Structure-Based Design of Novel Conformationally Restricted HIV

Chapter 12

Structure-Based Design of Novel Conformationally Restricted HIV Protease Inhibitors B . G . Rao, C . T . Baker, J. T . Court, D. D. Deininger, J. P. Griffith, E. E. K i m , J . L. K i m , B. Li, S. Pazhanisamy, F . G . Salituro, W . C . Schairer, and R. D. Tung

Downloaded by UNIV LAVAL on July 13, 2016 | http://pubs.acs.org Publication Date: July 7, 1999 | doi: 10.1021/bk-1999-0719.ch012

Vertex Pharmaceuticals Incorporated, 130 Waverly Street, Cambridge, MA 02139

In our efforts to discover a new generation of HIV protease inhibitors, which are structurally distinct and have minimal cross resistance to our current clinical candidate, amprenavir (VX-478), we designed a set of potent compounds bearing a novel backbone structure. Structural and modeling analysis of initial leads showed that these inhibitors bind in a strained conformation. We obtained dramatic improvement in binding by relieving the backbone conformational strain of the inhibitor. In this report, we will present our structure-based design approaches as well as the enzymatic results on these novel and highly potent inhibitors.

H I V - 1 protease inhibitors (Pis) have revolutionized treatment o f individuals with H I V and A I D S (/). Four P i s are already on the market and a fifth PI, amprenavir (with the chemical name of (3S)-tetrahydro-3-furyl N-((lS,2R)-3-(4-amino-Nisobutylbenzenesulfonamido)-l-benzyl-2-hydroxypropyl) carbamate, also formerly known as V X - 4 7 8 and 141W94) is undergoing advanced phase III clinical trials (2). amprenavir is a small molecular weight (506 Da), potent ( K i = 0.6 n M ; IC90 = 40 n M ) , and synthetically accessible H I V protease inhibitor that emerged from a focused application o f structure-based design approaches along in coordination with the disciplines o f medicinal chemistry and pharmacology (3,4). The chemical structure o f amprenavir is shown in Figure 1. In spite o f the availability o f potent regimens o f P i s in combination with reverse transcriptase inhibitors (RTIs), a cure is not in sight and patients may have to be on these drug regimens for long time. Additionally, the currently available drug regimens are not well-tolerated by many patients and are very difficult to comply with. Related to these issues is the problem o f drug resistance, which is a very compelling and immediate problem, since it can directly compromise the therapeutic efficacy o f a given treatment regimen. Therefore, there is a need to design a new generation o f Pis, w h i c h are more potent, easy to take and are not crossresistant to currently used Pis. Partaledis et al. (5) had shown using cell culture passage experiments that amprenavir is resistant to H I V - 1 protease with a unique set

© 1999 American Chemical Society

Parrill and Reddy; Rational Drug Design ACS Symposium Series; American Chemical Society: Washington, DC, 1999.

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of mutations at L I OF, M46I, I47V and I50V. It has also been shown that I50V is absolutely necessary for resistance against amprenavir by in cellular and enzymatic assays (6). W e have further characterized the biophysical basis o f the resistance o f these mutants by X - r a y crystal structure and computational methods (7). The results of this analysis is that the mutation o f He to V a l at residue 50 causes loss in hydrophobic interactions with amprenavir; mainly the loss o f interaction between the terminnal ( C D 1 ) methyl group o f 150 and the P2' aryl sulfonamide group o f the inhibitor contributes most to the loss o f binding. Based on these results, we have utilized the following four design concepts for the design o f next generation inhibitors: (a) Maintain strong interactions with the catalytic aspartates, (b) Maintain strong flap water interactions, (c) Space out P i and P i ' branching, so that inhibitors w i l l have less hydrophobic contact with the centrally located 150 and 184 side-chains, (d) M i n i m i z e interactions o f the P2/P2' groups with 150/150' side-chains. Linear Carbamate based Inhibitors The compound A i n Figure 2 is a close analog o f amprenavir with a K i o f < 0.1 n M . The shifting o f P i benzyl from the C a to the P i amide nitrogen spaces out the P i and P i ' branching. A l s o , such a compound is expected to maintain strong interactions with the catalytic dyad (D25 and D 2 5 ' ) and with the flap water. Therefore, the new compound B satisfies the first three design principles stated earlier. This compound showed good activity ( K i = 600 n M ) , but it is much less potent than the parent compound. A preliminary modeling study (see modeling details in the next section) o f this compound suggested that the benzyl group is too short to fill the P i pocket. The substitution o f P i benzyl with phenethyl lead to increased potency (Compound B l , K i = 40 n M ) . A l s o , this compound is more potent than its diastereomer B 2 ( K i = 170 n M ) , suggesting that the preferred configuration o f the central hydroxyl is the same as the parent compound. The chemical structures o f these two compounds are shown in Figure 3. However, the best o f these compounds is still > 400-fold weaker than the parent compound A . The new inhibitors are expected to be weaker due to two obvious reasons: (a) loss o f the hydrogen bond interaction with G 2 7 carbonyl o f the enzyme, since the P i amide has no hydrogen bond donor, and (b) added flexibility o f the main-chain and longer P i side-chain. But these factors alone are not expected to account for the >400 fold loss i n binding. Modeling

