Antivirals PDF

MMI 415/515 - Antivirals Vanessa Meier-Stephenson, MD PhD FRCPC March 9, 2023 1 I would like to acknowledge that we are located on Treaty 6 territory, the traditional and unceded lands of First Nations and Métis people on which we are learning and working today. 2 Outline • Antivirals – Curren...

MMI 415/515 - Antivirals Vanessa Meier-Stephenson, MD PhD FRCPC March 9, 2023 1 I would like to acknowledge that we are located on Treaty 6 territory, the traditional and unceded lands of First Nations and Métis people on which we are learning and working today. 2 Outline • Antivirals – Current state and challenges • Broad classes/Mechanisms of Action • 3 real-life examples: • Acyclovir • Saquinavir • Remdesivir • Summary and Points to Ponder • Importance of antivirals - Introduction to antivirals --. Current state and challenges of it - Broad classes of antivirals and mechanisms 3 Antivirals – Current state • Currently 118 antiviral therapies • 85 are monotherapies • 33 are combos • >50% of those are for HIV Tompa, et al, Int J Biol Macromol, 172;524-541 (2021) - Monotherapies  individual drugs Looking at different formulations of same drugs There are a lot of viruses that have no treatment Out of the 10 listed here HIV, HCV and HBV influenza 4 Antivirals – Challenges • Challenges: • Shutting down “viral machinery” = shutting down the cell • Every step of replication involves the host cell • Toxicities unacceptable (cf chemotherapy) • Some medically-important viruses are not safe to propagate (i.e., Ebola) or have no good animal model (i.e., HBV) • A drug must sufficiently suppress/treat or the virus to either clear or lower it until the immune system can take over* • Mutations, leading to resistance • Return of replication once tx stopped *exception: HCV and DAAs? - When you are shutting down the viral machinery you are trying to shut down the host cell that the virus uses to do its job Chemotherapy  trying to kill cancer cell which is a normal cell acting inappropriately Antivirals goal is target the virus more often than the host cell which is a challenge Examples of antivirals that are so good that you don’t require an operational immune system HCV is an exception Anything else if you do not have an immune system, it is really tricky and hard to clear a viral infection  it is about the binding time - For certain infections if you stop the viral infection, you will get rebound of the virus 5 Possible Antiviral Targets • • • • • • • Viral attachment Cell entry Uncoating Transcription Translation Viral assembly Viral release Take any step of the viral life cycle and make it is a potential target for therapy 6 Possible Antiviral Targets • • • • • • • Viral attachment i.e., enfuvirtide (HIV); palivizumab (RSV) Cell entry i.e., Amantadine (Influenza) Uncoating Transcription i.e., nucleotide analogues(HBV, HCV, HIV, HSV…) Translation i.e., Darunavir (HIV); rifampin Viral assembly (vaccinia) Viral release i.e., oseltamivir (influenza) • Integration i.e., Dolutegravir (HIV) - Integration  HIV has a specific step - Categories being any step of the viral infection, replication cascade that could be unique enough go after 7 3 examples Acyclovir Saquinavir Remdesivir 8 Acyclovir 9 Clinical Case • 36yoF engineer, otherwise healthy, brought in by partner • Confused, “couldn’t remember how to start the car leaving for work in the morning”, incoherent sentences. Had complained of H/A the night before – went to bed early. No recent illnesses, travel or sick contacts. • In ER, Temp 39.5, HR 115, BP 120/80. Confused, PEARL, no focal neurologic signs. Decreased LOC. • CT head – no mass lesions • Blood Cx, LP, tox. screen, metab w/u, HIV, syphilis • Empiric vancomycin, ceftriaxone and acyclovir 10 Clinical Continued… • CSF – • • • • 300 WBCs (90% lymphocytes); 0 RBCs; normal glucose and protein Neg bacterial cultures Viral PCR – positive for HSV-1 https://case.edu/med/neurology/NR/hsvsummary3.htm MRI- temporal lobe enhancements - The results of the lumbar puncture showed a lot of white blood cells - Increased lymphocytes  viral infection - The brain scan shows temporal lobe enhancement which is typical of HSV encephalitis 11 HSV encephalitits • Reactivation of HSV-1 (90% of cases) • Retrograde neuronal spread from infected ganglion • Temporal lobe predominance (trigeminal or olfactory nerve) • High morbidity and mortality – 60-70% untreated •Acyclovir reduces mortality by >50% to about 8-30% (depending on timing of