Patent Application: US-41583903-A

Abstract:
the present invention relates , inter alia , to the crystal structures of a phosphodiesterase 5 and pde5 / pde5 ligand complex and their uses in identifying pde5 ligands , including pde5 inhibitor compounds . the present invention also relates to methods of identifying such pde5 inhibitor compounds and their medical use . also contemplated by the present invention are crystals of pde5 / pde5 inhibitor complexes .

Description:
the term “ apo ” as used herein is taken to mean any protein ( or named protein ) that is detached from a / its ligand ( s ) and / or prosthetic group ( s ). the term “ active site ” as used herein is taken to include any site ( e . g . specific groups ) within a molecule ( and associated metal ions and / or hydration molecules ) where specific activity is undergone . such activity could include binding of a ligand to the site , catalysis of the molecule &# 39 ; s substrates by the site , recognition of a ligand by the site , etc . the term “ buffer ” as used herein is taken to include any solution containing a weak acid and a conjugate base of this acid ( or , less commonly , a weak base and its conjugate acid ). thus , a “ buffer ” as used herein resists change in its ph level when an acid or a base is added to it , because the acid neutralises an added base ( or , less commonly , the base neutralises an added acid ). the term “ precipitant ” as used herein is taken to include any substance that , when added to solutionm ( usually of macromolecules ), causes a precipitate to form or crystals to grow . the term “ complex ” as used herein is taken to mean a protein with ligand ( s ) bound and may be formed before , during or after protein crystallisation . the term “ soaking ” as used herein is taken to mean the addition of a solution containing a ( usually ) small molecule ( e . g . inhibitor ) to crystals of a protein to form a protein - ligand complex . the term “ co - crystallisation ” as used herein is taken to mean crystallisation of a pre - formed protein / small molecule complex . the terms “ mutant ”, “ variant ”, “ homologue ”, “ analogue ”, “ derivative ” or “ fragment ” in relation to the amino acid sequence of the crystal of the pde5 of the present invention include any substitution of , variation of , modification of , replacement of , deletion of or addition of one ( or more ) amino acids from ( or to ) the sequence providing the resultant pde5 is capable of being crystallised . the terms “ mutant ”, “ variant ”, “ homologue ”, “ analogue ”, “ derivative ” or “ fragment ” in relation to the nucleotide sequence coding for the pde5 of the crystal of the present invention include any substitution of , variation of , modification of , replacement of , deletion of or addition of one ( or more ) nucleic acid from ( or to ) the sequence providing the resultant nucleotide sequence codes for or is capable of coding for a pde5 which is capable of being crystallised . typically , for the “ mutant ”, “ variant ”, “ homologue ”, “ analogue ”, “ derivative ” or “ fragment ” in relation to the amino acid sequence of the crystal of the pde5 of the present invention , the types of amino acid substitutions that could be made should maintain the hydrophobicity / hydrophilicity of the amino acid sequence . amino acid substitutions may be made , for example from 1 , 2 or 3 to 10 , 20 or 30 substitutions provided that the modified pde5 retains the ability to be crystallised in accordance with present invention . amino acid substitutions may include the use of non - naturally occurring analogues . in relation to amino acid sequences , the term “ variant ” as used herein refers to additions , deletions or substitutions of amino acid residues comprised within the wild - type amino acid sequence or fragment thereof . preferably , a variant in relation to the amino acid sequence of the crystal of the pde5 of the present invention could include the deletion or substitution of the histidine ( his / h ) residue as shown emboldened and underlined in seq id no : 1 ( hrgvnnsyiqrsehplaqlyc h sime ), which sequence is comprised within the pde5 molecule of the crystal of the pde5 of the present invention . replacement of said histidine ( h ) residue is preferably by way of incorporating one or more amino acid residues ( other than histidine ), preferably wherein said amino acid residues are neutral or non - polar . more preferably , a variant in relation to the amino acid sequence of the crystal of the pde5 of the present invention includes the complete replacement of the loop region with a loop region ( or other equivalent amino acid sequence e . g . sub - domain ) from another protein , preferably a pde , more preferably pde4 , most preferably pde4b . alternatively , a variant in relation to the amino acid sequence of the crystal of the pde5 of the present invention includes the deletion or substitution of the amino acid residues plaq ( proline , leucine , alanine and