Overview
This lecture covers antiviral and antifungal drugs. For antivirals, it uses the
generic viral replication cycle (attachment, penetration, uncoating, gene
expression/genome replication, assembly, release/maturation) as a framework for
classifying drug mechanisms, then works through example agents for HIV,
influenza, herpesviruses, SARS-CoV-2 and hepatitis C. For antifungals, it
covers the clinical classification of mycoses by depth of tissue involvement,
the biological reasons antifungal drugs tend to be more toxic than antibiotics,
and the main antifungal drug classes grouped by target (ergosterol synthesis,
cell membrane, cell wall, nucleic acid synthesis).
Antiviral drug principles and the replication cycle framework
- Antiviral drugs inhibit virus replication by targeting specific viral
proteins; because targets are virus-specific, antivirals are specific to
particular viruses (unlike broad-spectrum antibacterials). - Resistance develops via mutation of the drug’s target protein.
- The viral replication cycle used to organise drug classes: (1)
Attachment, (2) Penetration, (3) Uncoating, (4a) Gene expression / (4b)
genome replication (linked by integration and polyprotein processing), (5)
Assembly, (6) Release and maturation. - Drug classes mapped to steps: attachment blockers act at receptor binding
(1); fusion inhibitors / uncoating inhibitors act at penetration (2) /
uncoating (3); nucleic acid synthesis inhibitors act at gene expression
(4a); integrase inhibitors (HIV) act at integration; protease inhibitors act
at polyprotein processing; NS5A inhibitors (HCV) act at assembly (5);
neuraminidase inhibitors and HIV protease inhibitors act at release/
maturation (6).
The lecture shows a detailed 13-step HIV replication cycle
diagram with drug-target boxes (fusion/CCR5 inhibitors at attachment/fusion,
NRTIs/NNRTIs at reverse transcription, INSTIs at integration, protease
inhibitors at maturation) but explicitly defers detail to separate HIV
lectures — it is presented only as a general reference framework here.
HIV attachment/fusion inhibitors and neutralising antibodies
- Maraviroc (attachment inhibitor): binds CCR5 on the host cell, preventing
gp120 from binding CCR5. - Enfuvirtide (fusion inhibitor): a peptide analogue of the fusion domain
of gp41; inhibits membrane fusion. - Sequence blocked: gp120/CD4 binding to conformational change to gp120/CD4
binding CCR5 (blocked by maraviroc) to gp41-mediated membrane penetration
(blocked by enfuvirtide) to membrane fusion. - Monoclonal neutralising antibodies as attachment inhibitors: bind viral
surface proteins (e.g. SARS-CoV-2 spike) to prevent binding to the entry
receptor (e.g. ACE2), blocking infection. Examples: Evusheld
(tixagevimab + cilgavimab), Ronapreve (casirivimab + imdevimab). - Immune escape: variants can escape monoclonal antibodies. Against later
Omicron subvariants (BA.2, BA.4, BA.5) most tested antibodies (imdevimab,
casirivimab, tixagevimab, cilgavimab, the sotrovimab precursor) showed
markedly reduced or undetectable neutralisation (>50,000 ng/mL, i.e.
effectively no activity). Bebtelovimab lost US emergency-use authorisation
because it does not neutralise Omicron subvariants BQ.1 and BQ.1.1.
Influenza: uncoating and release inhibitors
- Amantadine inhibits uncoating by binding the influenza A M2 protein.
Entry pathway: haemagglutinin-mediated binding to endocytosis to endosomal
acidification (H+ influx through M2) to fusion to uncoating (release of
vRNA). Amantadine blocks the M2-mediated H+ influx step that triggers
uncoating and release of the vRNP. Mutation in M2 causes resistance. - Neuraminidase inhibitors (Oseltamivir/Tamiflu, Zanamivir/Relenza)
prevent release of influenza A and B virions: normally neuraminidase cleaves
sialic-acid-containing receptor to release budding virions for further
rounds of replication; neuraminidase inhibitors bind neuraminidase, block
this cleavage, and prevent virion release, halting replication.
Herpesvirus nucleoside analogues
- Acyclovir: a deoxyguanosine (nucleoside) analogue. Requires activation
by viral thymidine kinase; cellular kinases then add the second and third
phosphates (mono- to di- to triphosphate). Acyclovir triphosphate inhibits
HSV DNA polymerase, blocking HSV DNA synthesis. Active against HSV-1, HSV-2
and VZV. Resistance can arise at either the thymidine kinase activation
step or the DNA polymerase inhibition step. - Ganciclovir: a nucleoside analogue active against cytomegalovirus (CMV,
human herpesvirus 5), which causes disease in immunocompromised hosts.
