Antiviral Drugs

Structure of viruses

structure of the influenza virus

•DNA-viruses: poxviruses, herpes, adenoviruses,
papillomaviruses.

Contain mostly double-stranded DNA, a small number
single-stranded DNA.

DNA viruses enter the cell nucleus and direct the
generation of new viruses. 


•RNA-viruses: influenza, measles, mumps, cold,

meningitis, poliomyelitis, retroviruses (AIDS, T-cell
leukemia), arenaviruses.

Contain largely single-stranded RNA (ssRNA).

RNA viruses do not enter the cell nucleus (except the
influenza virus).

RNA retroviruses uses the viral reverse transcriptase to
make a DNA copy of the viral RNA, which is then
integrated into the host genome.
Types of viruses
 Types of important viruses
Table 2 Classification of animal viruses with DNA genomes
Virus family Capsid shape Envelope Virion size (nm) DNA DNA length (kbp) Example
Andenoviruses Icosahedral No 80 ds 36 Human adenovirus
Herpesviruses Icosahedral Yes 200 ds 130–230 Herpes simplex virus
Poxviruses Complex Yes 300 ds 130–280 Smallpox virus
Papovaviruses Icosahedral No 45–60 ds 5–8 Human papillomavirus
Parvoviruses Icosahedral No 22 ss 5 kb Canine parvovirus
Hepadnaviruses Icosahedral Yes 42 ds 3 Hepatitis B virus
ds, double stranded; ss, single-stranded.

Table 3 Classification of selected animal viruses with RNA genomes

Virus family Capsid shape Envelope Virion size (nm) RNA RNA length (kb) Example
Picornaviruses Icosahedral No 27 ss; pos 7 Poliovirus
Togaviruses Icosahedral Yes 70 ss; pos 12 Equine encephalitis
virus
Flaviviruses Icosahedral Yes 45 ss; pos 11 Yellow fever virus
Bunyaviruses Helical Yes 100 ss; neg 14 Hantavirus
Orthomyxoviruses Helical Yes 100 ss; neg 14 Influenza virus
Paramyxoviruses Helical Yes 120–250 ss; neg 15 Measles virus
Rhabdoviruses Helical Yes 80 × 180 ss; neg 11 Rabies virus
Filoviruses Helical Yes Long filaments ss; neg 13 Ebola virus
Retroviruses Icosahedral Yes 90 ss; pos 9 HIV
Reoviruses Icosahedral Yes 80 ds 23 kbp Human rotavirus
HIV, human immunodeficiency virus; ds, double-stranded; ss, single-stranded; neg, negative; pos, positive.
 Virus Structure
Rabiesvirus
(Tollwut)

Poxvirus

Mumpsvirus
Poxvirus

T-virus Coliphage Herpes Simplex

Lambda

Adenovirus HIV Influenzavirus

upper respiratory infections

Tobacco Mosaic Simian Rhino

 The structure of viruses

tobacco virus

Herpes simplex

capsid
 Some disturbing figures about AIDS...(2013)
AIDS infected in 2000
In 2015, it was estimated that
36.7 million people lived with the
disease worldwide, and that AIDS
killed an estimated 1.1 million
people in 2015, including 210,000
children

Lifetime risk of dying of AIDS for 15-year-old boys

Regional HIV and AIDS statistics and features 2012
Adults and children Adults and children Adult prevalence Adult & child
living with HIV newly infected with HIV (15 49) [%] deaths due to AIDS

Sub-Saharan Africa 25.0 million 1.6 million 4.7% 1.2 million

[23.5 million – 26.6 million] [1.4 million – 1.8 million] [4.4% – 5.0%] [1.1 million – 1.3 million]

Middle East and North Africa 260 000 32 000 0.1% 17 000
[200 000 – 380 000] [22 000 – 47 000] [0.1% – 0.2%] [12 000 – 26 000]

South and South-East Asia 3.9 million 270 000 0.3% 220 000
[2.9 million – 5.2 million] [160 000 – 440 000] [0.2% – 0.4%] [150 000 – 310 000]

East Asia 880 000 81 000 <0.1% 41 000

[650 000 – 1.2 million] [34 000 – 160 000] [<0.1% – 0.1%] [25 000 – 64 000]

Latin America 1.5 million 86 000 0.4% 52 000

[1.2 million – 1.9 million] [57 000 – 150 000] [0.3% – 0.5%] [35 000 – 75 000]

Caribbean 250 000 12 000 1.0% 11 000

[220 000 – 280 000] [9400 – 14 000] [0.9% – 1.1%] [9400 – 14 000]

Eastern Europe and Central Asia 1.3 million 130 000 0.7% 91 000
[1.0 million – 1.7 million] [89 000 – 190 000] [0.6% – 1.0%] [66 000 – 120 000]

Western and Central Europe 860 000 29 000 0.2% 7600

[800 000 – 930 000] [25 000 – 35 000] [0.2% – 0.2%] [6900 – 8300]

North America 1.3 million 48 000 0.5% 20 000

[980 000 – 1.9 million] [15 000 – 100 000] [0.4% – 0.8%] [16 000 – 27 000]

Oceania 51 000 2100 0.2% 1200

[43 000 – 59 000] [1500 – 2700] [0.2% – 0.3%] [<1000 – 1800]

TOTAL 35.3 million 2.3 million 0.8% 1.6 million

[32.2 million – 38.8 million] [1.9 million – 2.7 million] [0.7% - 0.9%] [1.4 million – 1.9 million]

The ranges around the estimates in this table define the boundaries within which the actual numbers lie, based on the best available information.
Adults and children estimated to be living with HIV 2012

Western & Eastern Europe

Central Europe & Central Asia
860 000 1.3 million
North America [800 000 – 930 000] [1.0 million – 1.7 million]
1.3 million East Asia
[980 000 – 1.9 million] 880 000
Middle East & North Africa [650 000 – 1.2 million]
Caribbean 260 000
250 000 [200 000 – 380 000] South & South-East Asia
[220 000 – 280 000] 3.9 million
Sub-Saharan Africa [2.9 million – 5.2 million]
Latin America 25.0 million
1.5 million [23.5 million – 26.6 million] Oceania
[1.2 million – 1.9 million] 51 000
[43 000 – 59 000]

Total: 35.3 million [32.2 million – 38.8 million]