glutamine ) as emboldened and underlined in seq id no : 1 ( hrgvnnsyiqrseh plaq lychsime ). preferably , such substitution of amino acid residues utilises amino acids of similar charge to those substituted . in relation to nucleotide sequences , the term “ variant ” as used herein refers to additions , deletions or substitutions of nucleotides comprised within the wild - type nucleotide sequence or fragment thereof . the term “ fragment ” as used herein refers to any portion of the pde5 as defined in the present invention provided the resultant pde5 comprising said pde5 portion is capable of being crystallised . thus , the term “ fragment ” also includes pde5 , which comprises any portion of seq id nos : 1 , 2 , 3 , 4 , 5 , or 6 . for example , a specific fragment of seq id no : 3 ( full - length wild - type pde5 sequence ) according to the present invention could be seq id no : 2 ( wild - type pde5 catalytic domain ). an example of a specific fragment of seq id no : 2 ( wild - type pde5 catalytic domain ) according to the present invention could be seq id no : 1 ( pde5 “ loop region ”; hrgvnnsyiqrsehplaqlychsime ). an example of a specific fragment of seq id no : 6 ( full - length “ loop - swapped ” pde5 sequence ) according to the present invention could be seq id no : 5 (“ loop - swapped ” pde5 catalytic domain ). moreover , an example of a specific fragment of seq id no : 5 (“ loop - swapped ” pde5 catalytic domain ) according to the present invention could be seq id no : 4 ( pde4 “ loop region ”; hpgvsnqflintnselalmyndesvle ). the term “ analogue ” as used herein means a sequence similar to the amino acid sequence of the crystal of the pde5 of the present invention or of any one of seq id nos : 1 , 2 , 3 , 4 , 5 or 6 , but wherein non - detrimental ( i . e . not detrimental to the pde5 &# 39 ; s capability of being crystallised ) amino acid substitutions or deletions have been made . the term “ derivative ” as used herein in relation to the amino acid sequence of the crystal of the pde5 of the present invention , or of any one of seq id nos : 1 , 2 , 3 , 4 , 5 or 6 , includes chemical modification of pde5 . illustrative of such modifications would be replacement of hydrogen by an alkyl , acyl , or amino group . as used herein a “ deletion ” is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues , respectively , are absent . as used herein an “ insertion ” or “ addition ” is a change in a nucleotide or amino acid sequence , which has resulted in the addition of one or more nucleotides or amino acid residues , respectively , as compared to the naturally occurring pde5 . as used herein “ substitution ” results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids , respectively . conservative substitutions may be made , for example according to the table below . amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other : aliphatic non - polar g a p i l v polar - uncharged c s t m n q polar - charged d e k r aromatic h f w y the term “ homologue ” covers homology specifically with respect to structure and covers any structural pde5 homologue that is capable of being crystallised . with respect to homology of the amino acid sequences detailed herein , preferably there is at least 70 %, more preferably at least 75 %, more preferably at least 80 %, yet more preferably at least 85 %, even more preferably at least 90 % homology to seq id nos : 1 , 2 , 3 , 4 , 5 or 6 . more preferably there is at least 95 %, and most preferably at least 98 %, homology to seq id nos : 1 , 2 , 3 , 4 , 5 or 6 . with respect to homology of the nucleotide sequences coding for the amino acid sequences detailed herein , preferably there is at least 70 %, more preferably at least 75 %, more preferably at least 80 %, yet more preferably at least 85 %, even more preferably at least 90 % homology to the nucleotide sequences which code for seq id nos : 1 , 2 , 3 , 4 , 5 or 6 . more preferably there is at least 95 %, and most preferably at least 98 %, homology to the nucleotide sequences which code for seq id nos : 1 , 2 , 3 , 4 , 5 or 6 . the term “ homologue ” with respect to the nucleotide sequence of the pde5 as defined in the present invention and the amino acid sequence of the pde5 as defined in the present invention may be synonymous with allelic variations of the sequences . in particular , the term “ homology ” as used herein may be equated with the term “ identity ”. here , sequence homology with respect to , for example , the amino acid sequence of the crystal of the pde5 of the present invention can be determined by a simple “ eyeball ” comparison ( i . e . a strict comparison ) of any one or more of the sequences with another sequence to see if that other sequence has at least 70 % identity to the sequence ( s ). relative sequence homology ( i . e . sequence identity ) can also be determined by commercially available computer programs that can calculate percentage (%) homology between two or more sequences . a typical example