A New HIV Integrase Inhibitor

Antiviral Chemistry & Chemotherapy 2009 20: 79–85 (doi: 10.3851/IMP1397)

Pointer Elvitegravir: a new HIV integrase inhibitor

Kazuya Shimura1 and Eiichi N Kodama1,2*

1Laboratory of Virus Control, Institute for Virus Research, Kyoto University, Kyoto, Japan 2Present address: Division of Emerging Infectious Diseases, Tohoku University School of Medicine, Sendai, Japan

*Corresponding author: e-mail: [email protected]

Integration is a distinctive and essential process in the inhibitor, elvitegravir, is currently showing promise in Phase HIV infection cycle and thus represents an attractive anti- III clinical studies. Once-daily administration of elvitegravir viral drug target. Integrase inhibitors combined with other has a comparable antiviral activity to twice-daily of ralte- classes of drug might contribute to long-lasting suppres- gravir in HIV-1-infected patients. Here, we highlight the sion of HIV type-1 (HIV-1) replication for many patients. Of salient features of elvitegravir: its chemical structure com- the numerous potential integrase inhibitor leads that have pared with representative integrase inhibitors, mechanism been reported, few have reached clinical trials and only of action, in vitro and in vivo activity against HIV and other one, raltegravir, has been approved (in late 2007) for the retroviruses, and the effect of integrase polymorphisms treatment of HIV-1-infected patients. Another integrase and resistance mutations on its anti-HIV activity.

Introduction

Combinational use of anti-HIV drugs as part of highly human genome lacks functional homologues of HIV IN active antiretroviral therapy (HAART) has opened a new [5], consequently IN is an attractive target for the devel- era in the treatment of HIV infection. With HAART, HIV opment of new anti-HIV drugs. replication has been remarkably suppressed, and reduc- Many different molecules have been reported to tion of viral load to undetectable levels and decrease of function as IN inhibitors and some of them have been HIV-associated mortality have been successfully main- evaluated for their in vivo potency in animal and human tained [1]. To date, nucleoside/nucleotide reverse tran- trials; however, until recently, no IN inhibitors were scriptase inhibitors (NRTIs), non-nucleoside/nucleotide approved in clinical use. Raltegravir (RAL; Merck) was reverse transcriptase inhibitors (NNRTIs) and protease approved for the treatment of HIV infection in Octo- inhibitors (PIs) have been widely used in HAART. How- ber 2007 [6]. RAL shows potent activity in patients ever, long-term administration of the drugs to keep viral infected with HIV type-1 (HIV-1) with resistance to replication to minimum levels unfortunately induces the other classes of drugs, including NRTIs, NNRTIs and emergence of drug-resistant variants and leads to treat- PIs [7,8]. Elvitegravir (EVG), another IN inhibitor that ment failure [2]. Moreover, some mutations can con- is currently in late Phase III clinical trials, also shows fer cross- and/or multidrug resistance to one or more potent antiviral activity in HIV-1-infected patients inhibitors in the same class of agents [3]. To cope with [9,10]. Here, we describe both in vitro and in vivo anti- this issue, in addition to discovery of new reverse tran- viral activity and resistance profiles of EVG. scriptase (RT) and protease (PR) inhibitors without cross-resistance to existing drugs, the development of Similarities and differences in chemical novel therapeutic agents targeting virus-specific mol- structures ecules remains necessary. Integrase (IN) is one of three enzymes (including PR Compounds containing diketo acids or derivatives and RT) encoded by the viral genome and, like PR and thereof, such as γ-ketone, enolizable α-ketone and car- RT, is essential for the virus replication. IN consists of boxylic acid, were postulated to be the pharmacophore three functional domains: N- and C-terminal domains, of inhibitors for retroviral IN activity. Structures of dike- and a catalytic core domain (CCD). The CCD conducts totriazole (S-1360; Figure 1), diketotetrazole (5CITEP), the intrinsic function of IN, integration, which is a diketo-carboxylic acid (L-708,906), 1,6-naphthyridine- ligation reaction between viral and host DNA [4]. The 7-carboxamide (L-870,810 and L-870,812) have been