Estimated adult and child deaths from AIDS 2012

Western & Eastern Europe

Central Europe & Central Asia
7600 91 000
North America [6900 – 8300] [66 000 – 120 000]
20 000 East Asia
[16 000 – 27 000] 41 000
Middle East & North Africa [25 000 – 64 000]
Caribbean 17 000
11 000 [12 000 – 26 000] South & South-East Asia
[9400 – 14 000] 220 000
Sub-Saharan Africa [150 000 – 310 000]
Latin America 1.2 million
52 000 [1.1 million – 1.3 million] Oceania
[35 000 – 75 000] 1200
[<1000 – 1800]

Total: 1.6 million [1.4 million – 1.9 million]

About 6,300 new HIV infections a day in 2012

About 95% are in low- and middle-income countries

About 700 are in children under 15 years of age

About 5,500 are in adults aged 15 years and older, of whom:

almost 47% are among women
about 39% are among young people (15-24)
 Time course of the HIV infection

•Once HIV has killed so many CD4+ T cells that there are fewer than 200 of these cells per microliter of blood,
cellular immunity is lost. Acute HIV infection progresses over time to clinical latent HIV infection.
•In the absence of antiretroviral therapy, the median time of progression from HIV infection to AIDS is nine to
ten years, and the median survival time after developing AIDS is only 9.2 months
Scanning electron micrograph of HIV-1
budding from a cultured lymphocyte
The HIV virus structure and the HIV life cycle
 Structural Biology of HIV-1 Summers, J. Mol.
Biol. 285 (1999),1-32
SU gp120 NC nuclear capsid

MA matrix protein

CA capsid protein

TM (ectodomain)
membrane anchor
of gp160

IN integrase

RT reverse
transcriptase

PR protease
 The various stages during viral infection
HIV
HIV gp120
RNA

•Attachment of the virus to the host cell membrane via binding

Capsid core
Reverse
transcriptase
of molecules of the outer surface of the virion to a receptor
Fusion
Virus adsorption inhibitors

molecule on the host cell (protein or carbohydrate)

Integrase

Virus–c

•Penetration and uncoating of the virus and subsequent

Co-receptor
antagonists

CD4 receptor and

release of the virions into the host cell. Some viruses inject their
CXCR4 or CCR5
co-receptor proteins Uncoating material, others enter the cells intact (via endosomal entry) and
Integrase
Reverse
Reverse
transcriptase
transcription inhibitors
are uncoated inside.
inhibitors
DNA

•Retroviruses (e.g. HIV):reverse transcription of viral RNA into

(NRTIs,
NtRTIs,
NNRTIs)
Integration
(Strand transfer) DNA

Transcription
•Integration of the viral DNA into the host cell genome
CD4+
•Some viruses (e.g. HIV) use the cellular system for gene
T cell duplication and translation, while others (herpes) uses their own
system to produce mRNA coding for viral proteins (early genes)
Translation

Polypeptide • Synthesis and assembly of nucleocapsids (late genes):

Protease Proteolytic processing Synthesis of capsid proteins that self-assemble to form the
inhibitors by viral protease
capsid.
Viral proteins and
RNA assemble at
the cell membrane
• Virion release: Release of the naked virions by cell lysis, where
Budding
the cell is destroyed. Alternatively, the viruses with envelopes
can be released by a process known as budding, in which the
nucleocapsid is wrapped by the membrane and pinched off.
 Viral Entry
enveloped viruses non-enveloped viruses
 Virus receptors

Host cell receptor Virus

CD4 glycoprotein HIV

CCR5, MCP-1, RANTES HIV

chemokine receptor CXCR4 for

HIV
cytokine SDF-1

acetylcholine receptor rabies virus

complement C3d of B
glandular fever virus
lymphocytes

IL-2 receptor on T-lymphocytes T cell leukemia viruses

beta-adrenoreceptor infantile diarrhea viruses

MHC molecules adenoviruses; T-cell leukemia v.

Cell-Exit of Enveloped Viruses
Strategies for Antiviral Drug Development

• inhibition of virus adsorption

• inhibition of virus-cell fusion
• inhibition of the HIV integrase (HIV)
• inhibition of viral DNA or RNA synthesis
– inhibitors of viral DNA polymerase
– inhibitors of the reverse transcriptase
– acyclic nucleoside phosphonates
• viral protease inhibition
• viral neuraminidase inhibition (influenza)
• inhibition of IMP dehydrogenase
• inhibition of S-adenosylhomocysteine hydrolase
 Targets for Antiviral Drugs
Approach Target virus(es) Compounds approved Selected compounds in development
for the indicated target virus
Virus adsorption inhibitors HIV, HSV, CMV, RSV and Polysulphates, polysulphonates,
other enveloped viruses polycarboxylates, polyoxometalates,
chicoric acid, zintevir, cosalane
derivatives, negatively charged albumins
Virus–cell fusion inhibitors HIV, RSV and other HIV: AMD3100, TAK779 and T20
paramyxoviruses derivatives
Viral DNA polymerase Herpesviruses (HSV-1, -2, Acyclovir, valaciclovir, ganciclovir, Bicyclic furopyrimidine nucleoside
inhibitors VZV, CMV, EBV, valganciclovir, penciclovir analogues, A5021, cyclohexenylguanine
HHV-6, -7, -8) famciclovir, brivudin*, foscarnet
Reverse transcriptase HIV NRTIs: zidovudine, didanosine, Emtricitabine, amdoxovir
inhibitors zalcitabine, stavudine, lamivudine‡,
abacavir
NNRTIs: nevirapine, delavirdine, Emivirine, UC781, DPC083,
efavirenz TMC125 (R165335)
Acyclic nucleoside DNA viruses (polyoma-, CMV: cidofovir HBV: adefovir
phosphonates papilloma-, herpes-, adeno- HIV: tenofovir
and poxviruses), HIV, HBV
Inhibitors of processes associated HIV, HCV
with viral RNA synthesis
Viral protease inhibitors HIV, herpesviruses, HIV: saquinavir, ritonavir, indinavir, HIV: atazanavir, mozenavir, tipranavir
rhinoviruses, HCV nelfinavir, amprenavir, lopinavir Human rhinovirus: AG7088
Viral neuraminidase inhibitors Influenza A and B virus Zanamivir, oseltamivir § RWJ270201
IMP dehydrogenase inhibitors HCV, RSV Ribavirin|| Mycophenolic acid, EICAR, VX497
S-adenosylhomocysteine (–)RNA haemorrhagic fever
hydrolase inhibitors viruses (for example, Ebola)
* Brivudin is approved in some countries; for example, Germany.
‡
Lamivudine is also approved for the treatment of HBV.
§
In addition to zanamivir and oseltamivir, amantadine and rimantadine have been approved as anti-influenza drugs, but these compounds are targeted at the viral uncoating
process, not the viral neuraminidase.
||
Ribavirin is used in combination with interferon-α for HCV.
CMV, cytomegalovirus; EBV, Epstein–Barr virus; EICAR, 5-ethynyl-1-β-D-ribofuranosylimidazole-4-carboxamide; HBV, hepatitis B virus; HCV, hepatitis C virus; HHV, human
herpesvirus; HIV, human immunodeficiency virus; HSV, herpes simplex virus; IMP, inosine 5ʹ-monophosphate; NNRTI, non-nucleoside reverse transcriptase inhibitor; NRTI,
nucleoside reverse transcriptase inhibitor; RSV, respiratory syncytial virus; VZV, varicella-zoster virus.
 Togavirus (for example, Western RNA polymerase Several*
Equine encephalitis virus)