Activated by CMV phosphotransferase (UL97) to the monophosphate, then
cellular kinases produce the di- and triphosphate forms, which inhibit CMV
DNA polymerase (UL54). Resistance can arise at the UL97 activation step or
the UL54 polymerase step. - Nucleosides are unphosphorylated; nucleotides carry one or more phosphate
groups.
SARS-CoV-2 protease inhibitor (Paxlovid)
- Paxlovid = nirmatrelvir + ritonavir; ritonavir inhibits metabolism of
nirmatrelvir (boosts its levels), it is not itself an antiviral against
SARS-CoV-2 here. - Mechanism: after attachment/entry and translation of viral RNA into
polyprotein chains, the main protease (Mpro) cleaves the polyproteins into
functional viral proteins, feeding into genome replication. Nirmatrelvir
inhibits Mpro at this proteolysis step. - Efficacy: early treatment (within 3-5 days of symptom onset) in unvaccinated
high-risk patients reduced hospitalisation or death by 88% or more (Kaplan-
Meier cumulative incidence at day 28: 0.78% nirmatrelvir/ritonavir, n=1039,
8 events, vs 6.4% placebo, n=1046, 66 events; difference -5.62%, 95% CI
-7.21 to -4.03, P<0.001). - In a later study in vaccinated high-risk patients the benefit was smaller
and not statistically significant (0.95% Paxlovid vs 2.2% placebo, N.S.).
Hepatitis C direct-acting antivirals
- Natural history of untreated HCV over ~30 years: acute infection resolves
in 15-40% or becomes chronic in 60-85%; chronic infection progresses to
cirrhosis in 15-30% of cases, which can lead to death/transplantation or to
hepatocellular carcinoma (1-3% of cirrhotic patients), which can also lead
to death/transplantation. - HCV lifecycle in hepatocytes: virion binds entry receptors (LDLR, SRB1,
CD81, Claudin-1, Occludin) to clathrin-mediated endocytosis to RNA
uncoating/nucleocapsid release to polyprotein translation (with protease
cleavage at the ER membrane, target NS3B/protease inhibitors) to genome
replication in a membranous web (RNA-dependent RNA polymerase, target
NS5B/polymerase inhibitors) to NS5A-dependent replication complex (target
NS5A inhibitors) to assembly at the Golgi to release. - Direct-acting antiviral (DAA) classes: protease (NS3B) inhibitors (e.g.
telaprevir, boceprevir, paritaprevir); polymerase (NS5B) inhibitors, either
nucleotide (e.g. dasabuvir) or non-nucleotide (e.g. sofosbuvir); NS5A
inhibitors (e.g. daclatasvir, ombitasvir). Used in combination to prevent
resistance. - Earlier DAAs mainly targeted genotype 1; newer drugs are pangenotypic. High
cure rates (sustained virologic response, SVR, >90%), shorter treatment
courses (8-24 weeks), but high cost (~US$40,000-100,000 per course). - Pharmac-funded combinations: Maviret (pangenotypic) = glecaprevir (NS3B) +
pibrentasvir (NS5A); Harvoni (genotypes 1, 3, 4) = ledipasvir (NS5A) +
sofosbuvir (NS5B nucleotide inhibitor).
Classification of fungal infections
- Fungi are broadly divided morphologically into yeasts (budding round
cells, smooth/creamy colonies) and moulds (branching filamentous
structures with spore-bearing heads, fuzzy colonies). - Superficial mycoses: confined to the surface of skin/hair shaft, e.g.
seborrhoeic dermatitis, dandruff. - Cutaneous mycoses: involve skin, hair, nail or mucosa, e.g. dermatophytosis
(ringworm/tinea), candidiasis (thrush). - Subcutaneous mycoses: chronic, localised infections of skin and
subcutaneous tissue from traumatic implantation of soil saprophytes, e.g.
sporotrichosis, chromoblastomycosis, mycetoma. - Dimorphic systemic mycoses: caused by dimorphic fungi (mould form in the
environment, yeast form in the body), geographically restricted, primary
site of infection is pulmonary. - Opportunistic systemic mycoses: occur in immunocompromised patients, caused
by cosmopolitan fungi of very low intrinsic virulence, e.g. invasive
candidiasis, invasive aspergillosis, zygomycosis.
Antifungal drug targets and the toxicity problem
- Fungi are eukaryotes, and are more similar to human cells than bacteria are
(metabolically similar to human cells), so drugs that inhibit or kill fungi
tend to also be toxic to humans, giving antifungals a low therapeutic
index. - Exploitable differences from human cells are in cell membrane composition
and the presence of a cell wall. - Fungal cell wall/membrane structure (outer to inner): mannoproteins, then
beta-(1,6)- and beta-(1,3)-glucan, then chitin, then the phospholipid
bilayer of the cell membrane. - Key antifungal drug targets: the ergosterol synthesis pathway (via
squalene) in the membrane, beta-(1,3)-glucan synthase (cell wall
synthesis), and fungal DNA/RNA synthesis. - The key structural difference exploited by membrane-active antifungals is
that human cell membranes contain cholesterol while fungal cell membranes
contain ergosterol instead.
