See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/5544828 Novel HIV-1 reverse transcriptase inhibitors ARTICLE in VIRUS RESEARCH · JULY 2008 Impact Factor: 2.32 · DOI: 10.1016/j.virusres.2008.01.003 · Source: PubMed CITATIONS 68 READS 48 1 AUTHOR: Dirk Jochmans University of Leuven 33 PUBLICATIONS 1,094 CITATIONS SEE PROFILE All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately. Available from: Dirk Jochmans Retrieved on: 09 February 2016
Dirk Jochmans ∗Tibotec BVBA, Gen De Wittelaan L 11B 3, 2800 Mechelen, Belgium
Available online 4 March 2008
bstract
HIV-1 reverse transcriptase (RT) was the first viral enzyme to be targeted by anti-HIV drugs. Despite 20 years of experience with RT inhibitors,ew ways to inhibit this target and address viral resistance continue to emerge. In both licensed RT inhibitor classes, nucleosides (NRTIs) and non-ucleosides (NNRTIs), compounds with better resistance, pharmacokinetic and toxicity profiles are being developed. Second-generation NNRTIsctive against HIV-1 strains resistant to current NNRTIs are being clinically evaluated. Beyond the classical NRTIs, nucleoside analogs that areo longer obligate chain terminators but nevertheless impede reverse transcription or even lead to viral ablation after several replication cycles,re being studied. RT inhibitor research has also yielded additional mechanisms to block RT. Driven by new insights the RNase H field remainsn evolution. In addition, the binding of both substrates (deoxynucleotide and primer/template) to RT is now subject to competition by novel
nhibitors. Further development of aptamers bears promise for gene therapy but perhaps more importantly, reveals additional new platforms for theevelopment of small-molecule RT inhibitors. This promising research provides much optimism that RT inhibitors will continue to evolve withubsequent clinical benefit.
2008 Elsevier B.V. All rights reserved.
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eywords: HIV-1; Reverse transcriptase inhibitor; Mechanism of action; Inves
. Introduction
HIV-1 reverse transcriptase (RT) was identified early asn attractive target for antiretroviral therapy. AzidothymidineAZT), a nucleoside analogue RT inhibitor, was the first drugo be approved for the treatment of HIV infection (Mitsuya etl., 1985), and for some years had to be used as a monother-py. Later, additional classes of anti-HIV drugs were introducedllowing the design of drug cocktails that led to powerful andustained virus suppression. Currently, two classes of drugs thatarget HIV-1 RT are licensed for the treatment of HIV infec-ion: nucleoside/nucleotide RT inhibitors (NRTIs and NtRTIs,espectively) and non-nucleoside RT inhibitors (NNRTIs). Bothlasses are important components of what has come to be knowns highly active antiretroviral therapy (HAART). Atripla®, a co-ormulation of an NRTI (emtricitabine), an NtRTI (tenofovir),nd an NNRTI (efavirenz) was approved by the US Food andrug Administration (FDA) in 2006. As this combination pill
s well tolerated, effective and convenient (single tablet, onceaily), it could become the standard for first line anti-HIV ther-py, further demonstrating the value of the RT inhibitor class.
RT-directed therapy celebrated its 20th anniversary in 2007nd research continues to explore novel ways to inhibit thisnzyme and address viral resistance to current RT inhibitors.his review discusses innovative approaches, in particular thenes where recent progress has been reported.
. Nucleoside analogue RT inhibitors (NRTIs)
NRTIs were the first anti-HIV drugs discovered (Mitsuya etl., 1985), and currently the US FDA has approved seven NRTIsnd one NtRTI for the treatment of HIV infection. They all blockIV RT by chain termination. In this section, we will briefly dis-
uss the progress in clinical development and early discovery ofther chain terminators, including prodrug approaches. In addi-ion, we will elaborate on recent discovery work demonstratingovel mechanisms by which nucleoside analogues inhibit HIVeplication.
.1. Dideoxynucleoside analogues
Dideoxynucleoside analogues are NRTIs that lack both the′- and 3′-OH groups on their sugar moiety (Fig. 1). Furtherodifications include replacing the 3′-OH group with other
hemical functions (H, N3, . . .) or a complete elimination of
172 D. Jochmans / Virus Research 134 (2008) 171–185
Fig. 1. Different approaches in current (B–E) and novel (F–I) NRTIs. All molecules are depicted in their non-phosphorylated form. Intracellular phosphorylationto their triphosphate form is needed to allow incorporation and chain terminating activity. (A) As a reference the structure of a natural nucleoside (thymidine) isdepicted; (B) AZT is a thymidine analogue with a substitution at the 3′ position; (C) D4T is a thymidine analogue with a modified ribose that eliminates the 3′p ral baa elopmh e-AZT
tshatfi
tpi
amm
lpt
osition; (D) FTC is a cytosine analogue with a modified ribose and an unnatuphosphonate group; (F) ATC is a modified cytosine analogue currently in devas a borano-phosphonate; (I) 5′-�-borano-�,�-(difluoromethylene)triphosphat
he 3′-position by changing the ribose ring itself. In addition,ome NRTIs carry a modified N-base. The majority of NRTIsave broad-spectrum antiretroviral activity due to their action asntimetabolites of natural nucleosides. Based on this similarity,hey are incorporated into the growing DNA strand and terminateurther strand elongation. Hence, NRTIs are chain-terminatingnhibitors of viral reverse transcription.
Since NRTIs mimic natural nucleosides, they could poten-ially be incorporated in the host genome by cellularolymerases. Inhibition of the mitochondrial DNA polymerases an unwelcome consequence of treatment with some NRTIs. In
ontl
se; (E) PMPA, the parent-drug of TDF has an acyclic-ribose replacement andent; (G) �-borano-AZT-MP has an �-borano-phosphate; (H) �-borano-PMPAis a stable mimic of AZT-TP.
ddition, other mitochondrial processes like nucleoside uptakeay be disturbed. The resulting mitochondrial dysfunction is aajor side effect of NRTI use (Cossarizza and Moyle, 2004).For activity, NRTIs need to be phosphorylated intracel-
ularly to the 5′-triphosphate form. To circumvent the firsthosphorylation step, a phosphonate group at the 5′ position ofhe ribose was introduced. This imitation of the �-phosphate
f natural nucleoside-monophosphates (nucleoside-MP) ison-hydrolyzable and allows further phosphorylation to theriphosphate. Such molecules are also termed nucleotide ana-ogue RT Inhibitors or NtRTIs. To preserve bioavailability,
tRTIs are administered as prodrugs with the negative chargef the phosphonate group masked. An example of this class isenofovir disoproxil fumarate (TDF), the prodrug of tenofovir9-R-2-phosphonomethoxypropyl adenine; PMPA; Fig. 1E).