| Vir a l a nd c e llula r ta r ge ts for a ntivir a l a ge nts Rhabdovirus (Rabies virus)

Filovirus (for example, Ebola virus)
RNA polymerase
RNA polymerase
Several*
Several*

Virus Viral target Cellular target Comments

Hepacivirus (HCV) RNA polymerase,
RNA helicase,
–

Viral protease

Parvovirus DNA polymerase – Remains to be explored

Orthomyxovirus (Influenza) Matrix (M2) protein
Neuraminidase
–

Paramyxovirus (RSV) Fusion polypeptide Several*

Polyomavirus DNA polymerase – Remains to be explored
Coronavirus (SARS-CoV) Several‡ –
Reovirus (Rotavirus) – Several*
Papillomavirus DNA polymerase, E6, E7 – Remains
Retrovirus (HIV) to be explored Several§|| Several¶

Adenovirus DNA polymerase Cell-associated factors Remains to be explored

*Inosine 5’-monophosphate (IMP) dehydrogenase, S-adenosylhomocysteine (SAH) hydrolase
decarboxylase and cytosine 5’-triphosphate (CTP) synthetase. ‡Spike (S) protein, RNA polym
protease. §Fusion polypeptide (viral glycoprotein gp41), reverse transcriptase and viral proteas
α-herpesvirus Thymidine kinase – Target for activation
transcription transactivator (TAT). ¶Integration- and transcription-associated factors.

(HSV-1, HSV-2, VZV) DNA polymerase Target for inhibition

Helicase–primase Target for inhibition
β-herpesvirus Protein kinase – Target for activation
(HCMV, HHV-6, HHV-7) DNA polymerase Target for inhibition
Terminase Target for inhibition
γ-herpesvirus (EBV, HHV-8) DNA polymerase – Target for inhibition
Poxvirus (for example, variola and DNA and RNA polymerases Several* Remains to be explored
vaccinia viruses)
Hepadnavirus (HBV) DNA polymerase – Target for inhibition
(Reverse transcriptase)
Picornavirus Viral capsid – Target for inhibition
(Enteroviruses and Rhinoviruses) RNA polymerase Remains to be explored
Flavivirus (for example, yellow fever RNA polymerase – Remains to be explored
and dengue viruses)
Arenavirus (for example, Lassa) RNA polymerase Several* Remains to be explored
Bunyavirus (for example, RNA polymerase Several* Remains to be explored
Crimean–Congo)
Togavirus (for example, Western RNA polymerase Several* Remains to be explored
Equine encephalitis virus)
Rhabdovirus (Rabies virus) RNA polymerase Several* Remains to be explored
Filovirus (for example, Ebola virus) RNA polymerase Several* Remains to be explored
Hepacivirus (HCV) RNA polymerase, – Being investigated
RNA helicase,
Viral protease
Orthomyxovirus (Influenza) Matrix (M2) protein – Target for inhibition
Neuraminidase Target for inhibition
Paramyxovirus (RSV) Fusion polypeptide Several* Being explored
Coronavirus (SARS-CoV) Several ‡
– Being explored
Reovirus (Rotavirus) – Several* Remains to be explored
Retrovirus (HIV) Several §||
Several¶
Established or being
explored
 Virus–cell fusion Helper T-lymphocyte
Virus Reverse
adsorption transcription
RNA DNA
Viral envelope RNA DNA
proteins (gp160) Uncoating Reverse
transcriptase
CCR5 RNase H Chromosomal
DNA
CD4 protein
CXCR4
Integration Integrase
HIV (latency)

Transcription
Regulated by
Viral
Tat Nef RNA
Rev
Translation
Viral
proteins
Vif
RNA
Assembly

Budding

Drugs targeting
HIV viruses HIV protease
 •viral proteins
•cellular cofactors

•host cell inhibitors

•approved drugs
 Retroviral genes of the HIV virus

• gag (group-specific antigen): makes the cone-shaped viral shaped viral

capsid.
• pol(polymerase): codes for viral enzymes, (polymerase): codes for viral
enzymes, reverse transcriptase, integrase, and viral protease.
• env(envelope): makes surface protein gp120 and transmembrane gp41,
enabling HIV to fuse to CD4 cells.
• tat: eliminates the hairpin structure in RNA by phosphorylating CdK9/
CycT. This increases transcription and creates a positive feedback loop
• vpr: is involved in getting the viral RNA into the nucleus of the T cell. It
causes the cell to stop growing, stimulating an immune dysfunction.
• rev: creates a rev Response Unit that exports the HIVrev RNA into the
cytoplasm before it is spliced. It is also positive feedback loop.
• nef: down regulates CD4 on the T cell, inhibiting response to infectious
agents.
• vif: helps HIV infect other cells, though it is still unclear how. It is thought
that it interferes with the T cells proteins that modify RNA.
• vpu: enhances HIV release from the cell.
HIV-1 proteins

