Parameterization and Simulation of the Physical Properties of

Chapter 3

Parameterization and Simulation of the Physical Properties of Phosphorothioate Nucleic Acids 1

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Kenneth E. Lind , Luke D. Sherlin , Venkatraman Mohan , Richard H. Griffey , and David M. Ferguson Downloaded by UNIV OF SYDNEY on September 24, 2015 | http://pubs.acs.org Publication Date: November 26, 1997 | doi: 10.1021/bk-1998-0682.ch003

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Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455 ISIS Pharmaceuticals, 2922 Faraday Avenue, Carlsbad, CA 92008

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The physical properties of nucleic acid complexes containing phosphorothioate backbone modifications are studied using molecular mechanics and dynamics calculations. Parameters for the phosphorothioate oligonucleotide are derived from ab initio calculations in a manner consistent with the AMBER 4.1 force field database. The force field is applied to simulate the structural properties of hybrid DNA:RNA duplexes starting in both the A -andB-form geometries. The results show the phosphorothioate -DNA:RNA complex has an overall Α-form geometry with minor groove widths between A- and B-form. Although model compound calculations indicate the sulfur substitution increases torsional flexibility around the phosphorous, molecular dynamics simulations show the modification does not have a great effect on backbone geometry. The results are also compared with previous studies of standard DNA:RNA hybrid structures. While a wide range of sugar puckers are typically associated with the DNA strand of hybrid duplexes, the average structures reported here show C3'-endo puckering, suggesting phosphorothioate substitutions may influence sugar conformation.

Background Over the past few years there have been many advances in the design and characterization of antisense oligonucleotides for the treatment of various human diseases. These short, exogenous nucleic acid strands are designed to bind complementary messenger ribonucleic acids (mRNA) with high affinity and selectivity within the cell, thereby halting translation and/or promoting degradation of the mRNA strand by ribonuclease H (RNase H) ( 7 - 5 ) . Although © 1998 American Chemical Society

In Molecular Modeling of Nucleic Acids; Leontis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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Downloaded by UNIV OF SYDNEY on September 24, 2015 | http://pubs.acs.org Publication Date: November 26, 1997 | doi: 10.1021/bk-1998-0682.ch003

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MOLECULAR MODELING OF NUCLEIC ACIDS

naturally occurring nucleic acids are effective templates for the design of antisense drugs, their utility is limited by several factors. These include poor penetration through cellular membranes, degradation by naturally occurring nuclease enzymes, and low affinity for the target mRNA sequence (4,5). To overcome these problems, synthetic modifications typically are proposed to alter the nucleotide (4). Common strategies include modifications to the phosphate backbone, alteration of the sugar ring (especially the 2' position), and base substitutions. Although a wide variety of modified nucleic acids are now available that show high affinity for RNA as well as nuclease resistance, most cause a loss in RNase H activity toward the target mRNA strand (6,7). This appears to be linked to the structure of the modified-DNA:RNA hybrid. Several studies have shown that RNase H most likely recognizes the unique structural features of naturally occurring DNA:RNA hybrids (8-13). These duplexes tend to have an overall Α-form geometry, with unique differences in each strand. The R N A strand has characteristics of Α-form geometry, such as a northern sugar pucker, while the D N A strand has a unique form that is a mixture of both A - and B-type geometries. The shape of the minor groove may be especially critical, lying somewhere between ideal A - and B-form geometries, as seen by the positions of the phosphate groups and the width of the groove. It is hypothesized that these unique features of the hybrid duplex allow RNase H to distinguish D N A : R N A from pure B-form D N A : D N A and Α-form RNA:RNA geometries (8,13). In fact, it does appear that the enzyme recognizes and binds to the minor groove of the hybrid duplex. Some oligonucleotide modifications, however, do support RNase H activity. One of the first generation of antisense drugs developed, phosphorothioates (shown in Figure 1), falls in this category (14). While this

Figure 1. (a) Phosphate and (b) Phosphorothioate backbones.



