(5/2) Che Lecture: Medicinal Chemistry of Antiviral Agents I Drug Discovery and Development: A multifaceted, complicated, and lengthy process [Idea….12-15 years….Drug] When searching for new antiviral drugs, it is critical to understand the molecular biology and life cycle for the virus of interest. While it may difficult to produce candidate drugs, identifying targets of the virus for intervention will hasten the process. Common targets for viral agents include: Viral adsorption, Cell entry, Uncoating, Viral DNA/RNA synthesis, Assembly, Release

Developing a Candidate - Random Design: A total guess. Expensive, laborious… go play the Illinois Lottery while you’re at it - Analog Approach: A ‘Me2’ approach, modifying the structure of known drugs to apply its feature to your virus - Bio-structural approach: If the structure/active site is known, design a drug relative to that structure - Isolation from natural resources: Finding your drug under a rock or in a pile of dung in some Greenland glaciers Screening for Candidates - High-throughput In Vitro Screens: Cell-based, enzyme-based, receptor-based - Animal Models: If the in vitro results are promising, sure let’s try it out Identify leads, modify them, learn their mechanism, and optimize them to lead molecules.

Challenges in developing antiviral agents - Knowledge to the molecular level – is needed to define targets - Side effects – are common in antivirals, because it is difficult to target intracellular growth! - Resistance – is common due to the high tendency for mutation. This is a major drawback - Dangerous – viruses are more difficult to test in model systems - Labor intensive and expensive – drug design processes like high-throughput screening often prohibit development - Narrow window of time – is available to respond with treatment to viral infections

Nucleoside Analogs

- Structure: Nucleotide base + Sugar (ribose/deoxyribose) + Modifications - Prodrug Activation: Compounds of this class are prodrugs, requiring activation via a series of 3

metabolic phosphorylations by kinases at the hydroxy group of the sugar to the triphosphate form. - MoA: The triphosphates are incorporated into the growing DNA strand, but the modifications result in

chain termination. o Specificity: NOT Specific to viral DNA. They also affect mDNA of the mitochondria, \

resulting in toxicities. We currently do not have agents specific to viral DNA - PK: Being nucleotide mimics, nucleoside analog drugs are natural substrates for catabolic enzymes to

chop their glycosidic bond, therefore resulting in a short half-life and poor bioavailability.

Idoxuridine (IDU): [Thymine]-Based Nucleoside Analog - The first antiviral drug! (1963) • Antiviral Type: 5-iodo-substituted Nucleoside Analog • MoA: Idoxuridine is phosphorylated by thymidine kinase, thymidine monophosphate kinase, and thymidine diphosphate kinase in succession to the active triphosphate – which may then be added to the growing DNA strand and halt viral DNA synthesis. o Interruption of this pathway by Thymidylate synthase may occur to the monophosphate, which removes

the iodine and inactivates the drug

Edoxuridie and Brivudine: [Thymine]-Based Analog: Modified Nitrogenous Base Improved Specificity: Edoxudine (I- à Ethyl) and Brivudine (I- à bromovinyl) exhibit higher affinity for the viral thymidine kinase than for the cellular enzyme

o Improved bioavailability: Replacing the iodo- with strong-EWG (-F, CF3, -NO2) function to inhibit thymidylate synthase to provide protection from deactivation

Emtricitabine and Lamivudine: [Cytosine]-Based Analog: Modified Sugar Structure

- Improved Activity: Emtricitabine (HIV) and Lamivudine (HIV + HBV) - Structural Changes: Replacement of 3’-C with a Sulfur changes the deoxyribose to a 1,3-oxathiolane - MoA: These compound are reverse transcriptase inhibitors (NRTI) which, upon incorporation into the

growing DNA strand, immediately terminate DNA synthesis. Lacking a 3’-OH, formation of the essential 5’ to 3’ phosphodiester linkage is prevented

Entecavir: [Guanine]-Based Analog: Modified Sugar Structure - Structural Changes: Replacement of the furanose oxygen with a methylene - Improved Activity: Utilization of a carbocyclic ring allows the analog to overcome the degradation

issue faced by normal nucleosides. By switching out the oxygen, entecavir escapes hydrolytic degradation via glycosidic bond cleavage. Like the other analogs, entecavir is still converted intraceullarly to the triphosphate

o Improved Specificity: Through its competition with dGTP, entecavir inhibits HBV polymerase and is only a weak inhibitor of cellular DNA

- Indication: Used in the treatment of Chronic HBV Acyclovir, Ganciclovir, and Penciclovir: [Guanine]-Based Analogs: Modified Sugar Structure

- Structure: These compounds incorporated extensive modifications, although maintaining the essential functional groups (1) Hydroxy group ~sugar-like, (2) Nucleobase, (3) Substituted at N-9