Resistance to NRTIs and NtRTIs is discussed in a separateeview in this issue (Menendez-Arias, 2008).
.2. Apricitabine (ATC)
ATC is the most advanced NRTI in development. It is aeoxycytidine analogue ((−)-enantiomer of 2′-deoxy-3′-oxa-4′-hiocytidine) with an EC50 of 0.2-3.0 �M on wild-type HIV-1nd good coverage of clinical HIV-1 isolates from clades A–HFig. 1F; Mansour et al., 1995; Gu et al., 2006).
The resistance profile of ATC is of particular interest. Therug remains active on viruses carrying the M184V muta-ion as well as on AZT-resistant viruses containing multiplehymidine analogue-associated mutations (TAMs). The mostmportant mutations selected in vitro by ATC are K65R and75I (Wainberg et al., 2007). No mitochondrial toxicity haseen observed in vitro with ATC up to 200 �M (Gu et al.,006).
In a recent phase IIb trial, ATC showed good efficacy inatients harboring the lamivudine (3TC)/emtricitabine (FTC)-esistant M184V-containing virus (Anonymous, 2007). Inddition the drug seems to be well tolerated in the investigatedatient population as well as in healthy volunteers (Cahn et al.,006; Holdich et al., 2007). This suggests that ATC may belinically useful for the treatment of patients who have failedrevious 3TC- or FTC-containing regimens.
Studies confirmed that intracellular ATC-triphosphate (ATC-P) levels in peripheral blood mononuclear cells (PBMCs)ere reduced significantly by co-administration of 3TC or FTC
Holdich et al., 2007), due to both drugs utilizing the samentracellular enzyme for phosphorylation. This argues againstombining ATC with deoxycytidine analogues utilizing the samehosphorylation pathways for the treatment of HIV-1 infection.nother option would be to combine ATC with TDF or abacavir
ABC), but the selection of mutation K65R by ATC could limitts complementarity in such a combination.
.3. NRTI-monophosphate mimics
The restricted capacity of the intracellular phosphorylationachinery is a major limitation in the use of NRTIs. Only a
imited variation of chemical structures can be recognized andctivated, and sometimes the use of one NRTI saturates the sys-em causing antagonism in NRTI combinations. On the otherand, the use of NRTI-TP is unfeasible because of lack of sta-ility and cellular permeability.
Different approaches have been investigated to bypass therucial first phosphorylation step. Besides the use of a phospho-ate group as described above, investigators have successfully
eplaced an oxygen of the �-phosphate of deoxynucleoside-MPsy a borano group. These compounds remain good substrates forurther phosphorylation by cellular kinases (Fig. 1G and H) andhe subsequently formed triphosphates are potent inhibitors of
apra
h 134 (2008) 171–185 173
IV-1 RT (Meyer et al., 2000). An unexpected advantage ofhese �-borano-triphosphate-NRTIs is their activity on resistantT. Different mechanisms of NRTI-resistance, like increasediscrimination by K65R and M184V mutations or primer-nblocking in the case of TAMs, are counteracted when the-borano-phosphate-NRTIs are used. Further research showed
hat the presence of the borano group increases the rate of NRTI-P incorporation and as such decreases the level of resistance
Meyer et al., 2000; Selmi et al., 2001; Deval et al., 2005).nfortunately, none of these molecules has shown activity in
ell-based replication assays, most likely owing to a lack ofellular permeability.
In a similar approach, AZT-TP in which an �-phosphate oxy-en was replaced by a sulfur atom, was shown to be a goodubstrate for RT. The incorporated AZT-phosphorothioate actss a chain terminator and, because of the sulfur atom, resistsyrophosphorylysis mediated by ATP, in reactions catalyzed byZT-resistant RT (Matamoros et al., 2005).Recent studies investigated the effect of replacing an oxy-
en on the phosphonate group of tenofovir by a borano groupFig. 1H). The generated borano-phosphonates were found to beon-stable in cell culture and no antiviral effect was observed.t is unclear whether stability was the only issue since cellu-ar uptake and intracellular phosphorylation have not yet beenddressed (Barral et al., 2006). When the �-borano-phosphonatenalogue of tenofovir was converted chemically to its triphos-hate form, it showed a wild-type RT inhibition similar to theon-boronated analogue. As is the case with the phosphate ana-ogues, the introduction of a borano group in the phosphonatenalogues also suppresses HIV-1 RT resistance (Frangeul et al.,007).
.4. NRTI-triphosphate mimics
Approaches to generate mimics of the triphosphate moiety ofRTIs have also been investigated. Among the most promisingimics is the 5′-�-borano-�,�-(difluoromethylene)triphosphate
roup (Fig. 1I). Upon attachment to an NRTI core, the resultingolecule shows similar inhibition of RT activity as the canonicalRTI-TP together with a reasonable stability in serum (Wang
t al., 2004a; Boyle et al., 2005). No antiviral activity of theseolecules has been reported yet. Presumably they do not pen-
trate the cell membrane and a prodrug approach would beeeded. The sensitivity of cellular polymerases for these ana-ogues is still unknown.