initiation and risk factors) Risk factors for morbidity and mortality: • Age >30 • Immunocompromised • GCS 8 or less • Delays in receiving acyclovir (>2d) Tunkel, et al, Clinical Infectious Diseases 2008; 47:303–27 - The HSV virus causes cold sores or genital sores Spread from trigeminal neuron back into the brain Depends on the timing of the drugs mostly Acylovir is intravenous and can pass the blood brain barrier 12 Acyclovir 13 Acyclovir • Classification: • Acyclic nucleoside analogue of guanosine cf - The hydroxyl group is missing on the lower part on deoxyguanosine relative to acyclovir 14 Acyclovir (ACV) • Mechanism of Action: • ACV is tri-phosphorylated intracellularly to the active compound ACV-TP • Initial phosphorylation → viral thymidine kinase • High degree of selectivity for virus-infected cells • Di and tri-phosphorylation → cellular kinases • ACV-TP does 2 things: 40-100x higher concentrations in HSVinfected cells than in uninfected cells! • Potent inhibitor of viral DNA polymerase • DNA chain termination • No 3’ hydroxyl group (unlike PCV or GCV) ACV-TP gets incorporated into the DNA chain and inhibits the DNA polymerase and results in chain termination The first step is done by the viral thymidine kinase  the virus has its own kinase to get that first step done The next phosphorylation steps are done by the cellular kinases In cells that are infected with HSV  cellular kinases can upregulated High degree of cell selectivity of the virus for some of the cells and then it gets wrapped up so you get a lot of production in the beginning in small number of cells Advantage of acyclovir  activity in only the cells that are not getting incorporated 15 De Clercq, et al. Handbook of experimental pharmacology. (2009);189: 53-84. 1. Viral kinase 2. Next two steps are cellular 3. Gets incorporated In here and blocks DNA polymerase which causes chain termination 16 Acyclovir • Indications: • HSV and VZV infections • In vitro activity against EBV • Not useful in acute infectious mononucleosis • Suppression of frequently recurring: • Orolabial HSV  cold sores • Genital HSV • [CMV disease prophylaxis in transplantation] • Renal transplantation • HSCT prior to engraftment (high dose ACV) Barkholt L, et al, Transpl Infect Dis. 1999 Jun;1(2):89-97. - VZV causes chicken pox 17 If you were a herpesvirus, how could you avoid the effects of ACV? De Clercq, et al. Handbook of experimental pharmacology. (2009);189: 53-84. 1. Downregulate the activity of TK 2. Change binding sites of viral polymerase  mutate viral polymerase 3. Mutate TK If you are immunocompetent  the resistance is very rare 18 Acyclovir Resistance • Resistance (HSV and VZV): • Immunocompetent 0.1 - 0.7% • Immunocompromised 4 – 14% • Mechanism • Absent or reduced TK (95%) • TK deficient mutants are cross-resistant to other agents that require TK activation • TK deficient mutants have decreased virulence in vitro and are less neurovirulent • Altered TK (5%) • Does not phosphorylate ACV • Viral DNA pol mutations (1%) Virulence = wild type - Resistance is very rare when you are immunocompetent - Immunocompromised  more chances higher chances of resistance - HSV gets around by either downregulating or knockout its tyrosine kinase which makes it less efficient and is not as virulent hard one 19 CMV-Cytomegalovirus • Clinically important herpes virus cf • esp. Immunocompromised; Pregnancy • 60-70% people infected by adulthood • Has no viral thymidine kinase… • But it has a phosphotransferase kinase (PK) *Compared to ACV, GCV is 10-100x more potent inhibitor against CMV Takkar et al., J Transplant Technol Res 2013, S6 20 Ganciclovir • Spectrum: • CMV (10X potency of acyclovir) • Treatment and suppression of CMV retinitis • Treatment CMV ds in highly immunocompromised • Prophylaxis CMV ds in transplant patients • EBV (10X potency of acyclovir) • HSV/VZV (equal to acyclovir) • Human Herpesvirus 6 ? • Oral, intravenous, and intraocular - Has an extra hydroxyl group in the end is able to overcome the phosphorylation by the 21 CMV – GCV Resistance Attack – Counterattack! Can you think of a way to counter this resistance? • GCV resistance • UL97 mutation in CMV PK • UL54 mutation in CMV DNA polymerase Foscarnet Takkar et al., J Transplant Technol Res 2013, S6 1. The first phosphate attaches with phosphate transferase 2. Cellular enzymes are incorporated into the DNA 3. CMV is able to resist against this by mutating the first step so that phosphokinase with a single UL97 