Swanson et al., Cell 133

Antiviral Drugs on the Market in the US (2007)
Name Trade name C ompany Launched
Nucleoside or nucleotide reverse-transcriptase inhibitors
Zidovudine Retrovir GlaxoSmithKline 1987
Didanosine Videx Bristol–Myers Squibb 1991
Zalcitabine HIVID Roche 1992
Stavudine Zerit Bristol–Myers Squibb 1995
Lamivudine Epivir GlaxoSmithKline, 1998
Shire Pharmaceuticals
Abacavir Ziagen GlaxoSmithKline 1999
Tenofovir disoproxil fumarate Viread Gilead 2001
Emtricitabine Emtriva Gilead 2003
Non-nucleoside reverse-transcriptase inhibitors
Nevirapine Viramune Boehringer Ingelheim 1996
Efavirenz Sustiva, Stocrin Bristol–Myers Squibb, Merck 1998
Delavirdine Rescriptor Pharmacia & Upjohn, 1999
Agouron, Pfizer
Protease inhibitors
Saquinavir Invirase Hoffmann-La Roche 1995
Indinavir Crixivan Merck 1996
Ritonavir Norvir Abbott, GlaxoSmithKline 1996
Nelfinavir Viracept Agouron, Pfizer 1997
Amprenavir Agenerase, Prozei Vertex 1999
Lopinavir + ritonavir Kaletra, Aluvia Abbott 2000
Atazanavir Reyataz, Zrivada Bristol–Myers Squibb, Novartis 2003
Fosamprenavir Lexiva, Telzir Vertex, GlaxoSmithKline 2003
Tipranavir Aptivus Boehringer Ingelheim 2005
Darunavir Prezista Tibotec 2006
Entry inhibitors
Enfuvirtide Fuzeon Trimeris, Roche 2003
Maraviroc Celsentri, Selzentry Pfizer 2007
 Virus Adsorption Inhibitors
• polyanionic compounds such as polysulphates, polysulphonates,
polycarboxylates have been shown to bind to the V3 loop of the viral
envelope protein gp120, which is rich in Arg+ and Lys+ residues.
• Thereby, they hamper binding to heparan sulfate, widely expressed
on animal cell surfaces.
• Some polypeptides such as cyanovirin bind to the carbohydrate
portions of gp120, thereby inhibiting their interaction with the human
cell surface receptors CD4.
• Polyanions may become ineffective by mutations in the V3 loop
(K269E, Q278H, N293D), that make these strains resistant to such
treatments.

OSO3 SO3
n n

PVAS PVS

Viral adsorption inhibitors

 Virus-Cell Fusion Inhibitors
• in case of HIV gp120 interacts first with a co-receptor; for some
HIV strains its is the chemokine (C-X-C) motif receptor 4 (CXCR4),
for some the CCR5. These receptors usually act as receptors for
the chemokines SDF1 , RANTES or MIP5.
• Initial association of the HIV virions with the cells might be blocked
by antagonists of these receptors.

L33 W76

Y108

H Y37
N O T82
H3C
O N
CH3 5

TAK779

7
6

N HN 1
NH N
3 4
2

NH HN NH HN

AMD3100 Interaction of CCR5 with TAK779. A structural

Virus–cell fusion inhibitors
model of CCR5 complexed with TAK779 (FIG. 2) , viewed from
within the plane of the membrane12. The indicated cluster of
amino acids in the TAK779 binding site includes several
aromatic residues (Y37, W86, Y108) that might form
favourable interactions with the aromatic rings of TAK779.
 Mechanism of fusion of the human and viral membranes

•the fusion peptide

at the N terminus of
gp41 insert into the
cellular membrane.
This event triggers a
structural change,
that brings the two
membranes closer
together.


• several drugs
interfere with the
gp41-mediated fusion
process such as T20
(fuzeon, a 36 aa
peptide that mimics
the ectodomain of
gp41) or the so-called
5-helix, that binds to
the carboxy terminal
region of gp41.
Antibodies against the HIV-1 envelope spike
Antibody target sites on the HIV-1
envelope spike. Schematic
representation of the HIV-1 envelope
spike. Each of the monomer units of the
trimer is composed of a gp120 and
gp41 trans-membrane protein. The four
best-characterized broadly neutralizing
target sites are highlighted and include
the CD4-binding site (orange), the
glycan-associated epitopes on the
base of the V3 loop (purple), the V1/V2
loop (green), and the membrane-
proximal external region MPER on gp41
(gray). Electron micrograph–derived
illustration of the envelope spike
targeted by representative broadly
neutralizing F(ab)s shown approximately
to scale (bottom: NIH45-46, orange;
PG16, green; PGT128, purple; 2F5, gray).
Examples of first- and second-
generation bNAbs that target these
and other sites are color-coded and
indicated at the bottom.

Klein, Science, 341 (2013),1199

 Structure of Fuzeon

• Dose of Fuzeon for treating adults with HIV or AIDS is Fuzeon 90 mg (1 mL) injected
twice daily just under the skin
•Enfuvirtide therapy costs an estimated US$25,000 per year in the United States
 Inhibitors of viral DNA or RNA synthesis

• RNA viruses: First step is transcription of viral RNA into DNA by the viral reverse transcriptase. All
three phosphorylation steps must be carried out by cellular kinases.

Drugs as inhibitors of the reverse transcriptase
Non-nucleoside reverse transcriptase inhibitors

N N N

N N

O N NH

N HN N NH HN N NH
Br
NH2 N N

Etravirine Rilpivirine Dapivirine

•must be hydrophobic
•bind to an allosteric binding site that is adjacent to the substrate (nucleoside)
binding site.
•binding locks the adjacent substrate binding site into an inactive conformation.
•improved specificity for reverse transcriptase over DNA polymerases resulting in
less toxicity
•unfortunately, rapid resistance is developed
 Mechanism of action of the HIV integrase
•Integrase is essential for viral replication. No integrases are found in the host
cells.
•It is encoded in the POL gene of HIV, and generated by the HIV protease.
•It is a 32KDa protein with 3 domains: the amino-terminal domain (NTD), the
catalytic core domain (CCD) and the carboxy-terminal domain (CTD). The CCD
forms a dimer.
•It catalyses both 3’ processing of viral DNA in the cytoplasm, and insertion of
the viral DNA into chromosomal DNA in the nucleus