3. LIND ET AL.

Physical Properties of Phosphorothioate Nucleic Acids

change commonly is thought to have only a minor effect on the structure of the nucleic acid, phosphorothioates show a reduced binding affinity to complementary R N A (75). In addition, the affinity depends on the chirality of the phosphorothioate center. The R configuration (where the sulfur atom points into the major groove) has a higher affinity for the target R N A as compared to the S configuration (where the sulfiir points outward from the duplex) (16). Although phosphorothioates hold significant promise for the development of antisense therapies, the physical properties of these oligonucleotides, especially as they pertain to structure and function, are not well defined. Experimental structural data is simply not yet available for phosphorothioateD N A : R N A hybrids. In the present study, we examine the structural properties of phosphorothioate oligonucleotides using a combination of ab initio, molecular mechanics, and dynamics techniques. Model compounds are used to parameterize the modified backbone consistent with the A M B E R 4.1 force field database for simulating proteins and nucleic acids. The new force field is applied to simulate the average properties of a nucleic acid sequence taken from a library of antisense targets. Comparisons are made between phosphorothioate-DNA:RNA simulations started in both A - and B-form geometries. The results are further compared to experimental and theoretical studies of standard-DNA:RNA hybrids to identify structural features that may be induced by the modified oligonucleotide. Parameterization The model compounds used to parameterize the phosphorothioate backbone are shown in Figure 2. For completeness here, we have also re-evaluated the analogous phosphate fragment using the protocols reported by Cornell et al. in the development of the A M B E R 4.1 force field database (77). Partial atomic charges for the phosphorothioate fragment were initially derived from ab initio calculations at the HF/3-21G* level (18). These values were further evaluated by fitting the 6-31G* electrostatic potential with the RESP algorithm (19). The original set (performed before the availability of the RESP program) was virtually identical to the RESP-fit set which is not surprising considering the size of the fragments. The partial charges derived for the fragments are shown in Figure 2. These have been included with the standard residue charges given in the A M B E R 4.1 database. No other changes were made to the existing partial charges in the database. As might be expected, the sulfur substitution reduces the partial charge on the phosphorous center, slightly reducing the P-0 dipole moments of the modified center. The van der Waals parameters for the phosphate sp oxygen atom (standard type 02) and phosphorothioate sulfur atom (new type SD) were modified based on water docking studies. The HF/6-31G* optimized gaucheIgauche-fragmentwas aligned with a water molecule such that the P 0 or POS angle was bifurcated as shown in Figure 3. The water molecule was then manually adjusted along the water oxygen to phosphate non-bond vector while the rest of the molecule was held fixed to determine the energy as a function of distance. The 2

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(a) Downloaded by UNIV OF SYDNEY on September 24, 2015 | http://pubs.acs.org Publication Date: November 26, 1997 | doi: 10.1021/bk-1998-0682.ch003

^

\

+1.1659 F \ / "o -0.4954 ^

3

-0.6950 3

(b)

Figure 2. Model compounds used to parameterize the A M B E R 4.1 force field database. Partial atomic charges for (a) current A M B E R phosphate, and (b) new phosphorothioate for inclusion with existing nucleic acids.

Ο

Ο

Η/ Ή ο

Λ

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ο

Η/ Η

2

S ^ O

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ο

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CH, 3

(a)

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Figure 3. Water docking setup for (a) dimethyl phosphate and (b) dimethyl phosphorothioate models.

energy profile was then calculated using MP2/6-31G* single points and corrected to account for the monomer energies. This set of calculations was repeated using molecular mechanics with default van der Waals parameters taken from the A M B E R 4.1 database. To reproduce the profiles, the R* and epsilon values for 02 (of the phosphate fragment) were increased from 1.66 to 1.768 Â and from 0.21 to 0.25 kcal/mol, respectively. The SD R* and epsilon were started from the values for atom type S of the A M B E R 4.1 database and increased from 2.00 to 2.385 Â and from 0.25 to 0.50 kcal/mol, respectively. The resulting energy profiles are compared in Figure 4. These non-bonded parameters were subsequently applied to evaluate the torsional energy profile of the two model compounds. Following the methodology outlined by Cornell et al., the low energy conformers of the



3. LIND ET AL.


conformational energies calculated at the MP2/6-31G* level. This proved to be more difficult than we anticipated due to the asymmetry of the phosphorothioate fragment. In addition, the phosphorothioate energy surface is significantly flatter than the standard phosphate energy surface making direct comparisons of coefficients complex. The final energies are summarized in Table I. For all model compound calculations, bond stretching and angle bending terms were taken from the A M B E R 4.1 force field database. The missing terms involving the S-P bond and S-P-X angles were taken from the ab initio optimized dimethylester shown in Figure 2 were built and evaluated using both ab initio and molecular mechanics calculations. The four low energy forms, trans/trans (1807180°), transigauche+ (180760°), transi gauche- (1807300°), and gaucheIgauche- (3007300°) are shown in Figure 5. The conformers were optimized at the HF/6-31G* level. The evaluated MP2/6-31G* single point energies are reported in Table I. Although these values show slight differences with those reported by Cornell et al. for the dimethyl phosphate conformers, the trends are the same, with the gauche/'gauche conformer in the lowest energy form. Since we modified the 02 van der Waals parameters, the OS-P-OS-CT torsional coefficients for the standard phosphate backbone also required reparameterization. Based on the ab initio energies, the V I , V2, and V3 coefficients were adjusted starting from the default parameters taken from the A M B E R 4.1 database. In

•

Q M phosphate

—-•—-MM

phosphate

— A - - Q M phosphorothioate —-X—MM

phosphorothioate

Figure 4. MP2/6-31G* (QM) interaction energy and new molecular mechanics (MM) interaction energy for dimethyl phosphate and dimethyl phosphorothioate with a single water molecule.


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46

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(c) (d) Figure 5. Dimethyl phosphorothioate model fragments: (a) trans/trans conformation, (b) transigauche+ conformation, (c) trans/gauche- conformation, and (d) gauche-/gauche- conformation. Phosphate analogues of (b) and (c) conformers are identical due to symmetry.