designed, synthesized and demonstrated to inhibit consists of three sequential steps: firstly, two nucleotides HIV-1 IN as bioisosteres of a diketo acid motif. Such located at the 3′-terminal end of the reverse transcribed bioisosteres can be replaced with similar moieties. For HIV complementary DNA are removed (3′-processing); instance, the carboxylic acid could be replaced with secondly, the processed HIV genome is integrated into not only acidic bioisosters, such as triazole and tetra- the host chromosome (strand transfer); and lastly, the zole, but also by a basic heterocycle bearing a lone-pair gap between virus and host DNA is thought to be filled donor atom, such as a pyridine ring. The heteroaromatic by host repair machinery [18]. nitrogen in the pyridine ring mimics the correspond- Using an enzymatic strand transfer assay in vitro, ing carboxyl oxygen in the diketo acid as a Lewis base EVG has an enzymatic 50% inhibitory concentra- equivalent. The enolizable ketone at the α-position of tion (IC50) value of 54 nM [14]. EVG also inhibits the diketo acids can be replaced with a phenolic hydroxyl 3′-processing reaction, but the IC50 value of EVG for group, indicating that the α-enol form of each diketo this reaction is much higher than that of strand transfer acid is its biologically active coplanar conformation. (150–300-fold) [19]. These results indicate that EVG All bioisosteres of the diketo acid motif consist of the exerts antiviral activity primarily by selective blockage three functional groups that mimic a ketone, an eno- of the strand transfer reaction. lizable ketone and a carboxyl oxygen, and can have a An in silico docking simulation of IN with EVG has coplanar conformation (Figure 1). According to availa- been performed by Savarino [20]; however, the precise ble information, we designed and synthesized a series of binding site of EVG for IN has not yet been identi- compounds that have 4-quinolone-3-carbocyclic acid, fied as cocrystallization of IN with EVG has not been including EVG [11]. The 4-quinolone-3-carbocyclic reported. Recently, Marinello et al. [19] estimated the acid has two functional groups, a β-ketone and a carbo- binding mode of EVG using an in vitro oligonucleotide- cyclic acid, which are coplanar. Therefore, a coplanar based assay. The authors postulated that EVG preferen- monoketo acid motif in 4-quinolone-3-carbocyclic acid tially inhibits the strand transfer step as EVG recognizes could work as an alternative diketo acid motif, provid- the IN–DNA complex and binds the interface between ing novel insight into new structural designs and the 3′-processed viral DNA and IN. binding mode of this type of inhibitor. RAL (Figure 1) Most IN inhibitors, including EVG and RAL, prefer- also has a structurally similar moiety, with a pyrimidi- entially block strand transfer reactions; however, there none carboxamide as a diketo acid bioisostere [12]. are several compounds that inhibit another IN-catalyz- ing reaction, 3′-processing. For example, INH-I001/ Anti-HIV activity of EVG VNIII-083, one of the recently developed IN inhibitors, is a conceptually new β-diketo acid derivative and EVG shows potent anti-HIV activity to various HIV inhibits 3′-processing in addition to strand transfer strains that is comparable to other IN inhibitors, such as [21,22]. It is important to keep developing IN inhibi- L-870,810 and RAL [13]. EVG suppresses replication tors that target not only strand transfer reaction, but of various HIV-1 subtypes with 50% effective concen- also 3′-processing and other steps involving in integra- tration (EC50) values in the nanomolar to subnanomo- tion, such as IN interacting molecules. lar range, including clinical isolates that have NRTI, NNRTI and/or PI resistance mutations [14]. EVG also Clinical efficacy of EVG shows antiviral activity against HIV type-2 (HIV-2), such as ROD and EHO (subtypes A and B, respectively) Anti-HIV activity of EVG in HIV-1-infected patients strains with similar EC50 values to that observed for was initially evaluated in a Phase Ib clinical trial. In HIV-1 [14]. All 14 HIV-2 clinical isolates derived from this 10-day EVG monotherapy trial, 40 HIV-1-infected IN-inhibitor-naive patients were susceptible to EVG patients were administrated one of five dosage groups with EC50 values ranging from 0.3 to 0.9 nM [15]. It as follows: 200 mg twice daily, 400 mg twice daily, has been described that the fusion inhibitor, T-20 (enfu- 800 mg twice daily, 800 mg once daily or 50 mg EVG virtide), as well as most NNRTIs, minimally suppress plus 100 mg ritonavir once daily. Ritonavir was used for replication of HIV-2 [16,17]. In contrast to these inhibi- boosting the half-life of EVG. In the 400 mg twice daily tors, EVG is substantially active against HIV-2, indicat- group, all patients showed >1 log10 copies/ml reduction ing that EVG and other IN inhibitors are ideal for the of viral load and, moreover, half of the patients expe- treatment of both HIV-1 and HIV-2. rienced a >2 log10 copies/ml decrease (Table 1) [9]. In the Phase II clinical trial, >90% patients of both groups Mechanism of action of 50 mg plus ritonavir and 125 mg plus ritonavir