- Acyclovir (ACV) – Clinically and Historically significant, it started a new era of antiviral therapy (Elion) o Indication: Herpes viruses, varicella zoster, cytomegalovirus o Structure: Guanine derivative with an opened chain (removal of 2’,3’-C)

§ Hydroxy Group is required for activation by phosphorylation o MoA: Exerts high selectivity towards viral DNA Polymerase, greater efficacy and lower

toxicity. It requires metabolism by the viral thymidine kinase for addition of the first phosphate group, followed by host cell-mediated phosphorylation to the triphosphate. Absence of 3’-OH leads to chain termination

(5/4) Che Lecture: Medicinal Chemistry of Antiviral Agents II Big Theme: Knowing the Crystal Structure – Designing drugs based on the Structure-Activity Relationships (SAR)

- X-Ray Structure is a pre-requisite for any rational drug design. You need to know your target Prodrug Strategy: A biologically inactive derivative of a parent drug molecule that requires a chemical or enzymatic transformation within the body to convert to the active form. Using medicinal chemistry we can use prodrugs to enhance bioavailability through: • Enhancing intestinal absorption • Improving stability and solubility • Facilitating cell penetration •

- Ideal Prodrug o To be an ideal prodrug, the molecule should be inactive against pharmacological targets, chemically

stable across a pH range, highly soluble in aqueous environments, readily absorbed from the GI tract, resistant to hydrolysis during the absorption phase, and rapidly broken down to yield the active component post-absorption.

- Types of Prodrugs commonly seen in antivirals o Ester Analogs: Ester group provides enhanced lipophilicity to enhance absorption and cell penetration

§ Activation: Converted by esterases via hydrolysis to the active acid form § Example: Valine Ester of Acyclovir = Valacyclovir

Diacetyl esters of Penciclovir = Famciclovir (+ Xanthine Oxidase) o Nucleotide Analogs: Monophosphorylated and covered in protective R groups to cross the membrane

§ Activation: Once intracellular, enzymes remove the protecting groups and the phosphorylation cascade to the active triphosphate begins. The role of the protecting groups is to mask the polar O- of the phosphate groups

§ Example Tenofovir alafenamide/disoproxil à metabolized to Tenofovir Ribavirin Modification

- Once upon a time there was a guanosine ribonucleic analog named Ribavirin. Ribavirin was used to treat HepC, but what Ribavirin didn’t know was that it was also causing anemia as an adverse effect. So Rivavirin had its carboxamide switched to a carboxamidine and it was no longer toxic. It even had the benefit of attaining a new liver-targeting property and enhanced safety profile. The new drug was called Viramidine, happily ever after

Influenza (RNA Virus) - Antiviral Therapy: Currently, there is only one active class of

anti-influenza drugs being used called the à Neuraminidase Inhibitors (Zanamivir, Oseltamivir, Perimivir)

- Pathology: Following infection and replication, but prior to budding off, the viral particle must cleave to detach itself from the host cell. Viral neuraminidase on the virion membrane breaks the glycosidic bond at the sialic acid residue of the host-cell receptor.

- Neuraminidase Inhibitor MoA: By mimicking the structure of Sialic acid, neuraminidase inhibitors prevent detachment from the host cells, and therefore prevent the spread of the virus within the host, as it cannot dislodge.

- Oseltamivir (Tamiflu): The most clinically relevant influenza drug today, it was designed based off of the crystal structure of the neuraminidase-sialic acid complex that indicated sialic acid’s carboxylate interacted with key AA in the neuraminidase active site. Oseltamivir is considered a prodrug, once it’s ethyl ester (red circle) is cleaved for form the carboxylate (COO-), it then engages key Arg-residues, among other important hydrophilic interactions while in a conformation similar to the transition state.

Targets for Anti-Retroviral Therapy in HIV • Nucleoside Reverse Transcriptase Inhibitors (NRTI)

- MoA: Nucleoside analogs acting as prodrugs, requiring metabolic activation to the triphosphate to become active. Once the triphosphate is incorporated into the viral DNA, the lack of an available 3’-OH causes chain termination

- Zidovudine (AZT): The first HIV tx approved in the US, it is a simple nucleoside analog of Thymidine, which has the 3’-OH replaced with an Azido (N3) group. Like all other NRTI, it requires activation to the triphosphate for activity and chain termination.

o So yea, originally studied for antitumor properties, ended up working for HIV. Cool - So we have a buncha of nucleoside analogs, pyrmidine and purine-based analogs. But we also

have a nucleotide analog, mentioned previously briefly above – tenofovir is monophosphorylated… ooh ahh. But yea it still has the same fate relax

• Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTI) - The NNRTIs are a structurally diverse class of RT-inhibitors, but they are similar in that they are all designed to

fit into the RT active site. Recall, using X-Ray Crystallography, we have identified the active site and ‘essential’ AA residues. Therefore, we can simply design drugs to maximize interactions within the active site

