Introduction

1. GS-9148-Diphosphate (GS-9148-DP), an Active Metabolite of a Novel Adenine Nucleotide Analogue, is an Effective Inhibitor of HIV-1 Reverse Transcriptase (RT) KL White, JY Feng, AS Ray, G Laflamme, F Yu, M Tsiang, R Wang, M McDermott, MD Miller, R Mackman, and T Cihlar Gilead Sciences, Inc., Foster City, CA, USA

2. Introduction • GS-9148 (phosphonomethoxy-2’-fluoro-2’,3’-dideoxydidehydroadenosine, Fig. 1) is a novel nucleotide HIV-1 reverse transcriptase (RT) inhibitor (NtRTI) with a favorable in vitro resistance profile against a spectrum of HIV-1 variants with all classes of NRTI mutations1 • GS-9131, a mono-phosphonoamidate prodrug of GS-9148 (Fig. 1), exhibits good oral bioavailablity in vivo and effectively delivers GS-9148 and the active diphosphate metabolite (GS-9148-DP) into lymphocytes2

3. Objectives • Define the enzymatic mechanism of action of GS-9148-DP against HIV-1 RT • Determine the potency of GS-9148-DP against HIV-1 RT and its selectivity with respect to host DNA polymerases in vitro

4. Structures of GS-9131, GS-9148, and the Active Metabolite GS-9148 - Diphosphate Intracellular enzymatic hydrolysis O N H N H N H 2 2 2 Phosphorylation N N N E t O N N N O O O O O H N N N P O P P N P O N N P O N O O H O O O H O O OH OH O H OH Ph O F F F GS-9131 Prodrug GS-9148 Parent NtRTI GS-9148-diphosphate Active metabolite

5. Methods HIV-1 reverse transcriptase assays: • Steady state incorporation assay. Wild-type HIV-1 reverse transcriptase heterodimer was expressed and purified as previously described3. Steady state inhibition constants (Ki) were determined as previously described3 using activated calf thymus DNA as a template • Pre-steady state kinetics. Transient kinetic experiments were performed at 37ºC using a KinTek Instrument Model RQF-3 rapid-quench-flow apparatus as described4 • Gel-based chain-termination assay. A 31-nt 33P-DNA primer was annealed to a 50-nt DNA template. 6 pM primer/template was incubated with 60 pM HIV-1 RT in RT buffer, 10 µM of the 3 non-competing dNTP and varying concentrations of the competing dNTP and N(t)RTI for 10 min at 37°C • ATP-mediated excision assay. ATP-dependent nucleotide excision assays used a 19-mer DNA primer chain-terminated with either GS-9148 or tenofovir pre-annealed to a D36-mer template and HIV RT 4-TAM (AZT-resistant) mutant with 1 mM dATP, various concentrations of dCTP (0.2, 2 or 20 µM) and 3 mM ATP

6. Methods (cont’d) Host DNA polymerase assays: • Polymerase alpha and beta assay. A heteropolymeric template (78-mer)/primer (18-mer), DNA pol alpha or beta (Replizyme, UK) and various concentrations of inhibitor were incubated at 37°C and used to measure incorporation of [3H]dATP at 30 and 60 min • Polymerase gamma assay. Activated calf thymus DNA, human recombinant DNA polymerase gamma expressed in baculovirus (Provided by William Copeland, NIEHS, Research Triangle Park, NC), and various concentrations of inhibitor were incubated at 30°C and used to measure incorporation of [3H]dATP at 10 and 20 min

7. Results:GS-9148-DP is a Competitive Inhibitor of HIV-1 RT with Respect to dATP by Double Reciprocal Plot Analysis GS-9148-DP Tenofovir-DP 0 . 00 8 0 . 02 4 I = 2 µM I = 1 . 2 µ M I = 1 µM 0 . 02 0 0 . 00 6 I = 0 . 5 µ M 0 . 01 6 I = 0 . 6 µ M 0 . 00 4 0 . 01 2 I = 0 1/ V I = 0 . 3 µ M m a x 0 . 00 8 1/ V v v m a x 0 . 00 2 1/ 1/ 0 . 00 4 I = 0 0 . 00 0 0 . 00 0 -0 . 004 -0 . 002 0 . 00 0 0 . 002 0 . 00 4 0 . 006 -0 . 002 0 . 00 0 0 . 002 0 . 00 4 0 . 006 1 / [S ] 1 / [S ] Wild-type HIV-1 RT DNA polymerase activity was assayed by quantification of 3H-dATP incorporation using a heteropolymeric DNA template. The mode of inhibition of GS-9148-DP and tenofovir-DP with respect to dATP incorporation was assayed with increasing concentrations of inhibitors and showed a constant Vmax, demonstrating competitive inhibition

8. Steady State Inhibition Constants for Wild-type HIV-1 RT NRTI K (µM) a i GS-9148-DP 0.80 ± 0.07 T enofovir-DP 0.18 ± 0.03 dd A TP 0.021 ± 0.004 b c Carbovir-TP 0. 1 1 ± 0.03 b c AZ T -TP 0.038 ± 0.006 c d4 T -TP 0.06 ± 0.02 c 3TC-TP 2.0 ± 0.4 c FTC-TP 1.2 ± 0.4 c a. Ki values are means from ≥ 3 experiments ± standard deviation b. ddATP is the active metabolite of ddI; carbovir-TP is the active metabolite of abacavir c. Ki values were obtained from reference 3

