I. Sildenafil and Erectile
II. Mechanism by which Sildenafil Enables Erection
III. Clinical Use of Sildenafil
IV. Catalytic Domain of PDE5
V. Active Site of the Catalytic Domain
VI. Future Work
Viagra™ (Sildenafil)< > is a modern medical “miracle of science” that treats erectile dysfunction, allowing men to have an erection and maintain erection longer. Erectile dysfunction can be treated by relaxing the corpus cavernosum in the penis— the smooth muscle cavities that becomes engorged with blood during erection—and the arteriolar smooth muscle in the penis—muscles surrounding arterioles (blood vessels). The erectile smooth muscle must relax and the arterioles must dilate to allow the penis to fill with blood (Glossmann, et al., 1999).
The pathway that results in smooth muscle relaxation begins with the release of NO, a vasodilator—increases blood flow to muscle tissue by expanding blood vessel diameter (Lodato, 2001). The neurotransmitter, NO is released into the blood stream when penile nerves are stimulated, causing muscle relaxation (Glossmann, et al., 1999). When NO diffuses into these muscle cells, soluble guanylate cyclase is activated. This enzyme transforms GTP into cGMP (cyclic guanosine 3’,5’-monophosphate). cGMP is a smooth muscle relaxant (Lodato, 2001). cGMP works by the allosteric inhibition of myosin kinase I (Glossmann, et al., 1999). By inhibiting the activity of kinase I, myosin is unable to be phospohorlyated; therefore, smooth muscle contraction cannot occur (Gillen, pers. Comm.). However, cGMP is broken-down by phosphodiesterases (PDEs), thus decreasing the levels of active cGMP muscle relaxants in smooth muscle tissue (Lodato, 2001). Viagra inhibits Type 5 cGMP PDE; thus, drastically reducing the degradation of cGMP. Therefore, Viagra facilitates elevated levels of cGMP in erectile smooth-muscle cells; the resulting relaxation of these penile muscles and arteriolar muscles allows many men with erectile dysfunction to obtain and maintain erections (Terrett, et al., 1996).
Viagra is taken orally, and about 40% of the ingested drug enters the bloodstream. However, only 4% of the drug that enters the plasma is not protein-bound; thus, free to actively inhibit PDE-5. Unused portions of the drug leave the body through excretion. It is important to note that because this pathway effecting muscle relaxation occurs throughout the intestines, Viagra can effect digestion. Another potential complication of Viagra is priapism—this is a painful erection lasting anywhere between hours and days. This condition can occur if muscle relaxation continues for a prolonged period of time (Glossmann, et al., 1999). Some of the other side effects of Viagra have been traced to cross-reactivity with PDE6 and PDE11 (two phosphodiesterases that are closely related to PDE5). This cross-reactivity is thought to be responsible for side effects such as blue-tinged vision and back and muscle pain reported by some patients (Card et al. 2004).
The catalytic domain of PDE5 adopts a helical structure composed of 16 alpha helices and contains residues 537-860 of the PDE5 molecule< >. The catalytic domain composed of three subdomains (N-terminal domain , linker domain, and C-terminal domain) and a region called the disordered region < >. The N-terminal domain < > adopts a cyclin-fold topology and is composed of a core helix (alpha helix 3 < >) as a component of a 5-helical bundle < > including alpha helices 1, 5, 6, and 8 as well as three additional short helices < > (alpha helices 2, 4, and 7). The linker domain is formed by 2 long, antiparallel helices < > (alpha helics 9 and 10). These two helices are separated from the N-terminal domain by a region from Ile 665 to Leu 675 called the disordered region < > that cannot be determined because it is not visible on electron density maps. The C-terminal domain < > is a helical bundle composed of five long alpha helices < > (11, 12, 14, 17, and 18) and three short helices < > (13, 15, and 16).
The active site of PDE5 < > is at one end of the deep hydrophobic pocket formed by the interface of the three subdomains of the catalytic domain. The active site is located in the middle of the helical bundle in the C-terminal domain < >. The site measures about 330Å and is composed of 4 subsites: the metal binding site (M site), core pocket (Q pocket), hydrophobic pocket (H pocket), and lid region (L region) < > (Sung et al. 2003). Sildenafil < > binds to PDE5 through 3 different types of interactions:interactions with two metals ions contained in the active site mediated through water, hydrogen-bond interactions with protein residues, and hydrophobic interactions with residues lining the cavity of the active site(Card et al. 2004).
