2. • Brief overview of HIV/AIDS
• Very brief structure, entry, replication cycle, genome
• HIV Protease (HIV PR)
• Protease Inhibitors (PI)
• Development of PI
• Downsides of PI treatments
• Future of PI
OUTLINE
3. • Acquired immunodeficiency syndrome
(AIDS)
• Estimated 25 million people have died as a
result of AIDS
• AIDS is typically diagnosed 7-8 years after
initial infection
• Immune system has been fully
compromised
• Death is usually the result of an
opportunistic infection
• Human Immunodeficiency Virus 1 (HIV-1) is a
retrovirus and the primary cause of Acquired
immunodeficiency syndrome (AIDS)
• Origin
– SIVcpz
• Mode of Transmission:
– Blood
– Semen
– Vaginal fluids
– Breast milk
• Main target:
– CD4 lymphocyte
• Stages of HIV infection
– Acute
– Clinical
– AIDS
HIV/AIDS
4. • 1994 – AIDS becomes #1 CoD for
Americans age 25-44
• June 1, 1995 – FDA approves P.I.
and HAART
• 1995 – AIDS in US reaches 500,000
• 1997 – P.I. treatment causes viral
resistance increase
• 2000 – President Clinton declares
HIV/AIDS a threat to national
security
• June 5, 1981 – First reports of
immunocompromised gay men
• February 1, 1983 – Dr. Robert Gallo
suggests the retrovirus is
responsible
• January 1, 1985 – FDA approves
ELISA to detect HIVab
• January 11, 1985 – CDC reports
AIDS is caused by a “new” virus
• 1989 – AIDS in US reaches 100,000
TIMELINE OF HIV/AIDS
11. • Primary function: cleave long
polypeptides from translated gag and
pol genes.
• Inhibition of HIV protease results in the
release of immature and noninfectious
viral particles.
• Structure and function similar to
human renin and pepsin
• **Warning: Organic Chemistry Ahead**
HIV PR
Adamson, Catherine S. 2012
12. • C2 symmetric, 198-amino acid
homodimeric aspartyl protease
• Active site consists of two conserved
aspartic acid residues
– Asp-Thr-Gly motif
• Semi-open conformation
– Two models of activation
• Why is this important?
HIV PR
Credit: David R. Davies. The Structure and Function of Aspartic Proteinases. (1990)
13. HIV PR MECHANISM OF ACTION
http://upload.wikimedia.org/wikipedia/commons/0/00/Aspartyl_protease_mechanism.png
Nucleophilic Addition-Elimination Reaction
15. How do we
stop this?
https://www.youtube.com/watch?v=RO8MP3wMvqg
16. • Protease inhibitors are drugs that block a protease from making active
proteins
• Competitive inhibition
• Discovered primarily through structure-based drug design and a LOT of
organic/protein chemistry
• Goals
– Suppress viral load
– Reduce morbidity
– Maximize survival
– Restore and maintain immunological function
PROTEASE INHIBITORS (P.I.)
17. Protease Inhibitor
(1st Generation)
Dosage Structure
Saquinivir (1995)
Inverase
(Roche Labs)
SQV/r 1800mg/200mg
(600mg 3x/d)
With fatty meals
Ritonavir (1996)**
Norvir
(Abbott Labs)
2400mg/daily
(100, 200, 400mg 3x/d)
With or without food
Indinavir (1996)
Crixivan
(Merck)
IDV/r + EFV: 800mg every 8h
(100, 200, 400mg capsule)
Empty stomach
Nelfinavir (1997)
Viracept
(Agouron)
2,250mg/daily
(750mg 3x/d or 1250 2x/d)
With meals
19. • The development of P.I. involved a lot of organic chemistry, biochemistry
and, new at the time, protein crystallography and computer chemistry
• Structure-based and substrate-based
DEVELOPMENT OF PI
20. • The five first generation protease inhibitors were based on renin and
pepsin inhibitors (pepstatin)
• The idea was to create a substrate that can bind the active site as an
analogue, but be uncleavable.