.5. Other nucleoside analogues
.5.1. 4′-Substituted nucleosidesAnti-HIV activity of deoxynucleoside analogues with an
nmodified 3′-OH function but with a substitution at the 4′ posi-ion of the ribose was reported early on. The first substitutionsnvestigated were 4′-azido functions with 4′-azido-thymidine as
prototype compound. This nucleoside is intracellularly phos-horylated and incorporated in the nascent viral DNA duringeverse transcription. It inhibits HIV-1 replication with a potencynd selectivity similar to AZT (Chen et al., 1992; Maag et
174 D. Jochmans / Virus Research 134 (2008) 171–185
F
aamb(r1
dmdatawhrcnso2Euoa
atimfdi(vNc
2
iRiNfBi
Fig. 3. Structure of nucleosides in different enantiomeric conformations. (A)Tlt
itddt6aR
rctog3titmolncHap(
lAt
ig. 2. Chemical structure of 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA).
l., 1992). Because of the availability of the 3′-OH group, 4′-zido-thymidine is not an obligate chain terminator. Detailedechanistic studies showed that DNA chain elongation is only
locked after the incorporation of two 4′-azido-thymidine-MPChen et al., 1993). Early studies also identified the incorpo-ation of 4′-azido-thymidine-MP in cellular DNA (Chen et al.,992) posing a potential risk.
In addition, 4′-ethynyl substitutions were evaluated oneoxynucleoside analogues with an unmodified 3′-OH. Theost recently described compound is 4′-ethynyl-2-fluoro-2′-
eoxyadenosine (EFdA, Fig. 2). EFdA shows potent antiviralctivity (EC50 = 0.004 �M) and good activity on NRTI resis-ant strains. The compound is efficiently taken up by cellsnd the intracellular EFdA-TP concentrations are comparableith those reached by AZT-TP. Interestingly EFdA-TP shows aigher intracellular stability, generating a more persistent antivi-al effect than AZT or tenofovir. EFdA showed no inhibition ofellular polymerases, but incorporation into cellular DNA hasot yet been investigated (Nakata et al., 2007). The results of atructural analysis of the binding of EFdA and the mechanismf action of the compound were reported recently (Nakata et al.,007). In contrast with 4′-azido-thymidine, a single incorporatedFdA-MP acts as chain terminator. This could be explained bynfavorable interactions between the 4′-ethynyl group at the endf the inhibitor terminated primer and certain RT residues in thective site.
Other interesting deoxynucleoside analogues with 4′ alter-tions are 4′-methyl-thymidine and 4′-ethyl-thymidine. Similaro EFdA, 4′-ethyl-thymidine acts as a chain terminator uponncorporation at the primer-end. This is in contrast with 4′-ethyl thymidine that, when present at the primer-end, allows
or the incorporation of additional nucleotides although with aecreased efficiency. Both compounds block HIV-1 replicationn cell culture, but only in the presence of herpes simplex virusHSV) thymidine kinase; cellular kinases are not able to acti-ate the compounds. The compounds are effective against manyRTI drug-resistant RT variants, however, the M184V mutant
onfers resistance (Boyer et al., 2007).
.5.2. Locked-conformation nucleoside analoguesThe conformational properties of the ribose ring in the grow-
ng DNA chain provide an additional approach to developingT inhibiting nucleoside analogues. The ribose of nucleosides
n solution exists in two rapidly interchanging conformations:
(north) and S (south). Also in polynucleic acids, both con-
ormations can be observed resulting in, respectively, an A- and-form of the double helix. Crystal structures of RT bound to
ts template–primer show that at the primer-end, all nucleotides
lta2
he north and south conformation of 2′ deoxynucleosides are in a rapid equi-ibrium; (B) the complementary locked-conformation nucleoside analogues ofhymidine cannot interchange.
ncluding the next incoming deoxynucleoside-TP (dNTP) are inhe N-configuration. From nucleotide 6 or 7 on, the remainingouble helix contains riboses in their S conformation (Fig. 3). Asuring reverse transcription, all incorporated nucleotides haveo change shape from N to S when they move through position–7 in the elongating chain, it was speculated that nucleotidenalogues in which this transition could not occur would blockT (Boyer et al., 2005).
“Locked-conformation” nucleosides were generated byeplacing the ribose moiety with a cyclopentane ring fused to ayclopropane to lock the conformation. Depending on the posi-ion of the cyclopropane moiety, the ring could be locked in the Nr S conformation (Fig. 3). To avoid chain termination, the 3′-OHroup was preserved at a position equivalent to the deoxyribose′-OH. Again, for biochemical activity these molecules needo be phosphorylated to their triphosphate form. When usedn polymerization experiments, the compounds inhibit reverseranscription, not by direct chain termination, but by a poly-
erization block at 2–3 nucleosides after the incorporationf the N-locked nucleoside mimic. S-locked nucleoside ana-ogues showed no effect. Inhibition of HIV-1 replication couldot be addressed directly since cellular kinases do not signifi-antly phosphorylate N-locked analogues. But in the presence ofSV thymidine kinase that phosphorylates N-locked thymidine
nalogues, inhibition of HIV-1 replication could be observed,roviding a proof-of-principle for activity of such analoguesBoyer et al., 2005).
An additional reason to investigate locked thymidine ana-ogues was their potential activity on AZT-resistant strains. SinceZT resistance is associated with the excision of AZT-MP from
he primer-end (see below) and inhibiting locked nucleoside ana-
ogues are not located at the primer-end, it was anticipated thathey would remain active. This was confirmed in biochemicals well as in cell-based HIV replication assays (Boyer et al.,005).
D. Jochmans / Virus Research 134 (2008) 171–185 175
Fig. 4. Schematic overview of the primer-unblocking reaction and the mechanism of action of Np4N. (A) During nucleoside incorporation, HIV-1 RT binds thedNTP, and incorporates it at the primer terminus with the simultaneous release of pyrophosphate. (B) In the presence of TAMs the enzyme is capable to performthe opposite reaction. RT with the primer terminus in the nucleotide-binding site can bind ATP in a conformation in which the terminal two phosphate groupsoverlap with the pyrophosphate-binding site. This allows the enzyme to perform a reverse polymerization reaction causing the excision of the terminal nucleotidea his ext n reaH ch nuc
2
afcbftulttpfawAwb
dweEd
ss2a(tsrt
s a dinucleoside tetraphosphate. In case the primer-end is a chain terminator, this excision reaction are therefore associated with NRTI resistance. The excisioIV-1 RT causing the incorporation of nucleoside-MPs at the primer-end. If su
.5.3. Dinucleoside tetraphosphates (Np4Ns)The discovery of Np4Ns originates from studies on the mech-
nism of AZT resistance. During AZT therapy, TAMs wereound to be associated with resistance. However, when RTontaining these TAMs was compared to wild-type enzyme iniochemical polymerization assays, no change of susceptibilityor AZT-TP could be observed. Further investigations showedhat TAMs cause RT to perform a catalytic reaction that isnique for a polymerase (Meyer et al., 1998). When physio-ogical concentrations of ATP are added to the assay mixture,he TAM-mutated enzymes are capable of unblocking AZT-MPerminated primers. In this reaction, ATP acts as a pyrophos-hate donor that excises the chain-terminating AZT-MP byorming a dinucleoside tetraphosphate (Ap4AZT) and an extend-ble primer terminus (Fig. 4). While wild-type enzyme is only a
eak catalyst for this reaction, the presence of TAMs increasesZT-excision dramatically. The exact molecular mechanism byhich TAMs increase this primer unblocking activity has noteen determined, but docking studies indicate that TAMs are
rA
p
cision reaction rescues the primer for further elongation. Mutations increasingction is reversible (closed arrows), meaning that Np4N can act as a substrate ofleoside-MP is a chain terminator it inhibits further polymerization.