Antifungal drug classes
- Polyenes, e.g. Amphotericin B (introduced 1958): has greater avidity
for ergosterol than for cholesterol; binds ergosterol in the fungal
membrane, intercalates and forms pores, causing leakage of intracellular
cations (Ca2+, Na+, K+) and membrane disruption. - Ergosterol biosynthesis pathway (simplified): acetyl-CoA to farnesyl
pyrophosphate to squalene (Erg9) to squalene epoxide (Erg1, “squalene
monoxygenase,” inhibited by allylamines e.g. terbinafine) to lanosterol
(Erg7) to a 14-alpha-methylated sterol intermediate (Erg11, 14-alpha-
demethylase, inhibited by azoles e.g. fluconazole; this and downstream
steps also inhibited by morpholines e.g. fenpropimorph, amorolfine) to
further steps (Erg24, Erg2, Erg3, Erg5) to ergosterol (Erg4). - Azoles: deplete membrane ergosterol and impair membrane fluidity by
inhibiting 14-alpha-demethylase (Erg11), which also causes accumulation of
toxic 14-alpha-methylated sterols (in part via diversion through Erg3).
Imidazoles (e.g. miconazole) are for topical use only. Triazoles:
fluconazole has activity against yeasts but not moulds; itraconazole has
improved activity against moulds. - Azole resistance arises by mutation of 14-alpha-demethylase (causing
cross-resistance) or by overexpression of efflux pumps: MDR1
overexpression causes fluconazole resistance, and CDR1/CDR2 efflux pump
overexpression causes cross-resistance to azoles generally. - Allylamines, e.g. Terbinafine: inhibit ergosterol synthesis by
inhibiting squalene monoxygenase (Erg1). Concentrates in skin and nail
beds with relatively low bloodstream concentration, so use is restricted
to onychomycosis and cutaneous fungal infection. - Echinocandins, e.g. Caspofungin: target the fungal cell wall by binding
beta-(1,3)-D-glucan synthase, inhibiting synthesis of beta-(1,3)-D-glucan
polymers, a key structural cross-linking component of the cell wall in
some fungi; the resulting glucan-depleted wall is susceptible to osmotic
lysis. - Flucytosine (5-FC): selectively taken up by fungus-specific cytosine
permease and converted by fungus-specific cytosine deaminase to cytostatic
5-fluorouracil (5-FU) inside the fungal cell. 5-FU is then phosphorylated
to FdUMP, which inhibits thymidylate synthase (blocking DNA synthesis), and
separately to FUTP, which is incorporated into RNA in place of uracil,
causing RNA miscoding and inhibiting protein synthesis. Mutations in either
uptake enzyme cause resistance.
Self-test
- Describe the six steps of the generic viral replication cycle used in this
lecture to classify antiviral drug classes. - Distinguish the mechanisms of maraviroc and enfuvirtide as HIV entry
inhibitors. - Explain why monoclonal neutralising antibodies can lose efficacy against
new viral variants, using the Omicron subvariant example. - Describe how amantadine blocks influenza uncoating.
- Explain how neuraminidase inhibitors stop influenza virus spread from an
infected cell. - Describe the activation and mechanism of action of acyclovir, including
the step at which resistance can arise. - Distinguish acyclovir’s target virus and activation enzyme from
ganciclovir’s. - Describe how Paxlovid works and what its two components each contribute.
- What did the two Paxlovid trials described in the lecture find, and how did
vaccination status affect the result? - Describe the hepatitis C viral lifecycle and name the three DAA drug
classes with the step each blocks. - Why are DAA combinations used rather than a single agent for hepatitis C?
- Distinguish superficial, cutaneous, subcutaneous, dimorphic systemic and
opportunistic systemic mycoses, with an example of each. - Explain why antifungal drugs tend to have a lower therapeutic index than
antibacterial drugs. - Describe the mechanism of action of amphotericin B and why it targets
fungal but not human cells. - Describe how azoles impair fungal cell membranes and give one mechanism
of azole resistance. - Distinguish the antifungal targets of terbinafine, echinocandins and
flucytosine. - A patient develops onychomycosis. Which antifungal drug class would be
appropriate and why, based on its pharmacokinetic properties? - Compare how antiviral drugs and antifungal drugs each exploit differences
between the pathogen and the human host cell to achieve selective
toxicity.
