)
Parasites form amastigotesinside cells in a variety of tissues
May remain semi-quiescent for many years.
Drug treatment is unsatisfactory (nifurtimox, benznidazole)

Cruzain is a cysteine protease of t.cruzi.
K777 is a peptide memtic inhibitor or cruzain -
Curative effect in acute and non-acute models of infection
Efficacy against range of t.cruzi strains including banznidazole and nifurtimox resistant strains.
In final preclinical stages, trials being planned.