irectly involved in the interaction with the ATP substrate orith the adenosine portion of an intermediate formed during the
xcision reaction (Chamberlain et al., 2002; Meyer et al., 2002).xcision reactions and the effects of TAMs are discussed in moreetail in a separate review in this issue (Menendez-Arias, 2008).
The observation that HIV-1 RT can generate Np4Ns-inspiredcientists to investigate if the enzyme could also use them as aubstrate, that then could be turned into an inhibitor (Meyer et al.,006). Different Np4N analogues were synthesized by linkingdideoxynucleoside-MP (ddNMP) moiety to a nucleoside-TP
NTP) analogue (Fig. 5). A standard reverse transcription reac-ion in the presence of a suitable primer/template showed thatuch molecules indeed bind the RT active site, and incorpo-ation of the ddNMP moiety at the primer-end causes chainermination. The IC50s obtained with the different molecules
anged from 1.8 to >50 �M on WT RT and 0.13 to 1.3 �M onZT-resistant RT.The tetraphosphate linker allows bypassing the intracellular
hosphorylation step, thus decreasing the risk for interactions
176 D. Jochmans / Virus Research 134 (2008) 171–185
of Np
wf
awep
2
bhttfw
Hc(Dtintssti
imevdetected. Mutations in the virus samples at passage 11 showedan increase in purine–purine or pyrimidine–pyrimidine transi-tions but no purine–pyrimidine transversions, which confirmsthe mechanism of action of KP-1212 (Harris et al., 2005).
Fig. 5. Chemical structures
ith other NRTIs. On the other hand, the linker is an obstacleor stability and cellular permeability of the molecules.
The principle that dinucleoside tetraphosphates bind thective site of HIV-1 RT and that AZT resistance is associatedith hypersusceptibility to these molecules warrants further
xploration of analogous molecules with improved drug-likeroperties.
.5.4. Mutagenic nucleosidesThe high mutation rate of HIV-1 is well known. It has proba-
ly evolved as a mechanism to escape immune pressure, but nowas important clinical implications in the selection of resistanceo HAART. Mutagenic nucleosides aim at a further increase ofhis mutation rate to an extent that exceeds the error thresholdor virus replication. Proof-of-concept was first demonstratedith 5′-hydroxy-2′-deoxycytidine (Loeb et al., 1999).The most advanced mutagenic nucleoside for treatment of
IV-1 infection is KP-1461. It is a prodrug of KP-1212, alose 2′-deoxycytidine analogue with a different base-moietyFig. 6). KP-1212-TP is incorporated by HIV-1 RT in the nascentNA chain opposite to guanosine residues with an efficiency
hat is ∼10-fold lower than for dCTP incorporation. Althought decreases the incorporation of the next nucleosides, this isot the major mechanism of HIV-1 inhibition. More important,he incorporation of KP-1212 causes mutations during second-
trand DNA-synthesis where RT incorporates both the naturalubstrate (dGMP) and the purine mismatch (dAMP) opposite tohe KP-1212 moiety (Murakami et al., 2005). This mechanisms also confirmed in cell-based assays. Eleven passages of HIV-1
FK
4Ns that inhibit HIV-1 RT.
n the presence of 10 �M KP-1212 showed an increase in theutation rate by 55 and 91% in the HIV-1 RT-coding region and
nv gene, respectively. After 13 passages at this concentrationiral ablation was achieved, as viable virus could no longer be
ig. 6. Chemical structure of natural 2′-deoxycytidine, KP-1212 and its prodrugP-1461.
searc
oceo12miaoM
sAadtKfi
rrtppt
boiHriTsttobirrg
3
fivuwt
icc
OfStdor2
obIrvraftvdM
iioelotawitsdehqo
tcadpbritt4(
D. Jochmans / Virus Re
There is an obvious risk for side effects with mutagenic nucle-sides. Incorporation in the genomic or mitochondrial DNAould result in genotoxicity or mitochondrial toxicity. How-ver, different genotoxicity assays showed a negative (AMES)r only weakly positive (MLA, MNT) mutagenic activity of KP-212, which is comparable with approved NRTIs (Harris et al.,005). Despite KP-1212-TP being an efficient substrate for theitochondrial DNA polymerase �, cell-based studies showed no
ndication for mitochondrial toxicity. Possibly the exonucleasectivity of DNA polymerase � and/or metabolism or transportf KP-1212 into mitochondria play a role (Harris et al., 2005;urakami et al., 2005).A tempting benefit of using HIV specific mutagenic nucleo-
ides is the theoretical hurdle for the virus to become resistant.lthough there is only limited data available, KP-1212 remains
ctive on NRTI resistant strains. In vitro selection experimentsid not show an increase in resistance towards KP-1212. In con-rast, the harvested virus showed hypersusceptibility towardsP-1212 as well as AZT. This may be due to a reduced viraltness of the mutated virus (Harris et al., 2005).
Phase I clinical trials with KP-1461 are well advanced andecently a first-phase IIa trial was initiated to evaluate the antivi-al activity of the drug. In this study the safety, efficacy andolerability of KP-1461 will be investigated during a 124-dayeriod of monotherapy in HIV-1 patients who have failed multi-le antiretroviral regimens. The investigators expect to completehe trial in 2008.