and Structural Analysis of Linear Carbamates

Modeling. In order to understand the weaker binding o f the linear carbamate inhibitors, we modeled compound B l i n the active site o f H I V - 1 protease. The crystal structure o f compound A complexed with H I V - 1 protease (E. E . K i m , unpublished results) was used for modeling the bound conformation o f compound B l . The program Q U A N T A (Version 4.0b, Molecular Simulations Inc., Burlington, M A , 1992) was used for model building. Energy minimization was carried out with C H A R M M force field within Q U A N T A program. A s the prime-side o f compounds A and B l are common, the bound conformation o f A in the crystal structure was



187

F i g u r e 1. Chemical structure o f amprenavir (VX-478).

Compound A

I—A

F i g u r e 2. Chemical structures o f compound A (a close analog o f amprenavir, previously described as VB-11,328 in reference 6) and compound B (the new carbamate inhibitor, derived from compound A by shifting the P i benzyl from Coc position to the amide N o f the same residue).



188

F i g u r e 3.

The two stereoisomers (Compound B l and Compound B2) o f a

linear carbamate inhibitor with Phenethyl side-chain at P i .



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adopted for compound B l . The phenethyl and the T H F groups on the non-prime side o f compound B l was modeled into the S i and S2 pockets, respectively, by manipulating the main-chain and side-chain torsions. This model was energy minimized using two different approaches. In the first approach, the enzyme coordinates and the flap water were held fixed, and all atoms o f the inhibitor were minimized first by steepest descents method for 200 steps, followed by 1000 steps o f Adopted Basis Newton Raphson method i n Q U A N T A . In the second approach, the enzyme coordinates were fixed, but flap water and inhibitor were allowed to move during minimization with a distance constraint between flap water and the ?2 carbonyl to mimic a hydrogen bond. The two step minimization process was applied in this case also. The two approaches resulted i n two energetically similar models. The two models, superimposed with the parent compound A are shown Figures 4 and 5, respectively. Firstly, it may be noted that the compound B l does not make hydrogen bond with G27 carbonyl o f the enzyme in either models and it has two additional rotatable bonds. In model 1, the T H F group overlays quite well with the same group o f compound A , but the aromatic ring o f the P i group is pushed out o f the pocket. In the second model, the P i ring is closer to the same group o f compound A , but the T H F groups do not overlap as well. Further, the P2 carbonyl in model 1 is not oriented to make the flap water interaction, whereas it was forced to make flap water interaction in the second model. In either case, the inhibitor is likely to loose significant binding due to (a) non-optimal interactions o f P i and P 2 groups, (b) weak flap water interactions, (c) lack o f interaction with G 2 7 carbonyl group o f the enzyme and (d) addition o f two rotatable bonds. This analysis o f the models is consistent with the higher K i for compound B l . However, the modifications o f these inhibitors suggested from these two models yielded only moderate improvements i n binding (results not shown). S t r u c t u r a l A n a l y s i s . In view o f these difficulties i n understanding this series o f compounds by modeling, we attempted crystallization o f several potent compounds in this linear carbamate series. The crystal structures described i n this paper were obtained by the following procedure: Purified wild-type protease was refolded at 5 ° C by rapid dilution from 7 M urea, into a buffer containing 25 m M sodium formate, 50 m M D T T and a 5-fold molar excess o f inhibitor. The complex was concentrated and washed extensively in 15 m M sodium acetate, 5 m M D T T buffer ( p H 5.4). Hexagonal rod shaped crystals grew at room temperature i n about a week by vapor diffusion against an ammonium sulfate reservoir as described previously (3), i n space group P 6 i . A l l data were collected at room temperature from one crystal each o f the various complexes, using a Rigaku R-axis l i e image plate area detector (Molecular Structure Co., Woodlands, T X ) . In all cases, all measured reflections beyond 8.0 A resolution were included in the structure refinement, which was carried out using X P L O R (#). The complex structures were refined using the slow-cool algorithm with starting coordinates from an isomorphous structure o f HIV-1 protease i n complex with V X - 4 7 8 (3). The program Q U A N T A was used for model building. The first crystal structure obtained in this series o f compounds complexed with HIV-1 protease is o f VB-13,674 ( K i = 17 n M ) , which is a close analog o f compound



190

Figure 4. A stereo diagram of the first model of compound B 1 (thick line) overlapped with the crystal structure of compound A (thin line) in the ac tive site of H I V - 1 protease.