The M site is formed
by residues His 613, His 617, His 653, Asp 654, His 657, N662, M681, E682, D724,
L725, and D764, and contains two metal ions (one is zinc
and the other is thought to be magnesium)<
(Card et al. 2004). The site is surrounded by alpha helices 6, 8, 9, 10, and
12. The Zn ion is bound to the side chains of His
617, Asp 654, Asp 764, His 653 <
and two water molecules. The Mg ion is coordinated
by Asp 654 <
and five water molecules. Three of the water molecules form hydrogen bonds with
His 657, Asp 682, and His 685, and another forms a bridge between the two metal
ions (Sung et al 2003).
The Q pocket is the main interaction site for the binding of sildenafil and PDE5 and accommodates the pyrazolopyrimidinone group of sildenafil < >. Gln 817, Phe 820, Val 782, and Tyr 612 line the pocket < > (Sung et al. 2003). Sildenafil binds in the Q pocket in two different ways. First, sildenafil interacts with the “hydrophobic clamp” which consists of a pair of hydrophobic residues < > (Phe 820 and Val 782)(Card et al. 2004). Phe 820 engages the primary aromatic ring on sildenafil through an offset face-to-face interaction. Second, Gln 817 is H bonded (through a bidentate bond) to the pyrazolopyrimidinone group on sildenafil < >. It is the orientation of this glutamine that determines to a large extent which PDEs bind cAMP and which (like PDE5) bind cGMP (Card et al. 2004). Also, the Zn ion contained in the M site coordinates Tyr 612 and a water molecule < >, which in turn are hydrogen-bonded to an additional water molecule that is hydrogen-bonded to the pyrazole moiety of sildenafil (Sung et al. 2003).
The H pocket is lined
by residues Phe 786, Ala 783, Leu 804 and Val 782
and accommodates the ethoxyphenyl group of sildenafil
(Sung et al. 2003).
The L region narrows the entrance to the active site of PDE5 and surrounds the methylpiperazine group of sildenafil < > (Sung et al. 2003). The L region extends out and essentially caps the active site upon inhibitor binding demonstrating a large degree of conformational flexibility. It is formed primarily by Tyr 664, Met 816, Ala 823, and Gly 819 < >. The ‘lid’, which extends over the pocket of the active site is created by residues 662-664 which sit on and extended loop that reaches toward the active site and thus blocks a portion of the pocket < >. The methylpiperazine group on sildenafil extends past the lid and is thus exposed to the protein surface at the active site where it has hydrophobic interactions with the residues Tyr 664 and Met 816 < > (Card et al. 2004).
By elucidating the structure of the catalytic domain of phosphodiesterases, researchers are better able to develop therapeutic compounds that are specific to a single phosphodiesterase. Sequences between the 12 human PDEs known are highly conserved; thus, molecules can usually bind to more than one type of PDE which can cause serious side effects depending on the location and function of the PDE. Future research aims include using the structural information outlined here to develop PDE inhibitors that can specifically be used in pulmonary vasodilator therapy. This clinical use had been impossible due to low target specificity resulting in serious health complications (Lodato, 2001).
Card, G.L., England, B.P., Suzuki, Y., Fong, D., Powell, B., Lee, B., Luu C., Tabrizizad, M., Gillette, S., Ibrahim, P.N., Artis, D.R., Bollag, G., Milburn, M.V., Kim, S., Schlessinger, J., and Zhang, K.Y.J. (2004) Stuructural basis for the activity of drugs that inhibit phosphodiesterases. Structure, 12: 2233-2247.
Glossmann, Hartmut; Petrischor, Guenther; Bartsch, Georg. (1999). “Molecular mechanisms of the effects of sildenafil (VIAGRA(R)).” Experimental Gerontology, 34(3): 305-318.
Terrett, Nicholas; Bell, Andrew S.; Brown, David; Ellis, Peter. (1996). “Sildenafil (VIAGRATM), a potent and selective inhibitor of type 5 CGMP phosphodiesterase with utility for the treatment of male erectile dysfunction.” Bioorganic & Medicinal Chemistry Letters, 6(15): 1819-1824.
Robert F. Lodato. (2001). “Viagra for Impotence of Pulmonary Vasodilator Therapy?” Am. J. Respir. Crit. Care Med., 163(2): 312-313.
Sung, B, Hwang, K.Y., Jeon, Y.H., Lee, J.I., Heo, Y., Kim, J.H., Moon, J., Yon, J.M., Myun, Y. Kim, E. Eum, S.J., Park, S., Lee, J., Lee, T.G., Ro, S. and Cho, J.M. (2003) Structure of the catalytic domain of human phosphodiesterase 5 with bound drug molecules. Nature, 425: 98-102.
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