DEVELOPMENT OF PI
Virlgil, Scott C. “First Generation HIV-1 Protease Inhibitors for the Treatment of HIV/AIDS”. 2010.
23. • The inhibitors are analogues of
the substrates that PR would
normally recognize.
• Peptidomimetic transition-state
analogues
• Normal peptide linkage
– [—NH—CO—]
• Inhibitor linkage
– [—CH2—CH(OH)—]
P.I. MECHANISM OF ACTION
Lead Compound
Asp25---Asp125
X
25. • Monotherapy
– Protease inhibitor on it’s own (downsides)
• Combination Therapy
– 2 Protease inhibitors (boosted PI)
• ATV/r
– PI + NRTI, PI + NNRTI, PI + I.I., PI + BI
• HAART
– Combination therapy of PI + RTI + II
– Usually the most effective method of treatment
P.I. APPLICATIONS IN THERAPY
26. • Increased viral resistance
• Creation of new protease enzymes via integrase
• Expensive
• Time
• Drug Interactions
• Side effects
• Patient Compliance
• Genetic Polymorphisms
DOWNSIDES AND CHALLENGES
27. • Allosteric inhibitors
• The challenge will be to combat resistance
• Maturation Inhibitors – block the specific proteolytic processing of Gag
and Gag-Pol genes
– Bevirimat (BVM)
• 3-O-(3’,3’-dimethylsuccinyl) betulinic acid
• HIV-2 and SIV are not sensitive to BVM
– PF-46396
• 1-[2-(4-tert-butylphenyl)-2-(2,3-dihydro-1H-inden-2-ylamino)ethyl]-3-(trifluoromethyl)pyridin-2-one
• Still in trial phase, but promising
FUTURE OF PROTEASE INHIBITORS
28. • Acheson, Nicholas H. Fundamentals of Molecular Virology. 2nd ed. New Jersey: John Wiley & Sons, Inc, 2011. Print.
• Adamson, Catherine S. (2012). “Protease-Mediated Maturation of HIV: Inhibitors of Protease and the Maturation
Process.” Molecular Biology International 2012: 1-13.
http://scholar.google.com/scholar_url?url=http://downloads.hindawi.com/journals/mbi/2012/604261.pdf&hl=en&sa
=X&scisig=AAGBfm0kCsPPdYS6976mi6S1AhBRIQOO6Q&nossl=1&oi=scholarr
• Ali, Akbar et al. (2010). “Molecular Basis for Drug Resistance in HIV-1 Protease.” Viruses 2: 2509-2525.
http://scholar.google.com/scholar_url?url=http://www.mdpi.com/1999-
4915/2/11/2509/pdf&hl=en&sa=X&scisig=AAGBfm09kKIsQJtvP9i_iTPP-1GpgW16zA&nossl=1&oi=scholarr
• Cruciani, Mario. (2014). “Virological efficacy of abacavir: systematic review and meta-analysis.” Journal of
Antimicrobial Chemotherapy 69: 3169-3180. http://jac.oxfordjournals.org/content/69/12/3169.full.pdf+html
• Davies, David R. (1990). “The Structure and Function of the Aspartic Proteases.” Annual Review of Biophysics and
Biophysical Chemistry 19: 189-215. http://www.annualreviews.org/doi/abs/10.1146/annurev.bb.19.060190.001201
• Eron Jr., Joseph J. (2000). “HIV-1 Protease Inhibitors.” Infectious Diseases Society of America 30: S160-170.
http://cid.oxfordjournals.org/content/30/Supplement_2/S160.short
• Gaczynska, M., Osmulski, P.A. (2014). “Harnessing Proteasome Dynamics and Allostery in Drug Design.” Antioxidants
& Redox Signaling 21.17: 2286-2297. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4241894/
BIBLIOGRAPHY
29. • Hughes, Peter J. et al. (2011). “Protease Inhibitors for Patients with HIV-1 infection: A Comparative Overview.”
Pharmacy and Therapeutics 36.6: 332-343. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3138376/
• Lefebvre, Eric., Schiffer, Celia A. (2008). “Resilience to Resistance of HIV-1 Protease Inhibitors: Profile of Darunavir.”