Another way of increasing the mutation rate of HIV-1 woulde to use covert ribonucleoside analogues. Unlike deoxynucle-sides, ribonucleoside analogues are not directly incorporatednto the host genomic DNA, reducing the risk for side effects.owever, incorporation of ribonucleoside analogues cannot be
estricted to HIV-1 RNA, and the same amount of incorporationnto cellular RNA may happen and result in altered proteins.his may be only a transient aberration, because mRNAs havehort half-lives and proteins tolerate a wide variety of muta-ions. However, the potential effects of these nucleosides onRNA and rRNA structure are more difficult to predict. The ideaf covert ribonucleoside analogues has only been investigatediochemically, showing that certain nucleoside analogues cannduce mutations, both by misincorporation into RNA and byeverse transcription (Suzuki et al., 2006). An effect on viruseplication and/or cellular toxicity has not yet been investi-ated.
. Non-nucleoside RT inhibitors
In this section, we will discuss recent advances in the NNRTIeld. Important progress has been made with the clinical obser-ation that resistance against one NNRTI does not exclude these of newer NNRTIs in subsequent therapies. In addition, weill briefly discuss the novel NNRTI classes entering clinical
rials.
NNRTIs represent the second important group of HIV-1 RT
nhibitors with proven efficacy in the clinic. Although the nameould be interpreted as all RT-inhibitors with a non-nucleosidichemical structure, the group of NNRTIs is much more limited.
cma(
h 134 (2008) 171–185 177
nly compounds that bind to a specific pocket situated ∼10 Arom the enzyme’s polymerase active site are deemed NNRTIs.tructural biology showed that upon NNRTI binding, some dis-
ortion in the RT active site and in the primer grip occurs. Thisoes not dramatically change the binding affinity for the dNTPr the primer/template, but rather reduces the rate of incorpo-ation in a substantial manner (Spence et al., 1995; Xia et al.,007; Sluis-Cremer and Tachedjian, 2008).
Because of its potent activity on wild-type HIV-1 and its easef administration, efavirenz is the most prescribed NNRTI usedy patients in first-line antiretroviral therapy (Maggiolo, 2007).ts major drawbacks are certain adverse events, particularly neu-opsychiatric, and the nearly complete loss of activity when theirus selects the K103N mutation. Moreover, K103N confersesistance to licensed NNRTIs such as nevirapine, delavirdinend efavirenz. Selection of NNRTI-associated resistance occursrequently in the clinic and HIV-1 RT seems to be able to sus-ain multiple mutations in the NNRTI pocket without losingirus infectivity. Emergence of NNRTI resistance in the clinic isiscussed in another review in this issue (Martinez-Picado andartınez, 2008).At this moment, different novel NNRTIs are being evaluated
n clinical trials. Almost two decades of research focusing onmproving the resistance profile, led to the discovery and devel-pment of etravirine (De Corte, 2005) and rilpivirine (Janssent al., 2005) (Fig. 7), compounds that retain activity against aarge panel of mutant HIV-1 strains. Their exceptional spectrumf in vitro activity is thought to result from distinctive interac-ion points in the NNRTI pocket, and their torsional flexibilitynd compact structure that facilitates RT binding in numerousays, allowing easy adaptation to changes in the NNRTI bind-
ng pocket (Ren and Stammers, 2008). In large phase III clinicalrials, etravirine showed potent efficacy and a very favorableafety/tolerability profile in treatment-experienced patients withocumented resistance to currently approved NNRTIs (Lazzarint al., 2007; Madruga et al., 2007), the first time that an NNRTIas been shown to be sequenceable. This indicates that subse-uent to NNRTI-regimen failure, patients may still have furtherptions from the NNRTI class.
Another research effort for NNRTIs with an improved resis-ance profile resulted in the discovery of the benzophenonelass with GW-678248 as a prototype compound (Ferris etl., 2005). A N-propionyl sulfonamide prodrug of GW-678248emonstrated efficacy in NNRTI-experienced individuals inhase IIa clinical studies, but development was discontinuedecause of safety issues related to elevated liver enzymes andash (Boone, 2006). Different groups are currently investigat-ng modifications in this class of molecules that should retainhe antiviral activity while improving safety. Compounds inhis series reported to be in phase I clinical trials are VRX-80773 (Zhang et al., 2007) and R1206 (Klumpp et al., 2007)Fig. 7).
UK-453061 is representing a third chemical class currently in
linical development. The compound showed efficacy in a 7-dayonotherapy trial in NNRTI-naıve HIV patients, but more trials
re needed to demonstrate added value over current therapiesFatkenheuer et al., 2007) (Fig. 7).
178 D. Jochmans / Virus Research 134 (2008) 171–185
ucture
4
(tilpicowTopssw
itm
etasiusatk
Fig. 7. Chemical str
. Nucleotide-competing RT inhibitors (NcRTIs)
Recently a novel class of HIV-1 RT inhibitors was describedJochmans et al., 2006; Zhang et al., 2006). The chemical struc-ure of the prototype compound INDOPY-1 (=VRX-413638)s shown in Fig. 8. The molecule was obtained by screening aibrary of small molecules for anti-HIV activity and shows goodotency in cell-based HIV-1 replication assays (EC50 = 30 nM),n the absence of toxicity. The selectivity profile is unique asompared to current NRTIs and NNRTIs. INDOPY-1 is activen multiple species of Lentiviridae like HIV-1, HIV-2 and SIV,hich is different from the narrower antiviral spectrum of NNR-Is. In contrast to NRTIs, INDOPY-1 does not inhibit virusesutside the family of Lentiviridae. Subsequent cell-based and RT
olymerization assays pointed towards the reverse transcriptiontep as the target for inhibition. Because of the non-nucleosidictructure of the molecule it was expected that the compoundould act like an NNRTI. However, its unique selectivity and
Fig. 8. Chemical structure of INDOPY-1, VRX-413638.
ftrwin
nnRata
p(
s of novel NNRTIs.
ts activity on NNRTI-resistant strains were not consistent withhis hypothesis and warranted a more detailed exploration of the
echanism of action.RT enzyme kinetics tends to obey the laws of classical
nzyme kinetics, so this approach was extensively used to studyhe mechanism of action of NNRTIs and NRTIs. Where NRTIslways compete with the natural nucleotides that bind the activeite during reverse transcription, NNRTIs are non-competitivendicating that the inhibitor binds independently from the nat-ral substrate (Althaus et al., 1993; Ren et al., 2000). Forome NNRTIs increased inhibitor binding in the presence ofnucleotide was found and these NNRTIs are therefore referred
o as having a mixed non-competitive/uncompetitive bindinginetics (Fletcher et al., 1995; Maga et al., 2000). The kineticsor the binding mode of INDOPY-1 shows competitive inhibi-ion. The partial non-competitive component observed in oneeport (Zhang et al., 2006), remained marginal as comparedith the overall competitive character. Having different kinet-
cs compared to the NNRTIs, INDOPY-1 can be described as aucleotide-competing RT inhibitor.