Figure 5. A stereo diagram of the second model of compound B l (thick line) overlapped with the crystal structure of compound A (thin line) in the active site of H I V - 1 protease.



191

B l . VB-13,674 has pyridyl methyl carbamate at ?2 instead o f tetrahydrofuranyl carbamate in compound B l . The electron density o f the inhibitor at 2.2 A resolution is shown in Figure 6. A comparison o f this structure with the structure o f the parent compound shows that these two structures overlap quite well (Figure 7). Surprisingly, the P i group occupies almost the same position as the P i group o f compound A and also maintains the flap water interactions. O n both these counts, the two models are in disagreement with the crystal structure. Hence, it was not at once clear from the structure the reasons for weaker binding o f the linear carbamate based inhibitors with H I V - 1 protease. Further analysis o f the structure showed that the ?2? \ main-chain o f the inhibitor is quite distorted in the crystal structure: The Pl-P2 amide bond is twisted from planarity by about 35 degrees and the N - C - C - O H torsion is in an eclipsed conformation (Figure 7). These two factors in addition to the loss o f hydrogen bond o f the inhibitor with G27 carbonyl appear to be responsible for >400 fold loss in binding. A s models maintained the low energy conformation o f the two bonds, they did not predict the crystal structure conformation. The crystal structure revealed that the penalty for distortion o f the main-chain is less than the gain i n binding due to interactions with the flap water and the hydrophobic residues i n the S i pocket. This reality is obviously not captured by the modeled structures, reflecting the deficiency o f current force fields. This result, therefore, suggests that we have to be mindful o f such exceptions while using the docking and K i prediction algorithms using current force fields. Design of C y c l i c Lactam-based Inhibitors M o r e importantly for this study, these structural and modeling results lead us to new approaches in the modifications o f these inhibitors that would minimize the main chain distortions to gain binding. One o f these approaches is the design o f cyclic lactams: A close examination o f the crystal structure o f VB-13,674 showed that the carbamate oxygen o f the ?2 group and the C « methelene group o f the P i side-chain are close in space. This suggested that these two positions can be cyclized into a ring, thereby correct the amide distortion and decrease the flexibility o f the main-chain at the same time. W e modeled both 5- and 6-membered ring systems, as lactams, cyclic carbamates and cyclic ureas. A l l the cyclic models were energy minimized i n the active site o f HIV-1 protease using the procedures described earlier. O f all the models, 5membered lactam based inhibitors looked best in terms o f both conformations and energetics. The model o f a 5-membered lactam based inhibitor, minimized in the active site o f the enzyme, is overlapped with the crystal structure o f VB-13,674 in Figure 8. It is clear that the model overlays very well with the crystal structure: the backbone is not distorted, its carbonyl maintains flap water interaction and it offers possibilities for filling the S2 side different substituents on the ring. The first compound synthesized with a cyclic scaffold is, however, a cyclic carbamate C I (Figure9). This compound has a K i o f 1.2 u\M, and is as potent as the corresponding linear inhibitor C 2 ( K i = 1.6 p M ) (Figure 9). These results show that



192

F i g u r e 6. Diagram o f the 2|Fo| - |Fc| electron density around VB-13,674, a close analog o f compound B 1 .



193

F i g u r e 7. A stero diagram o f the overlap o f the crystal structures o f VB-13,674 (thick line) and compound A (thin line).

F i g u r e 8. A stereo diagram o f the overlap o f the model o f a cyclic carbamate (thin line), with the crystal structures o f V B - 1 3 , 6 7 4 (thick line).


194 Compound C I

-0CH3


°

o

Compound C 2

OCH3

F i g u r e 9. Chemical structures

o f a cyclic carbamate inhibitor, C I and the

corresponding linear analog, C2.

Figure 10. The 5-membered cyclic carbamates with different ?2 side-chains.


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novel scaffolding at the P i and ?2 lead to compounds with comparable or better potency than the linear carbamate inhibitors. The low potency o f the two molecules is due to lack o f any ?2 interactions. A s this cyclic carbamate scaffold does not provide any possibility to fill the S2 pocket, this series was not perused further. The 5-membered lactams were elaborated with different P2 side-chains, as shown in Figure 10. The K i values o f these compounds are given i n Table I. The compound

Table I. K i values of cyclic lactam based inhibitors illustrated in Figure 10.