of such a computer program is clustal . % homology may be calculated over contiguous sequences , i . e . one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence , one residue at a time . this is called an “ ungapped ” alignment . typically , such ungapped alignments are performed only over a relatively short number of residues ( for example less than 50 contiguous amino acids ). although this is a very simple and consistent method , it fails to take into consideration that , for example , in an otherwise identical pair of sequences , one insertion or deletion will cause the following amino acid residues to be put out of alignment , thus potentially resulting in a large reduction in % homology when a global alignment is performed . consequently , most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score . this is achieved by inserting “ gaps ” in the sequence alignment to try to maximise local homology . however , these more complex methods assign “ gap penalties ” to each gap that occurs in the alignment so that , for the same number of identical amino acids , a sequence alignment with as few gaps as possible — reflecting higher relatedness between the two compared sequences — will achieve a higher score than one with many gaps . “ affine gap costs ” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap . this is the most commonly used gap scoring system . high gap penalties will of course produce optimised alignments with fewer gaps . most alignment programs allow the gap penalties to be modified . however , it is preferred to use the default values when using such software for sequence comparisons . for example when using the gcg wisconsin bestfit package ( see below ) the default gap penalty for amino acid sequences is − 12 for a gap and − 4 for each extension . calculation of maximum % homology therefore firstly requires the production of an optimal alignment , taking into consideration gap penalties . a suitable computer program for carrying out such an alignment is the gcg wisconsin bestfit package ( university of wisconsin , u . s . a . ; devereux et al ., 1984 , nucleic acids research 12 : 387 ). examples of other software that can perform sequence comparisons include , but are not limited to , the blast package ( see ausubel et al ., 1999 ibid — chapter 18 ), fasta ( atschul et al ., 1990 , j . mol . biol ., 403 - 410 ) and the geneworks suite of comparison tools . both blast and fasta are available for off - line and on - line searching ( see ausubel et al ., 1999 ibid , pages 7 - 58 to 7 - 60 ). however , for some applications it is preferred to use the gcg bestfit program . although the final % homology can be measured in terms of identity , in some cases , the alignment process itself is typically not based on an all - or - nothing pair comparison . instead , a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance . an example of such a matrix commonly used is the blosum62 matrix — the default matrix for the blast suite of programs . gcg wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied ( see user manual for further details ). it is preferred to use the public default values for the gcg package , or in the case of other software , the default matrix , such as blosum62 . once the software has produced an optimal alignment , it is possible to calculate % homology , preferably % sequence identity . the software typically does this as part of the sequence comparison and generates a numerical result . as indicated , for some applications , sequence homology ( or identity ) may be determined using any suitable homology algorithm , using for example default parameters . for a discussion of basic issues in similarity searching of sequence databases , see altschul et al ( 1994 ) nature genetics 6 : 119 - 129 . for some applications , the blast algorithm is employed , with parameters set to default values . the blast algorithm is described in detail at http :// www . ncbi . nih . gov / blast / blast_help . html . advantageously , “ substantial homology ” when assessed by blast equates to sequences which match with an expect value of at least about 7 , preferably at least about 9 and most preferably 10 or more . the default threshold for expect in blast searching is usually 10 . other computer program methods to determine identify and similarity between the two sequences include but are not limited to the gcg program package ( devereux et al 1984 nucleic acids research 12 : 387 and fasta ( atschul et al 1990 j . mol . biol . 403 - 410 ). the amino acid sequence of the pde5 of the present invention present invention may be produced by expression of a nucleotide sequence coding for the same in a suitable expression system . in addition , or in the alternative , the protein itself could be produced using chemical methods to synthesize a pde5 amino acid sequence , in whole or in part . for example , peptides can be synthesized by solid phase techniques , cleaved from the resin , and purified by preparative high performance liquid chromatography ( e . g . creighton ( 1983 ) proteins structures and molecular principles , wh freeman and co ., new york , n . y ., usa ). the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing ( e . g . the edman degradation procedure ). direct peptide synthesis can be performed using various solid - phase techniques ( roberge j y et al , science , vol 269 , 1995 , pp . 