The fact that INDOPY-1 competes with the incomingucleotides is unique and indicates that despite its non-ucleosidic structure this inhibitor binds the active site of HIV-1T (Fig. 9). More biochemical studies support this mode ofction and resistance experiments showed that active-site muta-ions but not NNRTI resistance-associated mutations affect the
ctivity of INDOPY-1.
The M184V mutation is most important in the resistancerofile of INDOPY-1. It confers an intermediate resistance3–5×) and when combined with other active site mutations (e.g.
D. Jochmans / Virus Research 134 (2008) 171–185 179
Fig. 9. Proposed model for the mechanism of action of NcRTIs. (1) After binding the template/primer, RT (orange ellipse) translocates to clear the active site,s ind thp via ip xt inc
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uH inhibitors: the hydroxylated tropolones (Budihas et al., 2005).The chemical structure of the prototype molecule �-thujaplicinolis clearly different from the N-hydroxyimides and diketo acids.Nevertheless, it is expected that the inhibition mechanism is
liding one base upstream along the template/primer. (2) Hence, RT is able to byrophosphate. (4) NcRTI-1 (red sphere) reversibly binds the active site of RTyrimidine. ((2) and (4)) In binding the active site, NcRTIs compete with the ne
115F) it causes high resistance (>10×). Interestingly, K65Rs associated with hypersusceptibility towards INDOPY-1 andven neutralizes the effect of M184V. This makes the resistanceattern of INDOPY-1 exceptional in that all other RT inhibitorshich lose activity on M184V (3TC, FTC, ABC, . . .) are alsoegatively affected by K65R.
It is anticipated that structural biology efforts will lead todetailed analysis of the binding mode of INDOPY-1 in the
ctive site of HIV-1 RT. This may create a unique opportunity toevelop inhibitors, not only for blocking the HIV-1 RT but alsoo inhibit other polymerases.
. RNase H inhibitors
Although only limited progress concerning the identificationf novel RNase H inhibitors was reported lately, the advance-ent made in understanding the mechanism of action and lack
f anti-HIV activity of current inhibitors justifies some furtheriscussion on this subject.
RNase H inhibition has remained an elusive target in anti-IV research. It was one of the first enzymatic activities to be
haracterized, and many small molecules have been discoveredhat specifically block HIV-1 RNase H activity. Unfortunatelyardly any cell-based antiviral activity in the absence of toxicityas been reported (for a review, see Klumpp and Mirzadegan,006).
The most promising candidate is RDS1643 (Fig. 10). Thismall-molecule diketoacid inhibits RNase H activity and HIV-1eplication at 10 �M while toxicity is only apparent at 60 �M.urther studies would be needed to prove that the inhibition of
he viral replication is indeed due to inhibition of HIV-1 RNaseactivity (Tramontano et al., 2005).The active site of RNase H contains two-metal ions that
re ligated by the active carboxylate residues Asp443, Glu478,
Fi(
e next incoming dNTP. (3) This is followed by polymerization and release ofnteractions with the enzyme as well as with the primer-end that needs to be aoming dNTP.
sp498 and Asp549. Both metal ions are essential in bindinghe substrate and catalyzing the phosphodiester bond hydrol-sis. The diketo acid motif incorporated in RDS1643 is aell-characterized pharmacophore of two-metal-ion active-siteinding for different enzymes, e.g. HIV integrase (for a review,ee Zhao et al., 2007). The first RNase H inhibitors that wereesigned based on the two-metal-ion mechanism of RNase Hction were the N-hydroxyimides (Klumpp et al., 2003). Forhis class of compounds the binding mode could be confirmedy crystallization, indicating the potential of structural biologyn future RNase H inhibition research (Klumpp and Mirzadegan,006).
A high-throughput screening of a library of pure natural prod-cts led to the identification of another chemical class of RNase
ig. 10. Chemical structure of the most important classes of RNase Hnhibitors. (A) N-hydroxyimides, (B) diketaoacids (RDS1643), (C) tropolones�-thujaplicinol) and (D) the vinologous urea VU447.
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lso related to metal chelation or an alteration of the coordina-ion of the two-metal ions at the active site. Interestingly, while-thujaplicinol is amongst the most potent RNase H inhibitorsith an IC50 of 0.21 �M, no antiviral activity in cell-based HIV-1
eplication assays could be observed.Recent investigations into the mechanism of action of �-
hujaplicinol may shed light on why current RNase H inhibitorsack antiviral activity (Beilhartz et al., 2007a). Standard RNase
assays measure the enzymatic activity under classic steady-tate conditions that involve multiple cycles of catalysis withultiple events of primer/template dissociation/association.nder these conditions �-thujaplicinol, as well as the otherNase H inhibitors, potently prevent RNA cleavage. However,hen nucleotides are added to the reaction mixture, activityf �-thujaplicinol is no longer observed and the RNA cleav-ge patterns resemble the ones obtained in the absence ofnhibitor. Further experiments suggest that the enzyme goesnto the polymerization mode in the presence of nucleotides,nabling RT to form a more long-lived complex with theNA/DNA substrate. Under these conditions the nucleic sub-
trate is able to compete with the inhibitor in binding theNase H active site, dramatically reducing the potency of
he inhibitor (Fig. 11). More research is needed to prove thathe same mechanism plays a role for the other RNase Hnhibitors.