Name Dl D2 D3 D4 D5 D6

Rl H Me H Me H H

R2 H H Me Me Allyl Benzyl

K i (nM) 550 130 60 115 9 0.6

D l with only hydrogens at R l and R2 has a K i o f 550 n M . This compound is about 2-fold better in binding than compound C I ( K i = 1.2 u M ) . When R l or R2 is methyl, binding improved further by 4-9 fold. It is also clear that the methyl substituent (R2) trans to P i benzyl group offers bigger improvement in binding than the methyl at R l , cis to the P i benzyl group. Further elaboration o f R2 methyl to allyl jumped the binding by more than 10 fold in compound D 5 with K i = 9 n M . When allyl is substituted with benzyl at R2, the binding improved even more dramatically by about 15 fold i n compound D 6 with K i = 0.6 n M . This compound is as potent as amprenavir in terms o f enzymatic inhibition. The crystal structure o f D 6 in the active site o f H I V - 1 protease is shown i n Figure 11. It may be seen from the structure that it fills all the four subsites and maintains flap water interaction without any distortion o f the main-chain conformation. However, compound D 6 , which is novel and potent, does not satisfy one o f our gaols o f maintaining high level o f potency against 150V mutant o f H I V - 1 protease. In fact, the K i ' s o f compound D 6 against I50V and M46I/I47V/I50V mutants are 43 n M and 135 n M , respectively. This reduction i n binding o f this compound is as high as that o f V X - 4 7 8 against these two mutants (6). A s our structural analysis o f the mutants suggested (7) the reduction o f binding results from the interactions o f the P2' group with 150 side-chain. H e n c e , , the K i results against mutants are not surprising as the P2 part o f the new inhibitor D 6 is similar to that o f V X - 4 7 8 . f

Conclusions The structural data and modeling has been utilized successfully to discover H I V - 1 protease inhibitors with novel scaffolds with P i and P2 substituents. These compounds are very potent; the best one described has a K i o f 0.6 n M . There are a lot



196

F i g u r e 11. Crystal structure o f compound D 6 in the active site o f H I V - 1 protease. The inhibitor is shown i n thick sticks. A l l atoms are colored according to the atom-types ( C : green, O: red, N : blue, S: yellow). (Figure is printed in color in color insert.)



197

o f examples o f inhibitor optimization published i n the literature which show improvement i n binding due to addition/optimization o f hydrophobic, hydrogen bonding or electrostatic interactions. The present work is one o f the very few studies and perhaps the first one to show substantial increase i n binding accompanying the improvement i n inhibitor conformation. We have discovered several more potent inhibitors i n this series and related analogs with different P1-P2 scaffolds during the first phase o f our second generation HIV-1 protease inhibitor discovery program carried out solely at Vertex Pharmaceuticals. However, all these compounds have the same prime side substituents at P i ' and P 2 ' sites, much similar to amprenavir. A s one o f the goals o f the program is to discover inhibitor which are chemically distinct from amprenavir series, the second phase o f this program focused on changing the prime-side o f these new inhibitors. The second phase o f the program is being carried out i n collaboration with Glaxo Wellcome and the newer compounds w i l l be described elsewhere. Acknowledgments We would like to thank o f all the members o f HIV-1 protease discovery team at Vertex for their contributions, and D r . V i c k i Sato for her support and encouragement. Literature Cited

(1) Chrusciel, R. A.; Romines, K. R. Exp. Opin. Ther. Patents, 1997, 7, 111. (2) Painter, G. R.; Ching, S.; Reynolds D.; St. Clair M . ; Sadler B. M . ; Elkins M . ; Blum, R.; Dornsife, R; Livingston, D. J; Parteledis, J. A.; Pazhanisamy, S; Tung, R. D., Tisdale, M . Drugs of the Future, 1996, 21, 347. (3) Kim, E. .E.; Baker; C. T.; Dwyer, M . D.; Murcko, M . A.; Rao, B. G.; Tung, R. D.; Navia, M.A. J. Am. Chem. Soc., 1995, 117, 1181. (4) Navia, M . A.; Sato, V. L.; Tung, R.D. International Antiviral News, 1995, 3, 143. (5) Partaledis, J. A.; Yamaguchi, K.; Tisdale, M ; Blair, E. E.; Falcione, C.; Maschera, B.; Myers, R. E.; Pazhanisamy, S.; Futer, O.; Cullinan, A . B.; Stuver, C.; Byrn, R. A.; Livingston, D. J. J. Virol. 1995, 69, 5228. (6) Pazhanisamy, S.; Stuver, C. M . ; Cullinan, A. B.; Margolin, N . ; Rao, B. G.; Livingston, D. J. J. Biol. Chem., 1996, 271, 17979. (7) Rao, B. G.; Dwyer, M . D. ; Deininger, D. D.; Tung, R. D.; Navia, M . A.; Kim, E. E. Antiviral Therapy, 1996, 1(Suppl. 1), 13. (8) Brunger, A . T.; Krukowski, A.; Erickson, J. W. Acta Cryst.,1990, A46, 585.


Structure-Based Design of Novel Conformationally Restricted HIV

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