mutation 4. Similarly it can also mutate the DNA polymerase at UL54  which allows for the propagation of the CMV drugs Can you think of a way to counter this resistance? - Add the phosphate first instead of allowing the virus to add the first phosphate - A drug called cidofovir is a cytidine analog and it contains phosphate group already on the - Foscarnet is an analogue of organic phosphate group that is part of this With resistant CMV we do a combination of drugs 22 Foscarnet (PFA) • Classification: • Inorganic pyrophosphate analogue • Mechanism of action • Inhibition of: • Viral DNA pol (reversibly blocks pyrophosphate binding site and inhibits pyrophosphate cleavage from dNTP’s) • Reverse transcriptases • No enzyme is required for activation • Indications: • Herpesviruses • HSV 1&2, VZV, CMV, EBV, HHV-6, 7 and 8 • GCV resistant CMV • ACV resistant HSV and VZV • Retroviruses • HIV • HBV 23 Prodrugs Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases, 46, 577-595 - Prodrugs are drugs that need processing before they become their active form For example cidofovir needs adding of two phosphates before it becomes activated Brincidofovir needs to be cleaved Prodrugs is a way of administering a drug that is only deliverable by IV and turning it into one that can be given orally or in a way that it can be concentrated in the cell so that it only reaches that cell type 24 Back to the Clinical Case • Within 24hrs, rousable, conversing normally • Profoundly tired • Treated with IV acyclovir x 7d, then transitioned to po valacyclovir for further 2wks • Made a full recovery with no neurologic deficits. - Valacyclovir is a prodrug of acyclovir 25 Saquinavir 26 Who is this? - Brandon mercury died of HIV AIDS in 1991 27 - Before `99` we had didanosine and zalcitabine this medication helps with 28 Torian, et al, MMWR, 2011; 60(21);689-693 - Uptil 1995 this is the profile of how the HIV/AIDS diagnosis looked like - People became more aware, started wearing condoms a lower amount of people getting diagnosed with HIV - Dashed lines  AIDS death 29 A great example of Rational Drug Design! • Saquinavir Talele TT, Khedkar SA, Rigby AC. Curr Top Med Chem. (2010); 10: 127-141. 30 - A computer model rational drug design - It is a protease inhibitor and it was designed of a tetrapeptide transmission state of the molecule  analogue of the 4 amino acid structure that gets inserted in proteases 30 1. Entry of the virus 2. Reverse transcription and then it gets integrated into the genome where it starts to pop out the genomic RNAs that required for subsequent virion production 3. Proteases can act on one or two places or the translational part of the cleavage of the proteins 31 Discovery of the HIV protease • Viral proteins gag and gag-pol translated as fusion polyprotein • Sequencing comparisons with other retroviruses suggested it must also contain a protease1 • Contains a highly-conserved Asp-Thr-Gly motif similar to cellular aspartic proteases, therefore ?same mechanism2 • Cellular protease 200 a.a., but HIV protease only 99 a.a.dimer model proposed (1987)3 • Crystal structure solved (1989)4 PDB: 2HVP 1. Ratner, et al Nature 313:277-284 (1985). 2. Toh, et al (1985). 3. Pearl and Taylor, Nature, 329(6137):351-4 (1989). 4. Navia, et al, Nature. 337(6208):615-20 (1989); Redshaw, et al, “The Road to Fortovase…” in Proteases as Targets for Therapy pp 3-21, Handbook of Experimental Pharmacology book series, vol140, 2000 - Produces GAG protein all line up in the lipid bilayer - The protease clips all the edges around and it is now able to snap into a mature virion - Crystal structure  lead to the development of can we create something or a drug that is going to inhibit that dimerized protease 32 HIV maturation 33 Development of the 1st protease inhibitor • HIV protease cleaves N-terminal to Pro (something mammalian endopeptidases don’t do!) • Argued a transition state mimetic would bind with more stability T.A. Lyle, in Comprehensive Medicinal Chemistry II, 2007 - One thing about HIV proteases is that it cleaves at the N terminal to proline which other endopeptidases don’t do If there is a transition state mimetic there is a better chance of creating something with a stronger binding inside that pocket IC 50  750 for the initial drug down to 18 then to 0.4 When they are doing this model they are trying to have structure – activity quantitative relationship which is equating structure to the activity - This one is bigger and this one gets