Table I: Conformational energies for dimethyl phosphate and dimethyl phosphorothioate fragments.* 1

HF/ 6-31G*

Conformation

MP2/ 6-31G*

trans/trans 0.00 translgauche+ -1.28 trans/gauchegauche/gauche -1.57 a

kcal.

b

AMBER New

AMBER Original

Phosphate 0.00 0.00 -1.74 -1.60

0.00 -1.41

-2.85

-2.83

-2.86

HF/ 6-31G*

MP2/ 6-31G*

AMBER New

Phosphorothioate 0.00 0.00 0.00 -0.02 -0.27 -0.30 -0.59 -1.03 -1.04 -0.46 -0.89 -0.82

translgauche+ and transi gauche- values identical for phosphate due to

symmetry of P 0 ( O C H ) - . 2

3

2

contrast to the parameterization of the phosphate, the torsional coefficients for the phosphorothioate were initially set to zero, and fit using dihedral driver calculations with the SPASMS module of A M B E R . A n iterative process was performed in which we first measured the torsional profile due to van der Waals and electrostatics, and second adjusted V I , V2, and V3 to reproduce the



3. LIND ET AL.


geometries. The force constants were estimated from the standard 02-P and 02P-X parameters based on trends noted in ether and thiol parameters reported in the A M B E R 4.1 database. We used a simple percent reduction in sulfur versus oxygen stretching and bending which worked surprisingly well. The values were checked using a normal mode analysis with the N M O D E module of A M B E R . The predicted experimental frequencies were compared with ab initio frequencies calculated at the HF/3-21G* level. Good agreement was obtained using a uniform scaling factor of 0.89 for the ab initio values. The complete set of new parameters for the phosphate and phosphorothioate model compounds is given in Table II. The new atom type (SD) and parameters have been added to the A M B E R 4.1 force field database for simulating proteins and nucleic acids. For completeness, we have also included a comparison of geometrical parameters taken from optimized model compound structures using the updated A M B E R 4.1 force field and HF/6-31G* basis set calculations. These values are given in Table III.

Table II: New parameters for phosphate and phosphorothioate fragments.** Parameter van der Waals: 02 SD*

Value R*,A

ε, kcal/mol 0.2500 (0.21) 0.5000 (0.25)

1.7680 (1.66) 2.3850 (2.00)

Bonds:

2

^ kcal/(molA ) r?

SD-P

1.960

420.0

Angles:

2

Kq, kcal/(mol rad )

02-P-SD OS-P-SD Torsions: OS-P -OS-CT

SD-P -OS-CT

122.90° 108.10°

112.0 80.0 γd

C

Yn/2 1.00 (0.25) 0.25 (1.20) 1.25 (0.00) 0.15 1.05 0.25

e

n 3 2 1 3 2 1

0.0 0.0 180.0 0.0 0.0 0.0

a

b

Original values from A M B E R 4.1 force field (17) given in parentheses. New sulfur compared to standard sulfur atom of A M B E R . Magnitude of torsion in kcal/mol. Phase offset in deg. Periodicity of the torsion. c

d

e


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Table III: Selected Geometrical Parameters. MM


Dimethyl Phosphate r (OS-P) (Â) r (02-P) (Â)

a

1.62 1.48

1.64 1.47

θ (OS-P-02)

108.14°

107.41°

θ (OS-P-OS) θ (02-P-02)

103.12° 119.92°

99.63° 124.76°

1.62 1.48 1.96

1.63 1.47 1.98

θ (OS-P-02)

107.02°

108.13°

θ (OS-P-SD)

108.27° 103.44°

108.18°

Dimethyl Phosphorothioate r (OS-P) (A) r (02-P) (A) r(SD-P)(A)

θ (OS-P-OS)

121.42° θ (02-P-SD) A M B E R optimized geometry with new parameters. geometry. a

99.57° 122.32° b

HF/6-31G* optimized

Molecular Simulations Methods Starting Α-form and B-form structures of the sequence d[CCTATAATCC]r[GGAUUAUAGG] were model built using the N U C G E N module of the A M B E R 4.1 package (20). The D N A strand was created with an R configuration phosphorothioate backbone (designated as ps-DNA) and the R N A strand with a standard phosphate backbone. This setup causes the sulfur atoms to point into the major groove of the duplex. These structures were minimized in vacuo to relax the hydrogen atoms after which counter ions were added in the EDIT module of A M B E R to neutralize the net charge. The resulting structure was solvated with explicit TIP3P water molecules to surround the nucleic acid by approximately 9Â in each direction. The B-form structure contained 8210 atoms in a box approximately 53Â χ 40Â χ 40Â. The Α-form structure was composed of 7721 atoms and was contained in a box approximately 56Â χ 37Â χ 37 Α. Initially, the SANDER module of A M B E R 4.1 was used to minimize the water and counter ions to an energy convergence of

Parameterization and Simulation of the Physical Properties of

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