A novel RNase H inhibitor that was recently reported, theinologous urea VU477, maintains its inhibitory activity in theresence of nucleotides (Beilhartz et al., 2007b). Further inves-igations are ongoing, but the compound is suggested to have anllosteric mechanism of action.
There are observations that argue against the possible use ofNase H inhibitors in antiretroviral therapy. RT has the potential
o excise incorporated chain terminating nucleotides to rescuehe primer-end (Fig. 4), but under normal conditions RNase
degrades the RNA-template before excision can occur. Thisrreversibly inactivates the template and prevents further poly-
erization (Nikolenko et al., 2005). It could be conceived thatnhibition of RNase H activity will result in a longer-lived tem-late and therefore more opportunity for the enzyme to rescuehe primer-end by excision. This would represent an antagonis-ic interaction between RNase H inhibitors and NRTIs. Whileuch antagonism has not yet been tested, there are indirect indi-ations that RNase H inhibition decreases NRTI activity. InRTI-resistant strains, mutations in the connection subdomain
re found alongside mutations in the polymerase active site (con-ributing to excision). These connection subdomain mutationsre thought to influence proper aligning of the RNA substratelong the RNase H active site, thus causing inhibition of RNase Hctivity and increasing NRTI excision and resistance (Nikolenkot al., 2007). Next to the positive contribution of RNase H activityn the potency of NRTIs, this enzyme has also been implicated inhe proper functioning of APOBEC3G. APOBEC3G is a cellu-ar protein that restricts HIV replication by binding to the viral
enomic RNA during virus production, and by causing muta-ions in the nascent viral DNA during the next round of infectionAguiar and Peterlin, 2008). Although the detailed mechanismas not been determined, recent experiments show that RNase
ttt2
h 134 (2008) 171–185
activity is required for activation of APOBEC3G. Inhibitionf RNase H activity by small molecules would therefore coun-eract the innate antiviral response of APOBEC3G (Soros et al.,007). Both observations on RNase H activity warrant furthernvestigations before potential RNase H inhibitors could be usedn the clinic.
. Nucleic acids
Since RT uses nucleic acids as both primer and template,t is an interesting target for inhibitory nucleic acids. In thisection, we will describe recent progress in the characterizationf aptamer–RT interactions and a novel screening approach tond short oligonucleotides that specifically bind RT.
.1. Aptamers
Early after the discovery of the SELEX (systematic evolu-ion of ligands by exponential enrichment) technology, multipleT-binding nucleic acid species, engineered through repeated
ounds of in vitro selection, were discovered (Tuerk et al., 1992;chneider et al., 1995; Burke et al., 1996; and reviewed inames, 2007). For the most potent of these aptamers, the antivi-al activity was carefully investigated. It turned out that uponntracellular expression in infected cells, aptamers are incorpo-ated in virions during assembly, and block the next cycle ofnfection early in reverse transcription (Chaloin et al., 2002;oshi and Prasad, 2002). In the case of the three strongest bind-ng aptamers, there was no emergence of drug-resistant viruses,ffering a relevant argument for further development of theseolecules towards a gene therapy for HIV.The structure of an RNA aptamer bound on HIV-1 RT was
olved using X-ray diffraction although the obtained resolutionid not allow characterization of detailed interactions (Jaeger etl., 1998). Presumably, this RNA ligand inhibits RT by bindingo a site that partly overlaps the primer/template-binding site. A
ore recent look at different HIV clades and SIV showed limitedoverage by the RNA aptamers tested. Position 277 seems to bekey factor in RNA aptamer sensitivity of the different enzymes
ested (Held et al., 2007).Recently the interactions between the ssDNA aptamer
T1t49 and HIV-1 RT were mapped by mutational studiesnd hydroxyl radical footprinting techniques (Kissel et al.,007a). The results demonstrated that the aptamer binds therimer/template-binding site and extends into the polymerasective site of the enzyme. In contrast with the RNA aptamers thisNA aptamer shows broad clade coverage and remains sensitive
n the presence of Arg277 mutations (Kissel et al., 2007b).Using the same technology, a DNA thioaptamer that binds
ightly and specifically to the RNase H domain of HIV-1 RTnd inhibits RNase H activity in vitro, was found. Inhibition ofT polymerase activity was not addressed, but since this thioap-
amer is a 62-mer duplex its binding site very likely extends inhe polymerase active site. This may explain the potent inhibi-ion of HIV-1 replication in cell culture (Somasunderam et al.,005).
D. Jochmans / Virus Research 134 (2008) 171–185 181
Fig. 11. Proposed model for the lack of activity of RNase H inhibitors in antiviral assays. (1) Binding of RT to its primer/template allows the RNase H site to cutt tive sic in a mb active
ao
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he RNA template every 9–18 bases. (2) Current inhibitors bind the RNase H acleavage. (3) Upon addition of dNTPs, the primer/template binding is forcedinding of primer/template competes off the RNase H inhibitor in the RNase H
A better understanding of the detailed interactions betweenptamers and the enzyme may lead to new insights for the devel-pment of small-molecule RT inhibitors.
.2. Hexanucleotides
The use of HIV-1 RT as a model system to investigate whetherery short non-structured oligonucleotides could specificallynteract with a protein target, led to the discovery of the oligonu-leotide Hex-S3 (5′-TCGTGT-3′) (Mescalchin et al., 2006). Thisexanucleotide binds specifically to HIV-1 RT (Kd = 5.3 �M),nd cross-linking studies, competition experiments and molec-lar modeling indicate binding at the bottom of the p66 thumbubdomain overlapping with the nucleic acid binding cleft. Bio-
hemical analysis showed that Hex-S3 does not inhibit theolymerization or RNase H activity of RT. In addition, cell-ased assays could not demonstrate an inhibition during the earlyhases of the replication cycle. Still a potent effect of Hex-S3
tvwa
te and force the primer/template to change its trajectory thus protecting it fromore stable tertiary complex (enzyme-primer/template-dNTP). This increasedsite. (4) As a result, the RNase H activity is rescued.
n virus particle production as determined by p24 quantificationas observed. Most likely Hex-S3 binding on RT has a direct
ffect on virion assembly and causes a decreased p24 production,r virus maturation.