- Efavirenz (EFV): EFV is the final product of a long series of trial and error. It was designed after the ‘essential’ AA of the active site and uniquely includes many points of hydrophobic contact

• Protease Inhibitors (PI) - Good News – We know the crystal structure of the HIV Protease – so we can direct our drug design - PI Structure Types – While they are categorized by type, both mimic the substrate transition state

(1) Peptide-Like ‘Peptidomimetics’ - Atazanavir – Boom, the magic of using SAR (2) Non-Peptidal - Darunavir – A 2nd generation PI that was praised for having the most interactions

with the active site of the HIV protease. Tightly anchored into the active site • Integrase Inhibitors (INSTI)

- Good News – We know the crystal structure of the HIV Integrase – so we can direct our drug design. In fact, by doing our homework we had determined that there were critical Mg2+ and Mn2+ cofactors located in the active site pocket. Using this information, we designed drugs to chelate the Mg2+. Once chelated, the enzyme is inactive

Drugs of this class share two main similarities in their structure • 3 Oxygens in close proximity that function to chelate the Mg2+ • An Aromatic Ring used to establish hydrophobic interactions within the active site à This hydrophobic structure anchors the molecule onto the active site MoA: And therefore, these INSTI drugs function to inhibit the insertion of the viral DNA into the human genome. There are 3 drugs in this class: - Dolutegravir, Elvitegravir, and Raltegravir

• Entry Inhibitors - Good News – We know the structure and functionality of the gp120 and gp41 proteins of the HIV viral envelope

o gp120: Binds to the CD4 receptor of the T Cell, causing a change in confirmation that improves its affinity for and brings it closer to the CCR5 coreceptor. gp120 binds to CCR5 (or CXCR4 in resistant cells)

o gp41: Closely associated with gp120, gp41 penetrates the cell membrane and promotes fusion - Enfuviritide: A 36AA peptide ‘fusion inhibitor’ that partially mimics the gp41 structure, and displaces the viral

transmembrane gp41 and binds to gp41 to prevent conformational change related to fusion activity - Maraviroc: As it turns out, not only do we know the structure of the HIV virus, we also know our own structures.

The CCR5 coreceptor structure is known, and Maraviroc was designed to be its antagonist, hence blocking gp120 from associating with CCR5, hindering entry into the cell.

All of this is quite remarkable, so perhaps we should celebrate. While we’ve had great victories in combating viral infections, so far we only have developed drugs for 9 types of viral infections, most of which are focused on inhibiting replication. As a result, preventative vaccination remains the key to global control of viral infections.

- Take Home Point: Without X-Ray Crystallography technology, as pharmacists we would not have as many drugs to play with. So take the time to thank the medicinal chemists. Or better yet just study physics. Or go above and beyond and read the original Cosmos by Carl Sagan – just as awe-inspiring.

BEST OF LUCK!!!! Remember, the test will be cumulative. Likely only the basics for the past material, more intense for this section. If you want the exam I, II, or III lecture reviews I made – sure à (posen2@uic.edu) Below is the content I was going to use to make a practice test – my guess was that these would be questions on the actual test. But I do not have any time to make that. Peace Wenzler: Suspect GAS necrotizing fasciitis (needs the antitoxin thing) Wenzler: Recurrent ABSSSI Purulent – cultures or abx or i+d what is correct combo Wenzer: Bite from a dog, what is the best abx Schriever: Matching to one of the 4 criteria of emerging disease (or the purpose of IDPH reporting requirements) Shriever: ZIka – go to endemic area, advice patient’s family to wait X months (6)before trying to have a baby Bulman: Bone biopsy is gold standard. After a + probe to bone test, do a bone biopsy, don’t tx yet Bulman: Vertebral Osteomyelitis, bone biopsy and sensitivities show MRSA, TX and 8+ weeks therapy Bulman: mild diabetic foot infection Max: Which of the following is not a mode by which HIV is transmitted Max: Primary reason for HIV tx failure (infection, drug drug interactions, adherence, resistance) – adherence Max: Drug Resistance testing, when should we do drug ressitance testing (all of the above) Max: TDF vs TAF Max: PreP Max: What would be an appropriate option for HBV co-treatment Briars: Appropriate rationale for using abx therapy would be : Seveirty – higher temperature and higher pain Briars: Severe infection, PCN allergy, fail augmentin therapy, what do? (ceftriaxone IM qdaily x 3days) Briars: Duration of therapy Smith: Late to take your drug therapy, not terrible because you are on a protease inhibitor regimen Smith: Cross resistance EVG and RAL, slide 20 Smith: Unable to use INSTI, must use PI-based with NRTI, what one to use.