Computer-Aided Molecular Design - American Chemical Society

Chapter 3

Structure-Based Design of Human Immunodeficiency Virus-1 Protease Inhibitors Correlating Calculated Energy with Activity

Downloaded by FUDAN UNIV on February 15, 2017 | http://pubs.acs.org Publication Date: March 31, 1995 | doi: 10.1021/bk-1995-0589.ch003

M . Katharine Holloway and Jenny M . Wai Molecular Systems Department, Merck Research Laboratories, West Point, PA 19486

We have found that a simple calculated energy value, E , correlates well with the observed in vitro enzyme activity of a series of HIV-1 protease inhibitors. This correlation was derived employing a test dataset of 33 inhibitors with modifications at the P and P ' sites. It has proved valuable in the structure-based design of subsequent HIV-1 protease inhibitors which exhibit significant structural variation. In particular, it has been successful in a truly predictive sense, i.e. predictions of activity were made prior to synthesis. Several examples of this are illustrated, including a precursor (41) to a current clinical candidate, L-735,524 (42). inter

1

2

Of critical importance to the application of computer-aided molecular design is the accurate assessment of the relative energy of interaction between two or more molecules of interest. In the design of pharmaceuticals, these molecules may correspond to an enzyme and an inhibitor, a receptor and an antagonist, an antibody and an antigen, or a D N A helix and an intercalator. Ideally, one would like the energetic evaluation to be as rapid as possible, while maintaining a high level of accuracy such that useful predictions of activity may be made in advance of biological testing. In the absence of accurate energetic evaluation, only qualitative modeling is possible, which, while useful, cannot reliably address the question of binding affinity. Historically, several approaches have been employed to calculate and/or predict binding affinity. The free energy of binding can be calculated directly via Free Energy Peturbation (FEP) calculations (7). This approach has been reported to yield free energies accurate to ± 1 kcal/mol with respect to experiment. However, due to the amount of computer time required, these calculations are impractical for routine assessment of the binding affinity of proposed compounds. Other simpler approaches have included Comparative Molecular Field Analysis (CoMFA) (2) and the Hypothetical Active Site Lattice (HASL) (3) method. While these approaches are rapid relative to FEP, they involve correlation with multiple calculated properties, fields, or sites, and their accuracy can be limited by several factors, e.g. the size and diversity of the training set of structures and the choice of alignment for these structures.

0097-6156/95/0589-0036$12.00/0 © 1995 American Chemical Society Reynolds et al.; Computer-Aided Molecular Design ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

3.

HOLLOWAY &WAI

Structure-Based Design of HIV-1 Protease Inhibitors

We report herein a single simple predictor of relative binding affinity for HIV-1 protease inhibitors which has been highly effective in a structure-based design program.


HIV-1 Protease The HIV-1 protease is an aspartyl protease which is responsible for processing the polyproteins coded for by the gag and pol genes of the HIV-1 virus, thus leading to production of the HIV-1 structural proteins (e.g. pl7, p24, p7, and p6) and enzymes (reverse transcriptase, integrase, and the protease itself) (4). Inactivation of HIV-1 protease has been demonstrated to result in the production of noninfectious virions (5). Thus, HIV-1 protease inhibitors have been an attractive drug design target for treatment of the Acquired ImmunoDeficiency Syndrome (AIDS) (6). Structurally, the HIV-1 protease is a symmetrical homodimer, which contains 99 residues and one characteristic Asp-Thr-Gly sequence per monomer. Figure 1 shows two views of an X-ray structure of the native form of the HIV-1 protease (7). In the first view, one can clearly see two β hairpin loops at the top of the structure which are commonly referred to as the flaps due to their dynamic nature. These flaps are presumably open initially to allow diffusion of the substrate or inhibitor into the active site and subsequently close to form both hydrogen-bonding and hydrophobic contacts with the small molecule. The second view is looking through the flaps at the active site, with the binding cleft roughly from top to bottom. The two catalytic aspartates are located directly below the flaps on the floor of the active site.

Figure 1. Two views of the α-carbon trace of the native HIV-1 protease.