202 - 204 ) and automated synthesis may be achieved , for example , using the abi 431 a peptide synthesizer ( perkin elmer , boston , mass ., usa ) in accordance with the instructions provided by the manufacturer . additionally , the amino acid sequence of pde5 , or any part thereof , may be altered during direct synthesis and / or combined using chemical methods with a sequence from other subunits , or any part thereof , to produce a variant polypeptide . a recombinant construct of the catalytic domain ( e534 - n875 ) of human pde5 was expressed and the protein crystallised in complex with sildenafil and its structure determined by multi - wavelength anomalous dispersion ( hendrickson et al . 1989 ). at the time of structure solution this represented a novel fold , however , subsequently the structure of the catalytic domain of pde4b was published ( xu et al . 2000 ). a topological comparison of the pde5 catalytic domain with the structures in the protein data bank ( pdb ) does not reveal significant additional homology with other known protein structures except for the pde4 structure . comparisons between the two structures have been made ( fig1 shows a sequence and secondary structural alignment of the two proteins ). the structure and domain arrangement is virtually identical to that of pde4 , save that the second sub - domain highlighted in pde4 is only partially present in the pde5 structure , as detailed below . the structure is composed of a single domain of 15 α helices arranged in a compact fold ( fig2 ). within the overall domain , three sub - domains can also be defined . helices 1 ( h1 539 - 545 ) and 2 ( h2 551 - 554 ) lie on the exterior of the protein and comprise the n - terminal region of the construct . these two helices do not overlay with the equivalent ones ( h0 , h1 and h2 ) in the pde4 structure . this region is not well conserved across the pde protein family . helices 3 ( h3 568 - 582 ), 4 ( h4 584 - 588 ), 5 ( h5 592 - 604 ), 6 ( h6 615 - 631 ) and 7 ( h7 640 - 652 ) form the first sub - domain of the protein and are contained within the core of the protein . there is no observable electron density for helices 8 and 9 based on the pde4 nomenclature . helix 10 ( h10 684 - 694 ) is again on the exterior and forms the dimer interface within the structure . helices 10 and 11 ( h11 706 - 721 ) are the visible portion of the second sub - domain . helices 12 ( h12a 725 - 731 , h12b 733 - 741 ), 13 ( h13 749 - 765 ), 14 ( h14 772 - 797 ), 15 ( h145 813 - 824 ), 16 ( h146 826 - 836 ) and 17 ( h147 841 - 861 ) form the third sub - domain of the protein . it should be noted that in pde5 helix h12 is not a contiguous helix as in pde4 but is composed of two short helices with a kink in the middle and helix h15 is a contiguous helix in pde5 but not in pde4 . there are four molecules present in the asymmetric unit , each molecule contains chain breaks and density is not visible for the c - terminal portion of the construct ( see details below ). the four molecules can be defined as two copies of a dimer . molecule a ( no electron density observed for residues : 534 - 536 ; 665 - 681 ; 863 - 875 ) is associated with molecule d ( no electron density observed for residues : 534 ; 667 - 681 ; 865 - 875 ) and molecule b ( no electron density observed for residues : 534 - 536 ; 667 ; 865 - 875 ) associated with molecule c ( no electron density observed for residues : 534 - 53 ; 663 - 678 ; 863 - 875 ). the molecules within the dimer are related by a two - fold rotation with the interface being formed by association of helix h10 from molecule a and d . key to this dimer association is the presence of 2 zinc ions ( one associated with each monomer ). residue his 683 from one molecule and his 684 and asp 687 from the dimer partner co - ordinate each zinc ion . it is believed that the metal co - ordinated dimerisation is an artefact of crystallisation . the missing regions of structure in each molecule are believed to be due to the high flexibility of this part of the structure . further it is believed that there is significant cleavage of the protein in this region which gives rise to much of the flexibility . this region corresponds to helices h8 and h9 within the second sub - domain of the pde4 structure . each of the independently refined molecules in the structure contains one molecule of sildenafil bound within the active site . the active site lies mainly within the third sub - domain of the protein and is bounded by helices h15 , h14 , the c - terminus of h13 , and the c - terminus of h11 along with the loop region between h11 and h12a . the majority of the interactions between the inhibitor and the protein are hydrophobic in nature ; with only two direct hydrogen bonds observed ( fig3 ). the first is between n17 of the purine ring of the inhibitor and oε1 of gln 817 ( 2 . 