better binding 34 Development of the 1st protease inhibitor • Quantitative Structure-Activity Relationship (QSAR) • Molecular modeling and docking studies Beilstein, et al, J. Org. Chem. 12:2694–2718 (2016) ; Redshaw, et al, “The Road to Fortovase…” in Proteases as Targets for Therapy pp 3-21, Handbook of Experimental Pharmacology book series, vol 140, 2000 - Looking at which functional groups will be successful at that - HIV 1 and HIV2 proteases destroyed the different results from that leading to sacq. 35 A great example of Rational Drug Design! • Saquinavir • Structure-based drug design • Ligand-based design • QSAR models • Molecular docking studies A GAME-CHANGER! Talele TT, Khedkar SA, Rigby AC. Curr Top Med Chem. (2010); 10: 127-141. 36 - First protease inhibitor - Proteases incorporated in various combination therapies 36 37 Torian, et al, MMWR, 2011; 60(21);689-693 - In 1995 when we started developing the drugs, the black line AIDS deaths declined 38 Torian, et al, MMWR, 2011; 60(21);689-693 39 Half of the award will be shared by two French virologists, Françoise Barré-Sinoussi, 61, and Luc A. Montagnier, 76, for discovering H.I.V., the virus that causes AIDS. Conspicuously omitted was Dr. Robert C. Gallo, an American virologist who vied with the French team in a long, often acrimonious dispute over credit for the discovery of H.I.V. - The Nobel prize was awarded to two French virologists 40 As of Mar 8, 2023 6,870,000 https://www.businessinsider.com/coronavirus-deaths-how-pandemic-compares-to-other-deadly-outbreaks-2020-4 41 Remdesivir 42 Discovery of Remdesivir (GS-5734): Antiviral Activity against Ebola Gilead library of nucleosides and nucleotides Screening (CDC) Prodrug design (Gilead) GS-441524 Profiling (USAMRIID, CDC) Slide Courtesy of Dr. M. Götte GS-5734 (Remdesivir) Source: Gilead Sciences Inc. Warren et al, Nature, 2016 - This drug can target SARS cov2 - Originally developed against Ebola - Created a prodrug out of it  gets cleaved and processed after 43 Several therapeutics had been under development from prior smaller outbreaks --ZMapp --Remdesivir Further antibody cocktails were developed during outbreak --Mab114 (from ebola survivor) --REGN-EB3 (3 hmAb cocktail) S Mulangu et al. N Engl J Med 2019;381:2293-2303. 44 2019 CONCLUSION: Two Antibody Therapies were superior to Remdesivir in reducing mortality Slide Courtesy of Dr. M. Götte 45 Broad-Spectrum of Antiviral Activity of Remdesivir 2016/17 Virus Family Virus Filoviruses Ebola (Makona) 0.19 Ebola (Kikwit) 0.14 Bundibugyo 0.19 Sudan 0.24 Marburg 0.06 MERS 0.03 SARS 0.10 Nipah 0.05 Measles 0.04 Hendra 0.06 Dengue 0.25 Yellow fever 0.13 Zika 0.10 West Nile 1.0 Coronaviruses Paramyxoviruses Flaviviruses Source: Gilead Sciences Inc. Warren et al, Nature, 2016 Lo et al, Sci Reports, 2017 Sheahan et al, Sci Transl Med, 2017 Slide Courtesy of Dr. M. Götte EC50 (μM) Arenaviruses Lassa 4.5 Bunyaviruses CCHF > 50 Togaviruses Chikungunya > 20 46 The viruses at the bottom didn’t show activity 46 2018 Slide Courtesy of Dr. M. Götte - Susceptibility was related to actions on viral polymerase and for 47 January 2020 Slide Courtesy of Dr. M. Götte 48 HOLD UP! This is really quite interesting! …but why? 49 Coronavirus Profile: 120nm • Enveloped • Linear (+)-ssRNA • 32,000bp (largest of all the RNA viruses) • Encodes 14 ORFs **possesses PROOFREADING capabilities (Nsp14 exoribonuclease) SARS-CoV2 aka 2019-nCoV - NSP14 is able to proofread which is important for RNA viruses because it is such a strong strand and you don’t have a second copy - It is there to make sure that propagation and replication is happening for being infectious 50 • Primary translation: Large polyprotein, partly cleaved by 3CL-protease • Secondary translation: additional smaller proteins • Produces its RdRP through a frameshift - Out of the 14 different ORF one of them don’t get produced until the ribosomal frameshift to actually produce RdRP and produces a long polyprotein that needs to be cleaved by the 3 CL protease 51 HOLD UP! This is really quite interesting! …but why? ***Viruses that have the ability to proofread are typically resistant to any of the nucleotide analogues!