. Primer/template-competing RT inhibitors
Recently new small molecules interfering with therimer/template binding of RT were identified (Yamazakit al., 2007). The elegant experimental set-up used a ham-erhead ribozyme fused to an aptamer that binds the RT
rimer/template-binding site. The unbound aptamer–ribozymedopts a conformation in which the ribozyme is active and canleave a fluorescently (FRET)-labeled substrate. Upon binding
o RT, the aptamer adopts a different conformation that inacti-ates the ribozyme. The N,N′-diphenylurea derivative SY-3E4as selected by screening a small-molecule library using this
ssay (Fig. 12). SY-3E4 not only disrupts the aptamer binding
182 D. Jochmans / Virus Research 134 (2008) 171–185
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o RT but also competes with the primer/template binding, result-ng in inhibition of DNA-dependent DNA polymerase activityith an IC50 of 2.1 �M on HIV-1 RT. Although the aptamersed in the screening is specific for HIV-1 RT, SY-3E4 alsolocks RT from HIV-2 (IC50 = 9.4 �M) and Moloney murineeukemia virus (MoMLV) (IC50 = 21 �M), but has reduced activ-ty on the Klenow fragment (IC50 = 99 �M). Interestingly theNA-dependent DNA polymerase activity was less susceptible
o SY-3E4 inhibition (IC50 = 150 �M for HIV-1 RT). When theompound was used in a cell-based antiviral assay it showed anC50 of 5.3 �M with no toxicity detected up to 100 �M. Theusceptibility remained unchanged in the presence of mutationsssociated with resistance to current RT inhibitors, but furtheresearch is needed to clearly demonstrate that the inhibition ofIV infection in cell culture is indeed due to RT inhibition. Theinding interface of the aptamer molecule on HIV-1 RT is quitearge and contains multiple interactions making it difficult toredict the binding site of SY-3E4. One potential candidate is aavity in the aptamer-binding site, close to the p66/p51 interfacehat contains residues conserved in HIV-1 and HIV-2 (Yamazakit al., 2007).
Previously, other N,N′-substituted urea derivatives wereescribed as primer/template-competing RT inhibitorsSkillman et al., 2002). These molecules were found bytructure-based design and combinatorial medicinal chemistrynd were shown to compete with the primer/template forinding to RT. KM-1, the most active molecule of the seriesWang et al., 2004b) (Fig. 12), blocked both DNA- as well asNA-dependent DNA polymerase activity with IC50s below.1 �M, which is different from SY-3E4. Similar with SY-3E4,M-1 inhibits HIV-1 and MoMLV RT but not Klenow fragment
nd it inhibits HIV replication in cell culture (EC50 = 2.5 �M)ith no toxicity up to 100 �M. But also here an alternative
echanism of action of HIV inhibition cannot be excluded.ased on their chemical structure, selectivity profile andechanism of action one could speculate that SY-3E4 andM-1 have a similar binding site on RT. The extended structure
ttdc
peting RT inhibitors.
f KM-1 may explain its increased potency and activity onNase H.
The fact that two groups independently find similar moleculesith the same mechanism of RT inhibition should inspire fur-
her research on these molecules. The characterization of theirinding site should be a priority since this would allow structure-ased design to further optimize the molecules. Similar to otherovel RT inhibitors, their activity on strains resistant to currentNRTIs and NRTIs holds a potential value.
. Conclusion
HIV-1 RT is a familiar target with proven clinical efficacy.ovel agents in this class remain attractive, especially if they
void typical adverse event profiles of licensed inhibitors andaise the resistance threshold. Despite the potency of currentombination therapy, the emergence of resistance-associatedutations remains a major cause for treatment failure. The
dvent of RT inhibitors with higher genetic barriers to theevelopment of resistance and/or novel mechanisms of actionromises to further reduce the risk of resistance development,n particular as part of HAART.
In this review, we discuss several strategies to develop nexteneration RT inhibitors. Some of these novel approaches likeocked-conformation nucleosides, dinucleoside-tetraphosphatesnd small-molecule primer/template-competing RT inhibitorsre only in early phases of research. More knowledge has beenenerated on the �-boranophosphate NRTIs but developmentemains challenging since their current lack of cellular per-eability does not allow direct evaluation of antiviral activity
nd toxicity. Equally difficult to develop might be the RNaseinhibitors because of their mechanism of action and potential
nterference with NRTI and APOBEC3G activity. Also NNRTIs
hat would follow-up on etravirine and rilpivirine are believedo be hard to pursue as it becomes increasingly difficult toemonstrate an added value in the clinic. Moreover, to remainommercially interesting, co-formulation potential is necessary
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or novel NNRTIs. The development of new NRTIs and NcRTIsight be less challenging. Replacing one of the current NRTIs in
o-formulations is a possibility if tolerability, resistance profilend interactions with other NRTIs prove to be clean. Besides,he addition of such compounds to an existing NRTI backboneould be argued as a class-sparing regimen.
Since HAART has improved dramatically, therapeuticpproaches that go beyond blocking viral replication mayecome the focus in anti-HIV discovery for the coming decade.einforcing the immune response, purging latent HIV reservoirsr using gene therapy to further protect patient cells against HIVnfection are amongst the approaches currently explored. These of mutagenic nucleosides fits in such approach since theyay be combined for short periods with HAART causing havoc
n the virus population. Transforming part of the patient cell pop-lations with genes that fight off HIV infection holds promiseor continuing the research on RT-binding aptamers.
Continued research efforts have resulted in a wealth ofovel and innovative RT inhibitors with potential value asntiretroviral therapy. Compounds with improved toxicity, phar-acokinetics and resistance profiles are poised to join the ranks
f the two licensed classes of RT inhibitors. But beyond NRTIsnd NNRTIs, novel drug classes that block this target are beingnvestigated. NcRTIs, RNase H inhibitors, primer/template-ompeting RT inhibitors, and RNA-based drugs all offer newvenues for inhibition. Attacking RT from multiple direc-ions should seriously compromise mutational escape routes.
hen RT inhibitors with complementary resistance pro-les are combined, resistance development could be severelyestricted—maybe prove to be impossible. Furthermore, newrugs from novel drug classes offer additional life-saving treat-ent options for treatment-experienced patients.
cknowledgement
The author wishes to thank Luc Geeraert for his help in thereparation of this manuscript.
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