8 å ) and the second from the adjacent oxygen atom o16 of the inhibitor to nε2 of the same residue gln 817 ( 3 . 1 å ). carbon atom c12 of the inhibitor points into a small hydrophobic pocket formed by leu 765 , ala 767 and ile 768 . these residues together with phe 820 form a planar face to the binding site against which the purine ring of the inhibitor stacks . the opposite side of the purine packs against val 782 . the c5 propyl substituent form good van der waals contacts with val 782 and phe 786 and tyr 612 . phe 786 and leu 804 form additional hydrophobic interactions with the phenyl moiety of the inhibitor . the o - alkyl moiety occupies a small pocket bounded by ala 779 , phe 786 , ala 783 , val 782 , leu 804 , ile 813 , met 816 and gln 817 . the sulphonamide group points out towards the solvent whilst the piperazine ring is bounded by the extended residues 662 - 665 , although whether the conformation of this part of the structure is unaffected by the chain break is questionable . there is no direct interaction between the inhibitor and the zinc ion found in the active site . the structure confirms the competitive nature of the mode of inhibition of sildenafil by binding in the active site therefore blocking access for the cgmp substrate ( which has also been modelled — data not included ). only one zinc atom is present in the active site of this structure . this can be clearly identified as a zinc atom since the phases used to determine the structure were obtained from a three - wavelength zinc mad experiment . the anomalous signal observed clearly indicates the presence of a zinc ion . the co - ordination of the ion within the active site is also consistent with that expected for zinc . the metal is co - ordinated by his 653 ( nε2 - zn 2 . 0 å ), his 617 ( nε2 - zn 2 . 1 å ), asp 764 ( od2 - zn 2 . 2 å ) and also asp 654 ( od2 - zn 2 . 2 å ). these residues are completely conserved across the pde gene family . there is no evidence of a second metal ion in the active site . a possible reason for the absence of any second metal ion in the active site is the sequestering of the metal ion ( in this case a zinc , again confirmed by the anomalous signal ) to form the dimer interface . additionally there is the possibility that the residues likely to be involved in co - ordinating a second metal ion in the active site are not in the native conformation due to the proximity to the disordered region of the protein and the dimer interface . analysis of the catalytic domain protein by mass spectrometry and sds page gel electrophoresis ( data not shown ) shows that the protein is cleaved within the region not visible in the structure ( residues 664 - 682 ). high concentrations of protease inhibitors provide some stabilisation of the protein , allowing the above structure to be determined . an engineered form of the catalytic domain of pde5 has been produced where the 657 - 682 region of pde5 has been replaced by the same region in pde4 producing a chimeric construct , ( see fig1 for sequence alignment of this region ). the c - terminus of this construct is also truncated ( c - term 858 ) compared to the wild - type structure ( c - term 875 ). hereafter this engineered construct will be referred to as ‘ pde5 *’. this engineered protein has been shown to be stable to degradation by mass spectrometry and sds page gel electrophoresis ( data not shown ). the protein shows improved biophysical properties allowing an alternative purification protocol to be developed . the new protocol utilises binding to a blue sepharose column and specific elution with cgmp . the wild - type protein had been shown not to bind to this column probably due to the disorder of the structure around the protease cleavage site . this pde5 * protein was used to produce crystals with sildenafil which diffract to higher resolution and have no disordered regions . the protein has also been used reproducibly to produce crystals with further inhibitors which routinely diffract to 1 . 