decreased viral load by >1 log10 copies/ml, and mean Integration is the crucial last step for the establishment CD4+ T-cell counts were increased compared with the of irreversible viral infection. The integration reaction control comparator protease inhibitor (Table 1) [10].

Figure 1. Structures of integrase inhibitors

HN N O N O OH S-1360

HN HN N N N 5CITEP O OH

OH L-708,906

O O O O

N N L-870,810 X= H O S N N O O OH CH3O O CH3 L-870,812 X= N C C N

CH3 F

H C 3 O H CH N 3 N CH3 N O CH3 N N H N Raltegravir O (MK-0518) O OH

OH CH3 H

CH3

H3CO N Elvitegravir O (GS-9137/JTK-303)

F O OH

Structures of representative integrase inhibitors are shown. A key moiety, a diketo acid and its alternatives, which are believed to be important for efficient binding to retroviral integrase, are highlighted in red and aligned with a dashed line in blue.

Antiviral Chemistry & Chemotherapy 20.2 81 K Shimura & EN Kodama

Table 1. Clinical efficacy of elvitegravir

Phase Iba Phase IIb 50 mg plus Comparator 50 mg plus 125 mg plus 200 mg 400 mg 800 mg 800 mg ritonavir protease ritonavir ritonavir Outcome twice daily twice daily twice daily once daily once daily inhibitor once daily once daily

Sample size, n 6 6 6 6 6 63 71 73 Mean baseline viral 4.75 (0.25) 5.01 (0.46) 4.46 (0.42) 4.74 (0.49) 5.00 (0.40) 4.54 (0.80)c 4.47 (1.07)c 4.71 (0.81)c s d load, log10 copies/ml (± ) c c c Patients with ≥1 log10 copies/ml 67 100 100 50 100 61 91 92 reduction, % c c c Patients with ≥2 log10 copies/ml 17 50 50 0 50 51 69 76 reduction, % Mean change of CD4+ T-cell 97 (166) 25 (37) -73 (127) -7 (127) 91 (101) 28 (69)d 52 (90) 61 (97)d count, cells/mm3 (±s d ) aPhase Ib 10 days elvitegravir monotherapy clinical trial [9]. bPhase II 24 weeks, randomized, active-control clinical trial [10]. cData at week 24. dData at week 16.