• viral DNA is processed in the cytosol by 3’

att 3ʹ-processing
endonucleophilic cleavage creating the
reactive intermediate required for the strand
transfer

• the complex formed of processed viral

Strand transfer
DNA and integrase diffuses into the nucleus.
In the nucleus the reactive ends attack
chromosomal DNA across the major groove.
5ʹ 5ʹ 5ʹ 5ʹ Therein, the 3’ OH groups of the viral DNA
are ligated to the 5’DNA phosphate.
5ʹ

5ʹ
 The HIV Integrase

CCD

CTD
 HIV Integrase Inhibitors in an advanced Phase of
Development:
Diketoaryl (DKA) and DKA-strand-transfer selective inhibitors
Diketo aryls (Shionogi) Diketo acids Naphthyridine carboxamides (Merck)
O OH O OH
Cl
O N
O OH COOH N
H
N N
NH F
HN N N O R

5CITEP L-708,906

N O
O OH O OH H3C
O
O N N CH3
N3 COOH S O N
NH
N
R= O CH3
S-1360 MA-DKA L-870,810 L-870,812
F
F
O OH
N OH
COOH H
O
OH F L-731,988 O N
N N N
H H
N N OH
O N
O O F O O

Raltegravir (MK-0518)
Cl
Elvitegravir (GS-9137, JTK-303)
 Mode of action of DKA integrase inhibitors

a Donor site Acceptor site b c

•The donor site of the integrase
Host DNA
(Acceptor)
binds viral DNA and catalyses 3’
processing (a).
3ʹ-processing Strand transfer
3ʹ
•The integrase undergoes a
structural change that allows the
Viral DNA
(Donor) e Viral DNA (Donor)
binding of the chromosomal DNA
DKA
•The DKA’s bind selectively to the
Adenine
O
5ʹ
O acceptor site
d
OH
O
–
O O–
DKA •DKA chelate the metal in the
O Me2+ O catalytic site of the integrase in
O O the CCD domain formed by the
– Me2+
O catalytic residues D64, D116 and
N3
O
–
E152. Usually the metals
Integrase acidic
residues
(D64, D116, E152)
O coordinate to these residues and
the phosphodiester backbone of
the DNA substrate.
 Inhibitors of viral DNA or RNA synthesis

• DNA viruses: First step is

replication of viral DNA. Inhibitors
of the latter are nucleoside
analogues. These are sufficiently
similar to the “normal”
nucleosides, so that they can
serve as substrates for the viral
DNA polymerases. Since the
sugar lacks the 3’ hydroxyl group
they act as chain terminators:

Chain terminators for viral DNA polymerases
Acyclovir Cidofovir

(does not require the presence of a

viral thymidine kinase)
• The drugs are prodrugs, that can only be converted into the
phosphorylated forms by the viral thymidine kinase and hence do
not interfere with DNA synthesis in non-infected cells:
Prodrugs with improved bioavailability
 Inhibitors of the helicase-primase complex
(HERPES)
•The helicase-primase complex unwinds viral DNA at the replication fork
(helicase), and initiates DNA synthesis at both the leading and the
lagging strand (primase).

Leading strand O
UL42 H H
DNA N N
polymerase NH N
ICP8 N O N O
H 2N H 2N
S S
8
Helicase–primase N
5 52
complex (UL5, -8, -52) BI C 1 1 5 BS B I LS 1 0 3 B S

O
Lagging strand H
N
N
N O Me N
H 2N
+ TZP
S

BILS 179 BS

TZP derivatives
(thiazolyl-phenyl)

•TZP enhances binding of the UL5 and UL52 subunits to the strands, resulting
in inhibition of the helicase activity by interrupting the catalytic cycle.
Protease inhibitors
HIV- protease structure with bound darunavir
Most drugs against HIV protease are
transition state inhibitors

Q (L) N (F) Y P (I) V (S) V

N
N
H
O
O
Substrate
peptidic bond

N N
H
OH
O
Inhibitor
hydroxyethylene bond

Saquinavir Ritonavir Indinavir Nelfinavir Amprenavir Lopinavir

Atazanavir Fosamprenavir Darunavir

 HIV protease Inhibitors

H3C CH3
O O
O H O O
H N
N S N N N O N O O
N N N H H S
H N CH3 O OH S N
O OH H
NH 2 H3C OH N F
O NH CH3
O F
H 3C CH 3 F

CH 3

Saquinavir Ritonavir Tipranavir (U-140690)

hard gel capsules, Invirase ®
soft gelatin capsules, Fortovase ® Norvir ® Aptivus ®

CH3

N OH H3C CH3
O
H O O O O
N N O NH O S
S
N CH3 O
O N N O O N N
O HO H H
O NH HO N N OH CH 3 NH2 HO O S
H
H3C CH3 OH O
CH3
CH3 NH 2

Nelfinavir Amprenavir
Indinavir Darunavir (TMC-114)
Viracept ® Agenerase ®, Prozei ® Prezista ®
Crixivan ®

CH 3 OH
H O O OH O O
O N N NH H O O
H O S
N N N N O
H O N N
O O H H O N N
H
CH 3 H 3C CH 3 O O CH 3
O O NH2
P
HO OH CH3

Lopinavir Fosamprenavir
Atazanavir
combined with ritonavir at 4/1 ratio Lexiva ®, Telzir ®
Reyataz ®
Kaletra ®
 The concept of a transition state
inhibitor
• Transition-state inhibitors are designed to mimic the transition state of an
enzymatic reaction
• They are likely to bind stronger to the active site than the natural enzyme
substrates
• The successful transition-state inhibitor will be a transition-state isostere, that
mimics the tetrahedral centre but cannot be hydrolyzed.
 The Development of Saquinavir
• Starting from a viral polypeptide sequence that serves as a substrate to the
viral peptidase (Leu-Asn-Phe-Pro-Ile) and retaining the Phe-Pro segment


• Converting it to a 

transition-state isostere:




• Adding fragments to fill the S2 and S2’ pockets


and optimizing P1 and P2




• Add fragments to fill S3 subsite





• Increase size of fragment that binds


to the S1’ subsite



 Commercial PI Drugs

•first PI on the market

•rapid development of resistance
•low oral bioavailability
•strong binding to plasma proteins
Saquinavir (Roche)