Reynolds et al.; Computer-Aided Molecular Design ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

37

38

COMPUTER-AIDED MOLECULAR DESIGN

Qualitative Inhibitor Modeling


Initially, we began our modeling of HIV-1 protease inhibitors in a qualitative sense. They were docked in the active site of the native enzyme shown in Figure 1 using conformations which were based on X-ray structures of renin inhibitors bound in the active sites of fungal aspartyl proteases such as endothiapepsin (8) and Rhizopus pepsin (9). The first compound which was modeled was L-685,434 (1), an early lead, which was very potent in vitro (IC50 = 0.25 nM) but lacked cell potency (CIC95 = 400 nM) (10,11). R

1

Ο

1,R = H 2,R= O C H C H N ^ O 2

2

Based on the model of 1 in the active site, shown in Figure 2, it became clear that solubilizing groups which might affect the physicochemical and thus pharmacokinetic properties could be attached at the para position of the P i ' substituent. This led to an

Figure 2. L-685,434 (1) as modeled in the native enzyme active site. A molecular surface is included to illustrate the fit of the inhibitor in the active site cavity.


3.

H O L L O W A Y & WAI



inhibitor, L-689,502 (2), of comparable in vitro potency (IC50 = 0.45 nM) but enhanced cell potency (CIC95 = 12 nM) which was the first safety assessment candidate in this program (77). Unfortunately, it failed in safety assessment due to hepatotoxicity. When a subsequent X-ray structure was solved for the complex of 2 with HIV-1 protease (77), it was clear that we had accurately modeled the bound conformation for this inhibitor as shown in Figure 3. In the X-ray structure, the para substituent on the P i ' aromatic group does indeed point out away from the active site towards solvent. Its exposure to solvent is further supported by the fact that the morpholine group is disordered and thus is absent from this plot.

Figure 3. A comparison of the model of 1 (solid) and the X-ray structure of 2 (dashed). Semi-Quantitative Inhibitor Modeling Our success with qualitative predictions led us to try a more quantitative approach. We looked for a correlation not only between the modeled and X-ray structures, but also between the calculated energy and the observed in vitro activity. This was accomplished via the following protocol. Protonation states for the titratable enzyme residues were selected based on the fact that the enzyme assay is carried out at a pH of 5.5. Thus we chose to make all titratable residues charged, with the exception of Tyr59 and AspA25» orie °f P * °f catalytic aspartates (72). A representative set of inhibitors was then selected to form a test dataset. This included 16 inhibitors with modifications in P i ' and 16 inhibitors with modifications in P 2 , which are shown in Tables I and Π. Each group contained as wide a spread in activity and structure as possible. A model of 1 was constructed as described above and all subsequent models were derived from it. The inhibitor models were minimized in the enzyme active site using the M M 2 X force field (73), a variant of M M 2 (14). In all calculations the inhibitor was completely flexible and the enzyme was completely rigid. Dielectric constants of 1.5 for intramolecular interactions and 1.0 for intermolecular interactions were employed. m

e

au


39

40


Table I. Experimental IC50 Values and Calculated Enzyme»Inhibitor Intermolecular Energies for the P i ' Training Set of HIV-1 Protease Inhibitors.