9. Pre-Steady State Incorporation by HIV-1 RT Wild-type HI V -1 R T Substrate k (s ) K (µM) k /K (µM s ) -1 a a -1 -1 a pol d pol d d A TP 183 ± 9 41 ± 6 4.4 ± 0.7 GS-9148-DP 22 ± 2 194 ± 32 0. 1 1 ± 0.02 T enofovir-DP 25 ± 1 42 ± 4 0.58 ± 0.07 • kpol: maximum rate of incorporation • Kd: dissociation constant • kpol/Kd: incorporation efficiency • Values are mean ± standard deviation

10. GS-9148-DP Functions as a DNA Chain-Terminator s s s s s s P P P P T T T T No dNTPs dATP(10), + dNTPs No dATP, + dNTPs GS-9148-DP(10), No dATP, dNTPs , dN , dN AZT AZT - - T T P P TF TF V V - - D D P P , dN , dN GS-9148-DP (10 µM) dATP, dNTP dATP, dNTP P P o o T T (10 µM (10 µM ) ) (10 µM (10 µM ) ) s s N N s s TTP(10), + dNTPs No TTP, + dNTPs AZT-TP(10), No TTP, dNTPs TTP TTP s s P P s s P P T T o o 0), 0), TFV-DP(10), No dATP, dNTPs TP TP N N 1 1 N N TT TT P P ( ( µ µ M M ) ) ( ( dATP (µM) dATP (µM) , , Chain-termination activity of N(t)RTIs was assayed with wild-type HIV-1 RT using a heteropolymeric DNA template. Distinct chain-termination products were observed that corresponded to sites of dATP incorporation for GS-9148-DP and tenofovir-DP (TFV-DP) and TTP incorporation for AZT-TP. For all N(t)RTIs, chain-termination was most efficient with low concentrations of the competing dNTP P P dN dN + + 0.4 2 10 50 0.4 2 10 50 0.4 2 10 50 , , (10) (10) 10), +dNTP 10), +dNTP TP TP ( ( - - -DP(10), No dA -DP(10), No dA TTP TTP -9148-D -9148-D V V o o N N No dATP, +dNT No dATP, +dNT No dNTPs No dNTPs TTP TTP AZT AZT dATP(10), +d dATP(10), +d GS GS TF TF 50-mer C C T T C C A A C C G G A A C C T T C C C C A A G G A A A A G G 31-mer T T A A A A

11. Excision of GS-9148 by AZT-Resistant HIV-1 RT 0 . 7 Tenofovir GS-9148 0 . 6 ) 1 - Values are mean ± standard deviation n 0 . 5 mi ( Excision Rate ( min-1 ) 0 . 4 ATP-mediated excision of incorporated NtRTIs was assayed using AZT-resistant HIV-1 RT and increasing concentrations of the next complementary nucleotide, dCTP. The excision of NtRTIs was decreased when the concentration of dCTP was increased e Rat 0 . 3 0 . 2 0 . 1 Excision 0 0 . 2 2 2 0 [ d C TP ] ( µ M )

12. IC [µM] Inhibitor 50 a b g Pol Pol Pol HI V -1 R T GS-9148-DP 44 ± 21 > 200 > 200 2.8 ± 1.2 a T enofovir-DP 61 ± 23 51 ± 3.5 > 200 0.6 ± 0.08 dd A TP > 100 2.4 ± 0.6 1.0 ± 0.4 0.2 ± 0.1 Inhibition of Host DNA Polymerases by GS-9148-DP a. Mean ± standard deviation

13. Conclusions • The active metabolite of GS-9148 is an effective competitive inhibitor of HIV-1 RT with respect to dATP, acting as an obligatory DNA chain terminator • Once incorporated into an elongating DNA strand, GS-9148 appears to be poorly excised by HIV-1 RT with multiple AZT resistance mutations • GS-9148-DP exhibits a favorable selectivity against host DNA polymerases including DNA polymerase gamma • In addition to previously presented favorable in vitro pharmacological profile1,2, these data further support the development of the GS-9148 oral prodrug (GS-9131) for the treatment of HIV-1 infection

14. References • T Cihlar, A Ray, D Boojamra, L Zhang, H Hui, D Grant, K White, M Desai, N Parkin, and R Mackman. Abstr. 45, CROI 2006, Denver. • A Ray, J Vela, R Mackman, L Zhang, H Hui, R Pakdaman, A Carey, M Wright, G Rhodes, and T Cihlar. Abstr. 498, CROI 2006, Denver. • K White, N Margot, J Ly, J Chen, A Ray, M Pavelko, R Wang, M McDermott, S Swaminathan, and M Miller, AIDS 2005, 19:1751-1760. • W Kati, K Johnson, L Jerva, K Anderson. J. Biol. Chem. 1992. 267:25988-25997