Answers
Reveal answers
- Attachment, penetration, uncoating, gene expression (4a) and genome
replication (4b, linked via integration and polyprotein processing),
assembly, and release/maturation.- Maraviroc binds host-cell CCR5, blocking gp120 from binding CCR5 (an
attachment inhibitor); enfuvirtide is a peptide analogue of gp41’s
fusion domain that blocks the membrane fusion/penetration step.- Antibodies bind specific epitopes on the viral surface protein (e.g.
spike); mutations in that protein in new variants can prevent antibody
binding. Against later Omicron subvariants (BA.2, BA.4, BA.5) most
tested antibodies lost neutralising activity, and bebtelovimab lost its
US authorisation because it could not neutralise BQ.1/BQ.1.1.- Amantadine binds the influenza A M2 protein, blocking the H+ influx
through M2 in the endosome that is needed to trigger uncoating and
release of the vRNP; mutation of M2 causes resistance.- Neuraminidase normally cleaves the sialic-acid receptor holding new
virions to the cell surface. Neuraminidase inhibitors bind neuraminidase
and block this cleavage, so new virions cannot be released to infect
further cells.- Acyclovir (a deoxyguanosine analogue) is first phosphorylated by viral
thymidine kinase to the monophosphate, then by cellular kinases to the
di- and triphosphate; the triphosphate inhibits HSV DNA polymerase,
blocking HSV DNA synthesis. Resistance can arise at either the
thymidine kinase step or the DNA polymerase step.- Acyclovir targets HSV-1, HSV-2 and VZV and is activated by viral
thymidine kinase; ganciclovir targets cytomegalovirus and is activated
by CMV phosphotransferase (UL97), then inhibits CMV DNA polymerase
(UL54).- Paxlovid combines nirmatrelvir, which inhibits the SARS-CoV-2 main
protease (Mpro) and so blocks cleavage of viral polyproteins into
functional proteins, with ritonavir, which inhibits the metabolism of
nirmatrelvir to keep its levels effective.- In unvaccinated high-risk patients treated within 3-5 days, Paxlovid
reduced hospitalisation/death by 88% or more (0.78% vs 6.4% at day 28,
P<0.001). In a later study in vaccinated high-risk patients the benefit
was much smaller and not statistically significant (0.95% vs 2.2%).- Virion binds entry receptors (LDLR, SRB1, CD81, Claudin-1, Occludin),
enters by endocytosis, uncoats, and its RNA is translated into a
polyprotein cleaved by a protease (NS3B, target of protease
inhibitors); the genome is replicated by an RNA-dependent RNA
polymerase (NS5B, target of polymerase inhibitors) in a replication
complex organised by NS5A (target of NS5A inhibitors); the virus is
then assembled at the Golgi and released.- Combining DAA classes with different targets reduces the chance that
resistance mutations to all drugs arise together, preventing treatment
failure from resistance.- Superficial: skin surface/hair shaft, e.g. seborrhoeic dermatitis.
Cutaneous: skin, hair, nail, mucosa, e.g. tinea, thrush. Subcutaneous:
traumatic implantation of soil saprophytes into skin/subcutis, e.g.
sporotrichosis. Dimorphic systemic: geographically restricted dimorphic
fungi, pulmonary primary site. Opportunistic systemic: low-virulence
cosmopolitan fungi causing disease in immunocompromised hosts, e.g.
invasive aspergillosis.- Fungi are eukaryotes that are metabolically and structurally more
similar to human cells than bacteria are, so drugs that damage fungal
cells often also damage human cells, giving a low therapeutic index.- Amphotericin B has greater avidity for ergosterol than cholesterol; it
binds ergosterol in the fungal membrane, intercalates and forms pores,
causing cation leakage. It preferentially targets fungal cells because
their membranes contain ergosterol rather than the cholesterol found
in human membranes.- Azoles inhibit 14-alpha-demethylase (Erg11), depleting ergosterol,
impairing membrane fluidity and causing accumulation of toxic
14-alpha-methylated sterols. Resistance can arise from mutation of
14-alpha-demethylase or from overexpression of efflux pumps (MDR1 for
fluconazole resistance; CDR1/CDR2 for cross-resistance).- Terbinafine inhibits squalene monoxygenase (Erg1) in ergosterol
synthesis; echinocandins inhibit beta-(1,3)-D-glucan synthase in the
cell wall; flucytosine is converted intracellularly to 5-FU, which
inhibits thymidylate synthase (blocking DNA synthesis) and causes RNA
miscoding.- Terbinafine, because it concentrates in skin and nail beds with
relatively low bloodstream levels, making it suited to onychomycosis
while limiting systemic exposure.- Antivirals exploit virus-specific proteins (e.g. viral thymidine
kinase, viral protease, viral polymerase) that either do not exist in
human cells or differ enough from human enzymes to allow selective
inhibition. Antifungals exploit differences in fungal cell membrane
composition (ergosterol vs cholesterol) and the fungal cell wall
(absent in human cells), though because fungi are otherwise similar to
human cells this selectivity is much less complete, giving antifungals
a lower therapeutic index than most antivirals.