8 å resolution or higher , making it an improved protein for use in structure based drug design . the structure of the catalytic domain of pde5 * protein was determined by molecular replacement using the wild - type protein structure as a basis for the search model . this structure comprises 17 α helices and the overall fold is very similar to the wild - type structure with a number of important differences . the major difference in the structure is the presence of helices h8 and h9 composed of the swapped portion from pde4 , residues 657 - 682 . these helices fold in an identical way to that observed in the pde4 structure and complete the second sub - domain of the protein . the entire c - terminal region of this construct can also be built into the electron density leaving just three disordered residues at the n - terminus of this structure . this is likely to contribute to its enhanced properties for crystallisation . the pde5 * catalytic domain crystallises as a monomer with two molecules present in the asymmetric unit related by a translational shift . ( pde5 * has also been crystallised with other inhibitors of pde5 in space group p2 1 with one molecule in the asymmetric unit . the crystals have approximate unit cell dimensions a = 56 b = 77 c = 83 å α = γ = 90 ° β = 103 °). each of the independently refined molecules again contains one molecule of sildenafil in the active site . sildenafil occupies the same region of the active site as observed in the wild - type structure forming the same mainly hydrophobic interactions with the protein ( fig4 ). the same two direct hydrogen bonds are formed between gln 817 of the protein and inhibitor ( oε1 - n17 2 . 8 å and nε2 - o16 3 . 1 å ). the remainder of the inhibitor makes the same contacts with the sulphonylpiperazine again pointing out towards solvent . this is close to the engineered region of the protein but the piperazine ring forms no interactions with the ordered swapped region of the catalytic domain construct . this is an important factor when considering the use of this chimeric catalytic domain for drug design . another notable difference in the structure of pde5 * compared with that of wild - type pde5 , is the presence of two metal ions in the active site . as observed in the wild - type complex there is no direct interaction between the inhibitor and the zinc ion found in the active site . there is also no direct interaction between sildenafil and the second metal ion observed in this complex . this second metal ion is coordinated to asp 764 ( od 1 2 . 15 å ) and to a water network that stabilises the metal environment . due to the co - ordination geometry and the relative observed electron density , this second metal ion has been refined as a mg 2 + in accordance with a similar observation in the pde4 structure solution . this structural arrangement is in accordance with the proposed mechanism , where an oh − ion is derived from an h 2 o molecule ionised by the presence of divalent metal atoms bound in the active site ( goldberg et al . 1980 , francis et al . 1994 ). the phosphodiester bond between the phosphorous and the oxygen atoms at the 3 ′ position of cgmp is then hydrolysed via oh − nucleophilic attack . the present invention will now be described , by way of example only , with reference to the accompanying sequence listing and figures , in which :— seq id no : 1 shows the amino acid sequence of the loop region from pde5 . seq id no : 2 shows the amino acid sequence of the wild - type pde5 catalytic domain . seq id no : 3 shows the amino acid sequence of the full - length wild - type pde5 sequence . seq id no : 4 shows the amino acid sequence of the loop region of pde4 . seq id no : 5 shows the amino acid sequence of the loop - swapped pde5 catalytic domain ═ pde5 *. seq id no : 6 shows the amino acid sequence of full - length pde5 sequence comprising pde5 *. fig1 shows an alignment of pde5 ( upper sequence ) and pde4b ( lower sequence ) catalytic domains . positions and numbering of helices from the structures are marked for each . residues in bold show a sequence alignment for the engineered region . the sequence from pde4 has been used to replace the corresponding region in pde5 . this results in a residue insertion in this region . underlining highlight c - terminal region absent in pde5 *. fig2 shows a ribbon representation of the overall fold of proteins showing secondary structure elements . the inhibitor is shown in an all atom stick representation and the metal ions as spheres . ( a )= pde4b , ( b )= wild - type pde5 + sildenafil , ( c )=“ loop - swapped ” pde5 ( pde5 *)+ sildenafil . helices are numbered using pde4 structure as reference . helices h0 - h7 form sub - domain 1 , helices h8 - h11 form sub - domain 2 , and helices h12 - h16 form sub - domain 3 . fig3 shows a view of compound sildenafil bound to wild - type pde5 . fig4 shows a view of compound sildenafil bound to “ loop - swapped ” pde5 ( pde5 *). oligonucleotide primers were designed from the sequence of human pde5 ( accession number = ab001635 ). dna fragments were generated by pcr amplification from a full - length pde5 clone . the following oligonucleotides were used : pde5 5 ′ untagged oligo : ( seq id no : 7 ) cgtgaattcatggaggaagaaacaagagagctac pde5 3 ′ long oligo : ( seq id no : 8 ) cgttctagactatcagttccgcttggcctggccgctttcccc the pcr reaction was carried out for 30 cycles in a total volume of 50 μl in a solution containing 1 . 5 mm mgcl 2 , 200 μm dntps , 50 pmol of each primer and 2 . 