Through these trials, the potent activity of EVG was Q146P/S147G and H51Y/E92Q/S147G/E157Q) by demonstrated. Currently, a Phase III study of the safety a dose-escalating method with HIV-1IIIB [14]. Among and efficacy of ritonavir-boosted EVG (EVG/r) has been the selected mutations, T66I and E92Q, both of which initiated. The study is a multicentre, double-blind and were observed relatively early during the selection, randomized 48-week investigation. Participants receive mainly conferred EVG resistance with an EC50 change 150 mg EVG/r once daily or 400 mg RAL twice daily, of 37-fold and 36-fold increase, respectively. Garvey each with a background regimen, and are evaluated by et al. [27] selected T66(A/I/K)/V72A/E92(Q/V)/T124A/ the primary endpoint, which is defined as the achieve- A128T/P145S/Q146L/Q148(K/R)/V151I mutations ment and maintenance of confirmed HIV-1 RNA levels by increasing concentrations of EVG with HIV-1IIIB. <50 copies/ml [23]. Among these mutations, E92Q, P145S, Q148K/R and Recently, Gilead Sciences (Foster City, CA, USA) V151I mutations, which emerged at various time points announced that they were developing a CYP3A inhibi- of the selection, each showed >10-fold resistance to tor, GS-9350, which has no antiviral activity but selec- EVG. Goethals et al. [28] also induced the resistant var- tively inhibits CYP3A metabolism [24,25]. It has been iants and selected D10E/R20K/T66(A/I)/L74M/E92Q/ reported that EVG is first oxidized by CYP3A4 and H114Y/A128T/E138K/Q148R/S230R mutations by then glucuronidated by glucuronosyltransferase 1A1 dose-escalating and automated selection experiments and 1A3 in its metabolic pathway [26]; hence the with HIV-1IIIB. Among these mutations, T66I and E92Q, rationale for combining EVG with ritonavir, which both of which were observed in early passages of the in is known to be a CYP3A4 inhibitor (despite the fact vitro selection, and Q148R mutations conferred resist- that it was originally developed as a PI). It is expected ance to EVG (33-fold, 57-fold and 96-fold, respec- that coadministration of EVG with GS-9350, instead tively). Kobayashi et al. [13] observed the emergence of ritonavir, might reduce unfavourable adverse effects of T66(A/I/K)/V72A/E92(Q/V)/T124A/A128T/P145S/ caused by ritonavir. Moreover, Gilead Sciences intend Q146L/Q148(K/R)/V151I mutations in the IN-coding to use GS-9350 as part of a four-in-one single pill that region of HIV-1IIIB and phenotypic analysis revealed includes EVG and two NRTIs (tenofovir disoproxil that T66K, E92Q, P145S and Q148K/R mutations, fumarate and emtricitabine) [24]. They plan to initiate which were observed at various time points of the selec- a Phase II study in HIV-infected patients in the second tion, showed high resistance to EVG (>10-fold). These quarter of 2009. This four-in-one single pill will con- results indicate that mutations at positions T66, E92 tribute to the improvement in drug adherence. and Q148 were preferentially selected in these three in vitro inductions and confer high resistance to EVG as In vitro resistance profile primary mutations. Several experiments to select EVG-resistant variants In vivo resistance profile have been performed by us and other groups. The EVG-selected mutations are summarized in Figure 2. Recently, the Phase II clinical trial of EVG (GS-US-183- We selected resistant variants to EVG that contained 0105) has been completed. From the data of virologi- mutations in the IN-coding region (T66I/Q95K/E138K/ cal failure (VF) in patients administrated with EVG, the