AIDS Review 10: 131-142. http://www.ncbi.nlm.nih.gov/pubmed/18820715
• Ogden, Richard C., Flexner, Charles W. Protease Inhibitors in AIDS Therapy. New York: Marcel Dekker, Inc., 2007. Print.
• Sharp, Paul M., Hahn, Beatrice H. (2011). “Origins of HIV and the AIDS Pandemic.” Cold Spring Harbor Perspectives in
Medicine 1: 1-16. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3234451/
• Virgil, Scott C. (2010). “First-Generation HIV-1 Protease Inhibitors for the Treatment of HIV/AIDS.” Aspartic Acid
Proteases as Therapeutic Targets. 139-161. http://onlinelibrary.wiley.com/doi/10.1002/9783527630943.ch6/pdf
• Yang, Hailiu et al. (2012). “Effects of HIV-1 Protease on Cellular Functions and their Potential Applications in
Antiretroviral Therapy.” Cell & Bioscience 2.32: 1-8. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3490751/
• “What is HIV/AIDS?” AIDS.gov. 29 April, 2014. Web. Accessed 30 March, 2015 from https://www.aids.gov/hiv-aids-
basics/
BIBLIOGRAPHY
30. • “Protease Inhibitors.” AIDSmap. 2015. Web. Accessed 1 April, 2015 from
http://www.aidsmap.com/Protease-inhibitors/page/1729414/
• “HIV Protease.” YouTube. Uploaded 12 May, 2007. Web. Accessed 1 April, 2015 from
https://www.youtube.com/watch?v=UvElXNo_aPw
• “HIV Replication 3D Medical Animation.” YouTube. Uploaded 3 March, 2007. Web. Accessed 1
April, 2015 from https://www.youtube.com/watch?v=RO8MP3wMvqg“Stages of HIV Infection.”
AIDS.gov. 19 December, 2013. Web. Accessed 2 April, 2015 from https://www.aids.gov/hiv-aids-
basics/just-diagnosed-with-hiv-aids/hiv-in-your-body/stages-of-hiv/index.html
• “Retroviral Proteases.” Retroviral Proteases. N.d. Web. Accessed 2 April, 2015 from
http://www.cs.stedwards.edu/chem/Chemistry/CHEM43/CHEM43/RetroV/BiochemProject.html
• “Aspartic Protease Inhibitors.” Cambridge MedChem Consulting. 2012. Web. Accessed 6 April,
2015 from
http://www.cambridgemedchemconsulting.com/resources/hit_identification/aspartic_protease_in
hibitors.html
BIBLIOGRAPHY
Origin – HIV-1 was sequenced to SIV and found that they are similar. It has been determined that HIV-1 is a zoonotic infection likely transmitted in the early 1900s (there were reports in the 1900s of healthy individuals succumbing to opportunistic infections) from butchered chimpanzees infected with SIVcpz. Monkeys do not, however, succumb to the same clinical syndromes seen in humans infected with HIV-1. This suggests an adaptation through selection
Mode of Transmission – the virus enters the body through those listed modes through mucocutaneous areas that line the mouth, vagina, rectum, penis and upper GI tract
June 5, 1981 – marked depletion of CD4+ T-lymphocytes of otherwise healthy young men in Los Angeles/San Francisco.
Binding and fusion – HIV binds to a CD4 receptor and coreceptor on the surface of the cell. The glycoproteins involved are GP120 and GP41. GP 120 binds to CD4 and attaches. This will allow GP41 to fold out and insert its hydrophobic tip into the cell membrane. GP41 then folds over itself to bring the virus and cell together where they fuse and viral genetic material is released.
Reverse Transcription – Reverse transcriptase is one of three enzymes carried with HIV (protease and integrase being the other two) and begins the process of integration of genetic material with the host genome. Reverse transcriptase first takes one of the RNA strands from HIV and, through reverse transcription, makes a RNA/DNA double helix. This hybrid helix returns to the transcriptase where the RNA is stripped and replaced with DNA making a viral DNA double helix
Integration – Integrase cuts off a 5’ and 3’ end of the new viral DNA (sticky ends) and moves it into the nucleus where it becomes integrated with the host genome.