*** If you are able to proofread the strand you are making, typically you are not seeing it 52 Remdesivir—Mechanism of Action Adenosine monophosphate Remdesivir - The drug gets cleaved at those two points at the carboxylic acid group 53 February 2020 April 2020 Slide Courtesy of Dr. M. Götte - In 2020 they were able to determine the mechanism of action  whether the RNA 54 Favorable Competition with ATP ATP/RDV-TP = 0.35 A R U ATP/dATP = 975 ATP/Favipiravir-TP = 570 ATP/Ribavirin-TP >> 1000 UTP/Sofosbuvir-TP = 1056 Efficiency of incorporation of ATP/RDV-TP = 0.35 Slide Courtesy of Dr. M. Götte - Outcompetes the natural nucleotide and gets incorporated into the strand The relative preference for remdesivir over the other Gets incorporated more easily than the actual ATP By the time it gets into the third space 3 nucleotides later you end up with steric hinderance at S861 which renders the enzyme inactive 55 Delayed Chain-termination at Position i+3 i i+1 i+2 i+3 N R U Slide Courtesy of Dr. M. Götte 56 Steric Clash between Remdesivir and S861 Prevents Translocation into i+4? S861 N R i+4 U Slide Courtesy of Dr. M. Götte 57 Prediction of a Steric Clash Between S861 and Remdesivir Slide Courtesy of Dr. M. Götte Tchesnokov, et al., JBC, 295(47):16156 – 16165 (2021). The gel shows the termination of the transcript 58 Some more proof… Kokic, et al, Nat Commun 12, 279 (2021). https://doi.org/10.1038/s41467-020-20542-0 59 https://static-content.springer.com/esm/art%3A10.1038%2Fs41467-020-20542-0/MediaObjects/41467_2020_20542_MOESM4_ESM.mp4 - It has a proof reading mechanism typically if it is recognizes there is an issue it is going to be first stages - You can get rid of the drug resistance and it gets incorporated in the chain and the subsequent ones 60 Mixed Clinical Data? The data show that remdesivir was superior to placebo in shortening the time to recovery… Remdesivir, hydroxychloroquine, lopinavir, and interferon regimens had little or no effect on hospitalized patients with Covid-19, as indicated by overall mortality… - Showing that remdesivir is better than placebo and shortens the duration of covid - There was no difference in hospitalized patients of COVID 19 or not hospitalized - Lowers the viral load  has to do with the phases of the viral life cycle  the first weeks the virus is active and is the viral phase  subsequent weeks after is the infection phase so by the time and by the time you go to the hospital they are already in inflammatory phase and you start to get antivirals during inflammatory phase it is not going to work  poor oxygenation and edema - Why bother studying about remdesivir? So much has been uncovered, used analogies from different viruses 61 2020 Sep;98:290-293. ePub June 2020 RDV RDV - Remdesivir is all intravenous 62 Baracitinib XXXXXX Molnupiravir Nirmatrevlir (+Rit) 63 SUMMARY (1) Antiviral Targets • • • • • • • Viral attachment i.e., enfuvirtide (HIV); palivizumab (RSV) Cell entry i.e., Amantadine (Influenza) Uncoating Transcription i.e., nucleotide analogues(HBV, HCV, HIV, HSV…) Translation i.e., Darunavir (HIV); rifampin Viral assembly (vaccinia) Viral release i.e., oseltamivir (influenza) • Integration i.e., Dolutegravir (HIV) 64 SUMMARY (2) Acyclovir (and derivatives) • Guanosine analogue • requires 1st phosphorylation by viral TK • life-saving! Saquinavir • Modelled after a peptide transition state • Inhibits HIV’s protease Remdesivir • Adenosine analogue • Important cyano group causing steric hindrance in the RdRP 65 Points to ponder… • Remdesivir not as effective as hoped in the clinical setting. Why? Siddiqi and Mehra, J. Heart Lung Transplant, 2020 39(5):405-407. • A drug either works or it doesn’t; FDA approval doesn’t require mechanism to be known. So why study the mechanism of a drug like Remdesivir? - Human factors Not all viruses have vaccines Studying antivirals because there might be viruses that are emerging For chronic viral infection  some people don’t know how to respond Disease factor  Break through infections like shingles 66 Importance of Antivirals • Viral Factors • Not all viruses have a vaccine or are readily developable (i.e., HIV, Dengue) • Many viruses use similar tactics (may have cross-applications) • Chronic viral infections (i.e., HBV, HIV) • Human Factors • Not everyone is able to mount a response to vaccines (i.e., immunocompromised) • Not everyone is ABLE to get a vaccine (i.e., component allergies, etc) • Not everyone is WILLING to get a vaccine • Disease Factors • Acute illness - Onset of action! • Breakthrough disease despite prior vaccination (i.e., Herpes Zoster) • Accessibility (?) - Vaccines require a particular temperature and its easier to get vaccine to the hospital settings than getting them to more rural areas. 67 Questions? [email protected] 68