No

Ri

R2

R3

IC50 pIC 0 nM

2

H

0.25

2

H

7.7

OH

1 CH Ph H 3 CH Ph CH 4 CH CH CH Ph H 5 CH -4-CF Ph H 6 (E)-CH CH=CHPh H 7 H CH C6F 8 CH -4-CH Ph H 2

2

2

3

2

3

2

2

5

c

EAcPep

E 02

-108.3

-134.8

-145.1

8.1135

-106.3

-131.1

-140.4

0.19

9.7212

-113.0

-139.2

-143.6

H

0.26

9.5850

-114.0

-141.3

-149.6

H

0.23

9.6383

-113.4

-138.3

-147.1

H

0.6

9.2218

-115.3

-139.1

-149.4

a

5

9.6021

E at

b

N

d

5

H

0.29

9.5376

-109.1

-135.4

-146.5

9

CH -4-NH Ph

H

H

0.31

9.5086

-110.4

-137.1

-146.1

10 11 12 13 14 15 16 17 18

CH -4-N0 Ph

H

H

0.27

9.5686

-118.4

-147.7

-151.4

H

H

H

2934

5.5325

-98.6

-125.0

-129.2

CH -4-OHPh

H

H

0.16

9.7959

-110.7

-136.6

-149.7

CH CH=CH

2

3

2

2

2

2

2

H

H

27.5

7.5607

-102.9

-131.5

-137.8

CH -4-IPh

H

H

0.72

9.1427

-113.4

-140.4

-148.4

CH C(0)Ph

H

H

5.42

8.2660

-114.2

-141.3

-150.3 -144.9 -146.0

2

2

2

2

CH -4-pyridyl

H

H

0.53

9.2757

-111.6

-134.4

CH SPh

H

H

0.25

9.6021

-112.3

-138.9

2

2

CH -4-t-butylPh H -137.0 -150.9 H 0.17 9.7696 -113.3 apIC o = -log(IC5o) Întermolecular energy (kcal/mol) calculated in the native HIV-1 protease active site. Intermolecular energy (kcal/mol) calculated in the acetylpepstatin inhibited HIV-1 protease active site. Întermolecular energy (kcal/mol) calculated in the L-689,502 inhibited active site. 2

5

c

We then looked for a correlation between the calculated intermolecular component of the energy (Ei r) and the observed I C 5 0 (15), assuming that Ei r might be proportional to the enthalpy of binding (AHbind) and that the entropy of binding (ASbind) might be small or constant, thus giving us something which might be proportional to the free energy of binding (AGbind)* the value we were actually interested in. nte

AGbind = AHbind - TASbind


nte

3.



Table II. Experimental IC50 Values and Calculated EnzymeÎnhibitor Intermolecular Energies for the Ρ ' Training Set of HIV-1 Protease Inhibitors. 2

5

"Ν®


21

22

/(

9.53

E cPep

d

-94.3

-122.3

-131.5

8.0209

-98.2

-128.1

-132.9

34.25

7.4653

-97.4

-124.7

-135.5

690

6.1612

-103.1

-135.0

-134.1

161

6.7932

-96.6

-127.7

-130.8

66.3

7.1785

-106.1

-134.6

-139.3

212.42

6.6728

-91.7

-133.6

-144.0

OH

121.8

6.9144

-99.2

-130.6

-134.2

OH

0.7

9.1549

-109.9

-135.6

-146.7

0.18

9.7447

-109.2

-136.4

-145.5

40.5

7.3925

-94.5

-135.5

-134.5

OH HN

6.9431

C

E 2

20

pIC 0

a

A

19

ICS? nM 114

5

5 0

6

HN„

HN^

OH

23

OH

24 P H

Ο

3

HN,.

25

26

OH

*8 ό HN.

27

ΗΝ Λ Λ /

28 HN

OH ^

A

6° 29

OH HN.

Continued on next page


41

42


No

R

IC50 nM

30 31 32

HN

OD

HN^CH

3

pIC 0

a

E at

5

b

N

EAcPep

E 02

C

d

5

30000

4.5229

-88.4

-120.1

-124.1

130

6.8861

-94.4

-122.3

-129.8

146

6.8356

-96.5

-123.2

-134.1

0.1

10.000

-111.7

-139.3

-149.1

38.6

7.4134

-108.1

-133.2

-138.4

ό 33

OH HN >v OH


A

34

4

Ο H N ^

0

H

ô apIC50 = -log(IC o) Întermolecular energy (kcal/mol) calculated in the native HIV-1 protease active site. Intermolecular energy (kcal/mol) calculated in the acetylpepstatin inhibited HIV-1 protease active site. Întermolecular energy (kcal/mol) calculated in the L-689,502 inhibited active site. 5

c

Tables I and II give the Ej t values for the compounds in the test dataset as minimized in three different enzyme active sites, the native enzyme (Nat), the acetylpepstatin inhibited enzyme (AcPep) (76), and the L-689,502 inhibited enzyme (502) (77). We observed a very good correlation between Ej ter and pICso, Le. -log(IC5o), for all three as shown in Figure 4. Surprisingly, a good correlation was observed even with the native enzyme active site where the flaps are open, rather than interacting with the inhibitor, as illustrated in Figure 1. Both the R and the cross-validated R values are listed below for each correlation. In all three cases these are very comparable, indicating that no one datapoint is overly influential to the correlation; thus, the correlation should be predictive. n

er

n

2

2

Native: p I C = -0.15435(E ) - 8.069 R=0.8524, R =0.7265, crossvalidated RÔ.6910 50

inter

(1)

2

Acetylpepstatin inhibited: pIC 0 = -0.17302(E er) - 14.901 R=0.7623, RÔ.5811, crossvalidated RÔ.5244

(2)

L-689,502 inhibited: p I C = -0.16946(E er) - 15.707 R=0.8852, R =0.7835, crossvalidated R =0.7551

(3)

5

int

50

int

2

2


3.