Transcription – When the host cell is activated it reads the integrated viral DNA and uses the cells enzymes to create new viral enzymes which can be longer and more complex
Assembly – Protease cuts the longer HIV proteins (made by the host cell) into individual proteins. These will come together with the new viral genetic material and capsid proteins (also from protease) will be assembled into a new, but immature, virus
Budding – The new virus pushes itself out of the host cell taking with it part of the membrane and glycoproteins for infection of other cells.
The Pol domain encodes the viral PR, RT and IN proteins. Cleavage at this points by HIV protease will result in new infectious viral particles that are assembled by the Gag (Pr55) polyprotein.
This enzyme is responsible for the creation of new infectious particle and is sometimes referred to ask the bulldog enzyme.
Primary Function – When protease cleaves these polypeptides at specific points they become functional proteins. If cleavage doesn’t happen then the proteins will not function, thus the new viruses won’t mature to become infectious.ig
Figure – this figure shows the difference between an infectious and non-infectious HIV virus. Visualized by transmission electron microscopy and cryoelectron tomography. In the image on the right (infectious) there is a large red spot in the middle. That is the capsid that is made to hold the viral RNA particles as well as the infectious viral enzymes.
Active Site
The active site of HIV PR has two conserved aspartic acid residues on either side of the dimer. The catalytic residues Asp25 and Asp25’ (Asp125) are embedded in the convex hydrophobic core.
Semi-open Conformation
the opening of the two flexible flaps allows entry of the substrate peptide (or inhibitor), and upon closing these flaps coordinate the carboxamide groups flanking the scissle bond through a water molecule
Two proposed models of activation
The first model suggests a collision complex with HIV-1 protease in the open flap conformation as it enters the active site and induces the flaps to close.
The second model suggests the ligand approaches the HIV-1 protease in the semi-open flap and induces the flaps to adopt an open conformation to let the ligand bind.
Cleavage and activation are believed to be autocatalytic, but the details of this process are still not known.
Hydrolysis of Peptide bonds (Proteolysis)- the active site of these enzymes contain two highly conserved aspartic acid residues, one contributed from each lobe of the enzyme (Asp25 and Asp125). Each subunit is held together by hydrogen bonds with water as well as a pair of twofold related, antiparallel beta-hairpin structures. This is important when it comes to inhibitor design because the interactions with the enzyme and inhibitor will determine the enzyme conformation and whether it remains active or becomes inactive. Proteolysis is a very specific process where the enzyme will only cut where it wants to unlike acid hydrolysis where the polypeptide is cut everywhere.
These Phe-Pro, Tyr-Pro linkages are unique cleavage sites not subject to mammalian endopeptidase activity
Cleavage site sequences recognized by HIV PR on the Gag and Gag-Pol polyproteins. As you may or may not remember your amino acids there is a preference for cleavage points of amino acids that are hydrophobic and any aromatic compound is followed by a proline. These cleavage sites were determined by the N and C terminus points of mature viral particles. Efforts to predict where it would cleave is not known and may never be known since the diversity of retroviral proteases vary.
There is a common theme (general pattern) where most substrate sites where cleavage occurs has a branched amino acid at P2, a hydrophobic residue at P1, and an aromatic or proline at P1’. The exact reason why this happens is not fully known or documented.
Protease Inhibitors – this is an important step when it comes to treatment because this is where the proteins for making a mature and infectious virus happen. If this is blocked, then the subsequent virus will not mature and not be infectious
Structure-based Drug Design – This method of drug design generates target specific and potent inhibitors, but the downside is drugs developed by this method can only target one site at a time. Side-effects are also common with drugs made by this method such as: cellular toxicity, limited target choices, efficacy and applicability.