•although larger than Saquinavir displays better bio-

availability
•potent inhibitor of cytochrom CYP3A4 and hence shuts
down its own metabolism
•good in combination with other PIs
Ritonavir (Abbott) •strong binding to plasma proteins

•was designed based on Saquinavir

•better oral bioavailability
•less binding to plasma proteins
Indinavir (Merck)
HIV combination
therapy (HAART)
Highly Active AntiRetroviral Therapy

Indinavir (Merck)
 The principle of vaccination

mimic infection with a pathogen that is attenuated and safe

thereby establishing immunological memory that could respond
in case of an actual infection
• live attenuated virus vaccines (mumps, measles)
• use of killed viruses (e.g. the original Salk killed poliovirus vaccine)
• exposure of the immune system to recombinant viral proteins alone
(hepatitis B)

• the main tool in vaccination is the use of neutralizing antibodies. These

antibodies can bind the free virus, prevent viral entry into the host cell
(“neutralization”). Antibodies can also bind to infected cells triggering
their elimination via the host effector system.

• the second defense line is the cellular immune response, particular

cytotoxic T lymphocytes (CTLs). Immunization with the corresponding T-
cell-inducing vaccine generates a population of memory T cells, that will
rapidly expand upon infection. The CTLs kill infected cells by recognizing
foreign viral proteins.
 Vaccination

Milestones in the history of virology

Year Event
1796 Vaccination for smallpox (Jenner)
1892 First virus identified ( Tobacco mosaic virus ) (Ivanovsky)
1898 First animal virus identified, foot-and-mouth disease virus (Loeffler and Frosch)
1901 First human virus identified, Yellow fever virus (Reed)
1911 Viral aetiology for malignant sarcoma in chickens (Rous)
1915; 1917 Discovery of viruses that infect bacteria (Twort; d’Herelle)
1918 Worldwide influenza epidemic
1935 Crystallization of Tobacco mosaic virus (Stanley)
1939 First visualization of viruses in the electron microscope
1952 Introduction of cell cultures for growth of animal viruses (Enders; Robbins)
1954; 1956 Salk (inactivated) and Sabin (live attenuated) vaccines for poliomyelitis
1962–1970 Mumps, measles and rubella vaccines licensed
1969 Identification of retrovirus reverse transcriptase (Temin; Baltimore)
1977 World declared free of smallpox
1980–1990 Nucleic acid sequences of many viruses determined
1983 Identification of human immunodeficiency virus, the aetiological agent of
acquired immune deficiency syndrome (AIDS) (Montagnier; Gallo)
 Development of Vaccines

D i sease ( i nf ecti ous agent) V acci ne type Y ear D i sease ( i nf ecti ous agent) V acci ne type Y ear
S mal l pox L i ve-attenuated vi r us 1796
M easl es L i ve-attenuated vi r us 1963
R abi es L i ve-attenuated vi r us 1885
M umps L i ve-attenuated vi r us 1966
K i l l ed vi r us 1907
R ubel l a L i ve-attenuated vi r us 1969
Salmonella typhi K i l l ed bacter i a 1912
Meningococcus Purified capsular polysaccharide (tetravalent A, C, Y, W135) 1984
L i ve-attenuated ( T 21A ) bacter i um 1991
T i ck - bor ne encephal i ti s K i l l ed vi r us 1981
P ur i fied capsul ar pol y sacchar i de 1991
P neumococcus P ur i fied capsul ar pol y sacchar i de 1977 ( 14-val ent)
T uber cul osi s L i ve-attenuated bacter i um ( B C G ) 1921
1983 (23-valent)
D i phther i a T ox oi d 1939
H epati ti s B P l asma-der i ved k i l l ed par ti cl es 1981
Y el l ow f ever L i ve-attenuated vi r us ( str ai n 17D ) 1940
R ecombi nant par ti cl es 1986
T etanus T ox oi d 1949
Haemophilus influenzae P ol ysacchar i de vacci ne 1985
P er tussi s K i l l ed bacter i al cel l s 1953
C onjugated vacci ne ( toddl er s) 1987
A cel l ul ar ( J apan) 1981
C onjugated vacci ne ( i nf ants) 1990
R ecombi nant 1993
H epati ti s A K i l l ed vi r us 1994
A cel l ul ar ( U S A ) 1996
V ar i cel l a L i ve-attenuated vi r us 1995
Poliovirus K i l l ed vi r us ( S al k ) 1954
L yme di sease R ecombi nant 1998
L i ve-attenuated vi r us ( S abi n) 1961
Rotavirus L i ve-attenuated vi r us 1998
I nfluenza K i l l ed vi r us 1958
M eni ngococcus C C onjugated vacci ne 1999
D eter gent-spl i t vi r us 1968
P neumococcus 7-val ent conjugated vacci ne 2000
P ur i fied vi r us subuni t 1976
I nfluenza L i ve-attenuated, col d adapted 2003
A djuvanted vacci ne 1997
C hol er a K i l l ed bacter i a ( i njectabl e) 1960
L i ve-attenuated vi r us 1998
K i l l ed bacter i um+ C T B ( or al ) 1994
Live-attenuated, recombinant (CVD 103 HgrR) 1995
 Development of influenza vaccines

• Vaccines are attenuated (or killed) viruses or viral particles

• Vaccines are usually produced by culturing the virus in chicken
eggs.
• The vaccine is made from the whole killed virus by treating the
virus with ether or detergent to produce a subunit vaccine
• Unfortunately, influenza viruses that are pathogenic in humans
are hard to grow in chicken.
• A way to increase virus yields from eggs is reassortment by mixing
the new virus with a known virus that does grow well in chicken.
The hope is that the reassorted virus contains genes from the
rapidly growing strain, and the hemaglutinin and neuraminidase
surface antigens from the new virus.
• from identifying the swine influenza virus in April 2009 to approval
of an vaccine took only 6 months
• Faster methods for producing vaccines are using live-attenuated
vaccines or vaccines produced in cell cultures
Producing a vaccine using reversed genetics
 Problems for producing HIV vaccines
• the enormous sequence diversity due to a highly error-prone
reverse transcriptase makes it difficult to raise vaccines against
particular epitopes

• within days of expose to HIV viruses massive infection and loss of

memory CD4+ T cells occurs. The latter are critical for
coordinating effective immune responses