Structure-Based Design of HW-1 Protease Inhibitors


-80

I | I I I I I I I I I | I I I I I I I I IJ

ο

3-

-90

%

ο ο ο " ο ^ Ο CT

-100

Λ

0)

-120 T3 -


ι—H

Ο

-140

S

-150

•

Ο -

-130

V

f

11111111111111111111 r

-160

5

7

8

9

10

11

Observed pIC^ Figure 4. Plot of calculated enzyme»inhibitor intermolecular energy vs. experimental enzyme inhibition (pICso) for the training set of inhibitors, 1, 3-34 (circles = native enzyme active site, squares = acetylpepstatin inhibited enzyme active site, diamonds = L-689,502 inhibited enzyme active site).

2

4

6

10

8

12

14

Predicted pIC

50

Figure 5. Plot of predicted 0IC50 vs. observed pICso values for the predicted set of inhibitors. The line is one of unit slope, i.e. predicted PIC50 = observed pICso-


43

44



In fact, using these correlation equations, we were able to make predictions of activity for inhibitors prior to synthesis, i.e. true predictions and not post hoc explanations of activity. Because this was done over a period of time some of the predictions were made with the earliest correlation equation, that of the native enzyme, some with the acetylpepstatin inhibited enzyme, and some with the L 689,502 inhibited enzyme. The accuracy of these predictions is illustrated in Figure 5. Here, the line is one of unit slope, not a correlation line. There is only one significant outlier which will be discussed in more detail subsequently. It must be emphasized that we made many more predictions than are shown in Figure 5. Frequently, when our prediction for a compound was unfavorable, it was not synthesized; or when a prediction was favorable, the exact compound which was modeled was not synthesized, but rather an analog. Examples. In order to illustrate the power and the limitations of this simple approach, some specific examples of predictions are listed in Table ΙΠ. Like many others (77), we hypothesized that a symmetrical inhibitor might bind more tightly to the symmetrical active site of the HIV-1 protease. Thus, 35 was designed as a symmetrical version of 1. The activity of 35 was predicted using the earliest correlation equation and it is the outlier seen in Figure 5. We were concerned that our prediction was poor due to differences in binding between our model and experiment. However, when the X-ray structure was solved (78), this was obviously not the case, as shown in Figure 6. There are several other possible explanations for the overprediction of activity: (1) the use of the native enzyme model in Equation 1, rather than one of the inhibited enzyme models in Equations 2 or 3; (2) the presence of an additional hydrogen bond to the active site which would be overemphasized in a gas-phase molecular mechanics calculation; (3) the addition of significant favorable van der Waals interactions via the second aminoindanol moiety at the N-terminus; or (4) the existence of a higher barrier to obtaining the bioactive conformation necessary for binding. Of the four possibilities, the first may be eliminated since the activity predicted using the other two models is also exaggerated. However, factor (4) may play a key role, since 35 experienced a large decrease in its intramolecular enthalpy when minimized outside of the active site, an indication that the bound conformation may be significantly higher in energy than the global minimum.

Figure 6. A comparison of the modeled and X-ray structures of 35.


3.

HOLLOWAY &WAI


TABLE ΠΙ. Calculated EnzymeÎnhibitor Intermolecular Energies and Predicted and Observed p!C5Q Values for the Predicted Set of HIV-1 Protease Inhibitors. No Structure Ei ter Corr. Predicted Observed kcal/mol Eq.*> pIC o flCso 12.012 9.1612 a

n

c


5

4.9099

5.8965

8.7383

8.2676

9.8847

10.2676

d

7.2774

8.6518

8.1163

8.3058

Calculated intermolecular energy in the indicated active site. ^Correlation equation which was used for prediction based upon minimization in the corresponding enzyme active site.

Computer-Aided Molecular Design - American Chemical Society

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