in vivo resistance profile has been identified. T66I, E92Q Effect of IN polymorphisms on the efficacy and Q148K/R, which were confirmed as primary muta- of EVG tions for EVG resistance in vitro, were also observed among the VF patients (Figure 2) [29]. An amino acid One of the major obstacles to effective antiretroviral substitution of glutamine to histidine at position 148 therapy is the emergence of resistant variants. To maxi- (Q148H) was newly observed in vivo. N155H, which mize the antiviral activities of inhibitors, genotyping was not induced during in vitro selection by EVG, was assays are widely used to detect resistance mutations observed in vivo with a frequency of 39% of total VF [31]. In addition, prior to initiation of antiretroviral [29]. Importantly, E92Q, Q148H/K/R and N155H therapy, the presence of natural polymorphisms are also mutations were also observed in HIV-infected patients a considerable factor for efficacious therapy [32,33]. experiencing VF on RAL (Figure 2) [8,30]. These The IN genes of a total of 243 HIV-1 clade B infected results indicate a potential for cross-resistance between patients who had not received any IN inhibitors were these IN inhibitors. Indeed, it has been reported that analysed [34]. EVG-resistance-related polymorphisms viruses from EVG VF that acquire a high resistance to at positions 66 and 92 were not observed, whereas EVG show cross-resistance to RAL (>28-fold resist- Q148H and N155H polymorphisms were observed ance) in vitro [29]. The effect of resistant mutations on with a frequency of 0.4%, indicating that IN inhibitors the replication kinetics has also been evaluated in vitro (such as RAL and EVG) might show a reduced effect and in vivo. The in vitro fitness of HIV-1 harbouring on patients who are infected with HIV-1 harbouring EVG resistance mutations is relatively low: <20–86% these polymorphisms. In contrast to HIV-1, no EVG- reported by Shimura et al. [14] and 40–80% reported resistance-related polymorphism was observed in 11 by Goethals et al. [28]. A similar result was observed HIV-2 isolates (10 subtype A and 1 subtype B) [35] and in vivo: replication kinetics of viruses derived from 52 HIV-2 clinical isolates [15], although the number of EVG VF was 54% compared with wild type [29]. isolates tested was somewhat small.

Figure 2. Mutation map for integrase inhibitors /P 46L 48K/R 47G Q1 S1 Q1

In vitro selection Y 0E Y 14 28T 45S 38K L74M D1 R20K H51 V72A Q95K T124A A1 E1 E157Q S230R T66A/I/K E92Q/V H1 P1 V151I

NTD CCD CTD

150 212 288 T66A /I/K E92Q Q148H/K/R E138K S147G

In vivo N155H selection

Elvitegravir (EVG)-selected mutations are mapped onto HIV type-1 integrase. Mutations selected in vitro are cited from Garvey et al. [27], Goethals et al. [28], Kobayashi et al. [13] and Shimura et al. [14], and in vivo from McColl et al. [29]. Mutations that mainly contribute to EVG resistance are shown in red and those observed in both EVG and raltegravir in vivo are shown in bold. Three functional domains of integrase, N-terminal domain (NTD), catalytic core domain (CCD) and C-terminal domain (CTD), are indicated.

Antiviral Chemistry & Chemotherapy 20.2 83 K Shimura & EN Kodama

Table 2. In vitro antiretroviral and lentiviral activity of Acknowledgements integrase inhibitors This work was supported, in part, by a grant for the Antiviral activity, nM Promotion of AIDS Research from the Ministry of Virus EVG L-708,906 L-870,810 L-870,812 Health and Welfare of Japan (ENK) and a grant for MLV 5.8a NR 22a NR Research for Health Science Focusing on Drug Innova- FIV NR NR 2.4b NR tion from the Japan Health Science Foundation (ENK). SIV 0.5c 22,600d 3.2c, 7-15e 350f KS was supported by the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science, a50% inhibition of Molony murine leukaemia virus (MLV)-derived retroviral and Technology (Japan). KS is a research fellow of the vector as reported by Shimura et al. [14]. b50% inhibition of replication of feline immunodeficiency virus (FIV)-Pet as reported by Savarino et al. [37]. Japan Health Sciences Foundation. c50% inhibition of replication of simian immunodeficiency virus (SIV)mac239 as reported by Shimura et al. [14]. d50% inhibition of replication of SIVmac251 as reported by Pluymers et al. [38]. e95% inhibition of replication of as Disclosure statement reported by Hazuda et al. [39]. f95% inhibition as reported by Hazuda et al. [36]. EVG, elvitegravir; NR, not reported. The authors declare no competing interests.

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Accepted for publication 16 April 2009

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