• HIV viruses have developed strategies for avoiding immune

elimination. E.g. the accessory protein NEF down-regulates
molecules of the major histocompatibility complex (MHC), that
are important for T cell recognition of infected cells. Moreover,
the surface envelope proteins incorporate multiple features to
avoid antibody recognition. The spikes on the protein surface,
composed of the gp41 TM protein and the glycosylated gp120,
exhibit enormous sequence variability. The recognition surface is
buried under the sugar chains
 IMP dehydrogenase inhibitors

O NH

•IMP (inosine-5-
H2N N H2N N
N N
N N
HO HO PRA monophosphate)
O O
dehydrogenase is an
OH OH
Ribavirin
OH OH
Viramidine
essential enzyme in the
Nucleoside de-novo biosynthesis of
kinase
purine mononucleotides.
O IMP
ATP IMP dehydrogenase
H2N N
ADP

•Although it is a cellular
N Succinyl
N XMP AMP
P O
O target cells infected with
GMP AMP
OH OH viruses display an
Ribavirin-MP GDP ADP increased need for DNA/
GTP ATP
RNA synthesis.

O RNA synthesis •Used to treat hepatitis

O
infections in combination
O
with interferon-α.
HO
O
OH

Mycophenolic acid
 S-adenosylhomocysteine (SAH) hydrolase
inhibitors

• SAH is a key enzyme for methylation reactions

that depend on S-adenosylmethionine (SAM) as
the methyl donor. Methylations are required for
the maturation of viral mRNA. NH2
N
N
• SAH prevents accumulation of itself by auto-
HO
hydrolysis.
N X

• Inhibitors of SAH lead to accumulation of SAH

resulting in inhibition of the methylation reaction. HO OH
(X=N or CH)
• Cellular target, but high activity in infected cells.
SAH hydrolase inhibitor
• Used to treat poxviruses, ebola fever
 Interferons

• interferons belong to the class of cytokines involved in cell-cell

signaling, particularly relating to the early detection of microbial
invasion
• IFNα and IFNβ are produced by most human cells in response to a
viral infection
• production of interferons is mediated through activation of toll-like
receptors
• its production is transient and requires stimulation by viruses, microbial
products and other inducers
• their action is mediated through the JAK/STAT and other signaling
pathways
• they induce expression of 2,5-oligoadenylate synthase, which in turn
produces oligoadenylate that activates ribonuclease L, resulting in
the degradation of single-stranded RNA, and triggers cellular
apoptosis
• IFNs are used to treat hepatitis B and C viral infections
The influenza lifecycle and drugs
 Inhibition of Virus Uncoating (Flu)

a
• Adamantane derivatives block
the migration of protons into the
interior of the virions within
endosomes, thereby preventing
the pH shift required for uncoating.
They act by blocking the M2
(matrix 2) channel. 

Unfortunately, rapid resistance
Ala-30 against them occurs.
Gly-34

His-37
H H

NH2• HCl
Trp-41 H NH2• HCl H
CH 3
H H
Amantadine Rimantadine
 Viral Neuraminidase Inhibitors (Flu)
• The influenza virus binds to the target cell via interaction of its surface
glycoprotein, haemagglutinin, with the host-cell surface receptor, that
contains sialic acid. Another viral glycoprotein, neuraminidase, cleaves
off the terminal sialic acid, allowing the virus to leave the cell once the
virus has replicated:






• Inhibiting the viral Neuraminidase prevents the virus from leaving the cell
and infecting others.

Host cell Budding virus

Neuraminidase cleaves receptor

Neuraminidase inhibitor
Haemagglutinin
Nucleus

Release of
new virions
Receptor Neuraminidase
Nucleus
containing
sialic acid Virion
 Commercial Neuraminidase Inhibitors

H2N NH O H2N NH H2N NH

HO HO O O
HN OH HN HN
OH OH OH OH
HO HO
H H H
O O O O HN OH Rʹ R
H H R
H3C N F3C N O NH H NH
OH OH CH 3 CH 3 O O
O HO O HO CH 3
CH 3 CH 3
DANA FANA Peramivir Cyclopentane Cyclopentane
RWJ-270201 derivative derivative

H2N NH CH 3
HO OH O
H H3C O
HN H2N OH
HO O O O
H3C O O H H H H
HN OH F 3C N OH
H3C N
O OH HN N N
O O CH 2 R N NH O O H H O
H3C HN NH 2
NH 2 CH 3 O N
H3C Rʹ O H3C O
NH CH 3 CH 3
Zanamvir Oseltamivir Cyclopentane amide Pyrrolidine derivative A-315675
(Relenza) (Tamiflu) derivative A-192558
The structure of the surface protein Hemaglutinin

haemagglutinin trimer complexed monomer unit

with N-acetylneuramic acid (in CPK form)
Complexation mode of α-Neu5Ac
Development of Neuramidase inhibitors
From the proposed catalytic mechanism (based on a X-ray
structure of the enzyme in complex to sialic acid) a transition-
state inhibitor was developed:
 (Relenza)

neuramic acid

Tamiflu has better bioavailability (is the ethylester prodrug, cleaved by

esterases). Can be administered orally.
 Tamiflu is a good mimic of α-Neu5Ac2en

OH
HO
O
O
HO OH Glu276

NH
OH
O
Neu5Ac2en Arg224

O
O
HO

NH
NH2
O
Oseltamivir (free acid)
Influenza pandemics
 Flu Epidemics

H1N1 H2N2 H3N?