** no longer used as monotherapy and is strictly used to boost other PI and block cytochrome P450 activity
Clinical Efficacy
Atazanavir
Structure-based vs substrate based – SUBSTRATE: The idea was that peptide substrate analogues would be replaced with noncleaveable, transition-state isotere. This follows the mechanism of action by both HIV and cellular proteases under the acid-base chemistry to mimic the hydrated amide that represented the intermediate in amide hydrolysis catalyzed by the two active site aspartic acids. STRUCTURE: crystallography allowed for the realization that HIV PR is a homodimer with twofold symmetry that showed a new generation of inhibitors were needed that didn’t rely on structure or peptide mimicry.
First five generations
The isosteres of the amide hydrolysis transition states were studied. Pepstatin is a natural transition state mimic
Figure
These are the 6 structures of peptidomimetic subunits used for HIV protease inhibitors. Each spot outlined in grey represents the cleavage site for PR (non-cleavable in this case). These are structures that did not break through the process of proteolytic hydrolysis.
Hydroxyethylamine is the structure that was used for Saquinivir…the first approved PR inhibitor. This greatly increase water solubility and facilitated oral absorption
There were over 100 different compounds synthesized following methods similar to this one. The multitude of combination attempts made subnanomolar enzyme activity almost impossible to discover. Additions such as extension of the C-terminal end with a P’2 Ile-NHiBu group lost activity. The preference for (R)-stereochemistry of the hydroxyl group in 6 was much more dramatic. That is why compound 10 was greater than that of 11.
#8) beta-naphthoyl derivative
#9) quinolone-2-carboxamide
Stereochemistry played a huge part in this as well such as with compound 10 was 200-fold more effective than compound 11.
The first scheme didn’t work out all that well. It made bioavailability low and potency ineffective
The second scheme was the scheme used to produce the lead compound for saquinavir:
1) Esterification with (SOCl2, MeOH) and carbamate protection (ClCO2Me, H2O) of L-Phe
The inhibitor linkage is a hydroxyethylene group
Resistance –the virus evolves to accumulate a multitude of mutations within the protease that prevent Pis from binding to the protease. More than half the residues within the protease mutate in different combinations and lead to drug resistance. The protease is still able to recognize and process the natural substrate sites in the gag and Gag-pol polyproteins, while no longer being effectively inhibited by competitive drug molecules
Creation – If protease is blocked and no other HIV replication machinery is blocked the integrase will successfully implant viral DNA. This will code for the host cell to create new protease enzymes with a structure different (possibly) from the original.
Expensive – The average cost to a patient for these drugs (protease inhibitors) is around $20,000 annually
Time – The P.I. must be taken 3 times a day at precise times.
Drug-Drug Interactions – There are numerous accounts of bad drug-drug interactions when using protease inhibitors as both a monotherapy and combination therapy. Some PI and NNRTI counteract each other. PI and other medications for immunocompromised patients don’t work. Patients on cholesterol medication (Lipitor) must be given the lowest dose possible.
Side effects – fever, diarrhea, nausea, vomiting, abdominal pain, rash, fatigue and headaches. Although these symptoms vary between drugs they are common.
Patient Compliance – As with any drug treatment regimen it is important to take the precise dosage at the precise time for the duration of the treatment. When it comes to HIV/AIDS patients must have >95% compliance in order for the infection to be contained.
Genetic Polymorphisms – PI are metabolized by the CYP 3A4 isoenzyme family, but some PIs have been shown to inhibit this enzyme as well. Patients who have a polymorphism of the CYP 3A4 gene are sucepible to side effects such as inhibition of bilirubin conjugation leading to hyperbilirubinemia. Polymorphisms of apolipoproteins, cholesteryl ester transfer proteins
Allosteric Inhibitors – attempt to bind and induce a permanent conformational change.
BVM specifically inhibits CA-SP1 cleavage late in the Gag cleavage cascade. Biochemical studies have demonstrated an accumulation of uncleaved CA-SP1 intermediates in cells treated with BVM. Structure of the BVM binding site is not known. It is accepted that the CA-SP1 region of Gag adopts an alpha-helical conformation via computer modelling prediction software.