•33’000 deaths in US
PB1
HA PB1
NA HA

1918 1957 1968

H1N1 H2N2 H3N2
Spanish Asian Hong Kong
H3N2

•50’000 000 worldwide •50’000 deaths in US

1977
1950s H1N1
Russian
H1N1 H1N1

•H5N1 (bird flu): originated in Hong Kong 1977, and re-appeared 2003. Highly pathogenic. Severe
respiratory infections with high mortality. (261 deaths so far)
•H1N1 (swine flu): originated from Mexico
Components of the H1N1 virus

•belongs to the class of influenza A viruses

•genome is composed of eight segments of single-stranded,
negative-sense RNA, that each encode 1 or 2 proteins (NP=
nucleoprotein; PB components of the polymerase complex)
 Pathogenicity
•Human influenza viruses preferentially bind to sialic acid that is linked to galactose
by an α2,6 linkage (SAα2,6Gal)
•this is matched by corresponding carbohydrates on epithelial cells in the human
trachea
•avian viruses preferentially recognize SAα2,3Gal in the intestinal tract of waterfowl
(birds)
•differences in receptor-binding specificities are determined by the amino acids in
the HA receptor binding pocket
•HA cleavage efficiency is essential for viral activity. Low pathogenic avian viruses
and non-avian influenza viruses posses a single Arg at the cleavage site. Highly
pathogenic H5 and H7 viruses posses several basic amino acids at the HA cleavage
site.
Table 2 | Determinants of viral pathogenicity
Protein Position Pathogenicity Swine-origin H1N1 viruses Function
Low High
PB2 627 Glu Lys Glu Replicative ability in some mammals, including
humans
701 Asp Asn Asp Nuclear import kinetics affecting replicative
ability in mice
PB1-F2 66 Asn Ser Truncated ( 11 aa) Induction of apoptosis
HA Cleavage site Single basic aa Multiple basic aa Single basic aa HA cleavage
NS1 92 Asp Glu Asp Unknown (interferon response?)
C terminus Arg-Ser-Glu-Val, Glu-Ser-Glu-Val 11 C-terminal aa truncated Unknown
deletion
aa, amino acid(s).
 History of the swine flu
(2009)

Table 1 |
Date Event
Mid-February Outbreak of respiratory illness in La Gloria, Veracruz, Mexico 31
12 April Mexican public health authorities report outbreak in Veracruz to the PAHO
15 April CDC identifies S-OIVs in the specimen of a boy from San Diego, California
17 April CDC identifies S-OIVs in the specimen of a girl from Imperial, California
21 April CDC alerts doctors to a new strain of H 1N1 influenza virus
23 April The Public Health Agency of Canada identifies S-OIVs in specimens from Mexico
24 April WHO issues Disease Outbreak Notice
27 April International spread and clusters of human-to-human transmission prompt WHO to raise the pandemic alert from phase3 to 4
29 April WHO raises the pandemic alert from phase 4 to 5 (human-to-human spread in at least two countries in one WHO region)
21 May 41 countries report 11,034 cases, including 85 deaths
The recent Ebola Outbreak
• The Ebola virus belongs to the class of
Filoviruses
• The family consists of the genera
Marburgvirus and Ebolavirus
• Filoviruses consist of a single-stranded
linear RNA genome
• After entry into the host cell it is
transcribed to generate polyadenylated
mRNA


• the gene has the following structure:


3′

 NP 35 40 sGP/GP 30 24 L 5′

• np (nucleoprotein), virion protein 35,

30,24,40, (vp35,vp40,vp30,vp24),
glycoprotein (gp) and polymerase (L) 

Source: WHO
 GP1,2
VP35 VP40/VP24
L

VP30

NP

Feldmann H. N Engl J Med 2014;371:1375-1378.

Source: WHO
 The development of Ebola vaccines
These include replication-deficient adenovirus vectors, replication-competent
vesicular stomatitis (VSV) and human parainfluenza (HPIV-3) vectors, and virus-like
nanoparticle preparations.

• hAd3-ZEBOV, developed by GlaxoSmithKline (GSK) in collaboration with

the US National Institute of Allergy and Infectious Diseases (NIAID). hAd3 is
an attenuated version of a chimpanzee adenovirus (cAd3) that has been
genetically altered so that it is unable to replicate in humans.The cAd3
vector has a DNA fragment insert that encodes the Ebola virus
glycoprotein,

• VSV-EBOV, developed by NewLink Genetics and Merck Vaccines USA in

collaboration with the Public Health Agency of Canada . Currently, Phase
II and Phase III clinical trials for VSV-EBOV are underway in Guinea and
Sierra Leone. This vaccine is based on the vesicular stomatitis virus (rabies-
like) which was genetically modified to express a surface glycoprotein of
Zaire Ebola virus

Both vaccine candidates have been shown to be safe and well tolerated
in humans.
Source: WHO/Wikipedia
 The development of Ebola drugs
Favipiravir, also known as T-705 or Avigan, is an experimental antiviral drug
with activity against many RNA viruses. The mechanism of its actions is
thought to be related to the selective inhibition of viral RNA-dependent RNA
polymerase

ZMapp is an experimental biopharmaceutical drug comprising three chimeric neutralizing

monoclonal antibodies under development as a treatment for Ebola virus disease.

Components:

Amino Lipid
TKM-Ebola siRNA cocktail adjusted to
Guinea strain, IV once daily infusion over Structural Lipid
two hours
PEG - Lipid

Nucleic Acid

40-140 nm diameter
Brincidofovir (CMX001) is an experimental antiviral drug being developed
by Chimerix of Durham, NC, for the treatment of cytomegalovirus,
adenovirus, smallpox, and ebolavirus infections. Conjugated to a lipid, the
compound is designed to release cidofovir intracellularly, allowing for
higher intracellular and lower plasma concentrations of cidofovir,
effectively increasing its activity against dsDNA viruses, as well as oral
bioavailability. Source: Wikipedia
source: presentation Peter Horby, Oxford, UK
 The Hepatitis viruses
Hepatitis implies injury to liver characterized by presence of inflammatory cells
in the liver tissue.

• Hepatitis A, (formerly known as infectious hepatitis), is caused by Hepatitis A

virus, which is most commonly transmitted by the fecal-oral route via
contaminated food or drinking water. Every year, approximately 10 million
people worldwide are infected with the virus.The Hepatitis virus (HAV) is a
Picornavirus; it is non-enveloped and contains a single-stranded RNA
packaged in a protein shell.The virus spreads in conditions of poor sanitation
and overcrowding.

•Hepatitis B virus is a DNA virus. Transmission results from exposure to infectious

blood or body fluids containing blood. Several vaccines have been developed
for the prevention of hepatitis B virus infection. These rely on the use of one of
the viral envelope proteins (hepatitis B surface antigen or HBsAg).

•The hepatitis C virus (HCV) is spread by blood-to-blood contact. No vaccine

against hepatitis C is available. An estimated 150-200 million people worldwide
are infected with hepatitis C. Current treatment is a combination of pegylated
interferon alpha (brand names Pegasys and PEG-Intron) and the antiviral drug
ribavirin.