5 units of expand dna polymerase ( roche , east sussex , uk ). each cycle was 94 ° c ., 1 min , 50 ° c ., 1 min and 72 ° c ., 2 mins . the final amplified dna fragments for both constructs were separated on a 1 % agarose gel and purified using a qiaquick gel extraction kit ( qiagen , west sussex , uk ). the fragment was then digested using ecori and xbai , and ligated into pfastbac1 ecori / xbai - digested vector ( life technologies , paisley , uk ). the ligation was carried out at 12 ° c . for 16 hours . the ligation mix was then electroporated into e . coli ( top 10 ) ( invitrogen , gronigen , the netherlands ). clones containing the desired insert were selected by using 2yt plates containing 100 μg / ml ampicillin and checked using endonuclease digestion for presence of correct size insert . dna sequence analysis was carried out by lark ( saffron waldon , uk ). recombinant bacmid dna was produced by transforming e . coli dh10bac ™ with pfastbacl :: pde5 catalytic domain ( 534 - 875 ) plasmid dna . this was carried out according to the method shown in the bac to bac ™ baculovirus expression manual ( life technologies , paisley , uk ). pcr analysis was used to verify successful transposition to the bacmid using puc / m13 amplification primers ( invitrogen , gronigen , the netherlands ). generation of primary baculovirus stocks was carried out by transfection using sf - 9 insect cells . bacmid dna containing the correct insert was mixed with cellfectin ™ transfection reagent ( life technologies , paisley , uk ) and added to a monolayer of sf - 9 insect cells using sf - 900ii serum free medium ( invitrogen , gronigen , the netherlands ). following 72 hours incubation at 27 ° c . the supernatant was harvested as the initial baculovirus stock . this stock was amplified by adding the initial virus stock into a suspension of sf - 9 insect cells at 1 × 10 6 cells / ml in 1 litre erlenmeyer flasks ( coming life sciences , new york , usa ), at an agitation of 125 rpm at 27 ° c . after 6 days post - infection the supernatant was harvested by centrifugation and stored at 4 ° c . as the working virus stock . the titre of this working stock was determined by conventional plaque assay analysis as in the bac to bac ™ baculovirus expression manual ( invitrogen , gronigen , the netherlands ). protein expression was optimised in erlenmeyer flask cultures using sf - 9 and t . ni high5 insect cell cultures looking at different multiplicity &# 39 ; s of infection ( moi ) and harvest times , the optimal conditions found were then scaled up into fermenters . the fermenters used were autoclavable applikon 3 litre stirred vessels controlled using applikon 1030 biocontrollers . inoculum of t . ni high5 cells was initially prepared from shake flask cultures . the fermenter was inoculated with 5 × 10 5 cells / ml , with an initial working volume of 1 . 8 litres made up in excel 405 serum free medium ( jrh biosciences , kansas , usa ). temperature was controlled at 27 ° c ., dissolved oxygen concentration controlled at 60 % and ph was measured but not controlled . oxygen concentration was controlled throughout . agitation was set at 150 rpm with a double impeller system of marine impeller within the culture and rushton impeller at the liquid / headspace interface . aeration was continuous to the headspace at 0 . 5 l / min . when the viable cell density reached 2 × 10 6 cells / ml the culture was infected using an moi of 1 from the titred baculovirus working stock . harvest time for the culture was 48 hours post - infection . this was achieved by centrifugation at 2000 g for 15 mins ; the insect cell pellet was then stored at − 80 ° c . prior to purification . oligonucleotide primers were designed from the sequence of human pde5 (═ pde5a1 isoform ; accession number = ab001635 ). dna fragments were generated by pcr amplification from a full - length pde5 clone . the following oligonucleotides were used : pde5 5 ′ his6 oligo ( seq id no : 9 ) cgtgaattcatgcatcatcatcatcatcatcttctggttccg cgtggatctgcgcccgaggaagaaacaagagagctac pde5 3 ′ long oligo ( seq id no : 8 ) cgttctagactatcagttccgcttggcctggccgctttcccc the pcr reaction was carried out for 30 cycles in a total volume of 50 μl in a solution containing 1 . 5 mm mgcl 2 , 200 μm dntps , 50 pmol of each primer and 2 . 5 units of expand dna polymerase ( roche , east sussex , uk ). each cycle was 94 ° c ., 1 min , 50 ° c ., 1 min and 72 ° c ., 2 mins . the final amplified dna fragments for both constructs were separated on a 1 % agarose gel and purified using a qiaquick gel extraction kit ( qiagen , west sussex , uk ). the fragment was then digested using ecori and xbai , and ligated into pfastbac1 ecori / xbai - digested vector ( life technologies , paisley , uk ). the ligation was carried out at 12 ° c . for 16 hours . the ligation mix was then electroporated into e . coli ( top 10 ) ( invitrogen , gronigen , the netherlands ). clones containing the desired insert were selected by using 2yt plates containing 100 μg / ml ampicillin and checked using endonuclease digestion for presence of correct size insert . dna sequence analysis was carried out by lark ( saffron waldon , uk ). methods to generate the recombinant baculovirus were as those for wild - type pde5 catalytic domain ( see example 1 ). expression optimisation again showed t . ni high5 insect cells to give the best expression . therefore baculovirus expression in fermenters was carried out using the same procedures as for the previous construct . the pde5 * construct was produced by using overlap extension pcr where the following oligonucleotides were used : ( seq id no : 7 ) a : cgtgaattcatggaggaagaaacaagagagctac ( seq id no : 10 ) b : caaagaaagttctgaatttgtgttgatgagaaactgattggagactc caggatgatccaaatcgtggcttag ( seq id no : 11 ) c : atcaacacaaattcagaacttgctttgatgtataatgatgaatctgt gttggaacaccatcattttgaccag ( seq id no : 12 ) d : cgttctagactatcattctgcaagggcctgccatttctg initial dna fragments were generated using oligonucleotides a + b and c + d with the same template dna as for the wild - type pde5 catalytic domain construct . the pcr reaction was carried out for 30 cycles in a total volume of 50 μl in a solution containing 1 . 5 mm mgcl 2 , 200 μm dntps , 50 pmol of each primer and 2 units of expand dna polymerase ( roche , east sussex , uk ). each cycle was 94 ° c ., 1 min , 50 ° c ., 2 min , and 72 ° c ., 3 min . in the second round of pcr , dna products from pcr a + b and c + d were used as template dna with the oligonucleotides a + d used to amplify the full - length construct . the pcr conditions were the same as the initial pcr reaction . this generates a construct with the pde4 swapped region and a c - terminal truncation ( c - term 858 ) as compared to pde5 catalytic domain ( c - term 875 ). methods to generate the recombinant baculovirus were as those for wild - type pde5 catalytic domain ( see example 1 ). expression optimisation again showed t . ni high5 insect cells to give the best expression . therefore baculovirus expression in fermenters was carried out using the same procedures as for the previous construct . the pde5 * construct in e . coli was produced by using pcr where the following oligonucleotides were used and the template dna being pfastbac1 :: pde5 * plasmid dna ( sequence verified ), produced in example 3 . the pcr reaction was carried out for 30 cycles in a total volume of 50 μl in a solution containing 1 . 5 mm mgcl 2 , 200 μm dntps , 50 pmol of each primer and 2 . 5 units of expand dna polymerase ( roche , east sussex , uk ). each cycle was 94 ° c ., 1 min , 50 ° c ., 1 min and 72 ° c ., 2 mins . the final amplified dna fragment was separated on a 1 % agarose gel and purified using a qiaquick gel extraction kit ( qiagen , west sussex , uk ). the fragments were then digested using nde1 and xho1 , and ligated into pet21 c ( novagen , nottingham , uk ) nde1 / xho1 - digested vector . the ligation was carried out at 12 ° c . for 16 hours . the ligation mix was then electroporated into e . coli ( top 10 ) ( invitrogen , gronigen , the netherlands ). clones containing the desired insert were selected by using 2yt plates containing 100 μg / ml ampicillin . plasmid dna was also checked using endonuclease digestion for presence of correct size insert . dna sequence analysis was carried out by lark ( saffron waldon , uk ). the correctly sequenced plasmid dna was then electroporated into e . coli bl21 ( de3 ) ( novagen , nottingham , uk ) for expression . expression was carried out in 7 litre applikon fermenters using 5 litre 2yt broth containing 100 μg / ml carbenicillin as the medium . agitation was set at 1000 rpm using a double rushton impeller assembly and aeration to the sparger at 2 litres / min . the fermenter was inoculated with an overnight shake flask culture grown at 37 ° c . and 200 rpm , the inoculation density was 1 % vol / vol . the fermentation was ph controlled at 7 . 2 using 20 % vol / vol nh 4 oh solution and temperature initially set to 37 ° c . when the od 600nm reached 1 . 5 the temperature set - point was reduced to 25 ° c . and then the culture was induced with iptg at a final concentration of 1 mm . the fermentation was then harvested 4 hours post - induction by batch centrifugation ( 8 , 000 rpm for 10 minutes ). the final pellet was then frozen (− 80 ° c .) to await subsequent purification . pellet from the fermentation was resuspended into 10 mls lysis buffer per gram wet cell weight and mechanically broken using a continuous cell disrupter ( constant systems , warwickshire , uk ) at a pressure of 20 kpsi . the lysis buffer consisted of 50 mm tris hcl ( ph 7 . 2 ), 100 mm nacl , 1 mm dl - dithiothreitol ( dtt ) containing edta - free complete protease inhibitor cocktail tablets ( roche , east sussex , uk ) and 10 μm epoxysuccinyl - 1 - leucylamido -( 4 - guanidino ) butane ( e - 64 ) ( sigma , dorset , uk ; catalogue no . e - 3132 ). the lysate was chilled and centrifuged at 14000 g for 45 min to remove cell debris . all purifications were subsequently carried out using an akta explorer purification system ( amersham pharmacia , buckinghamshire , uk ). the supernatant was applied to a 50 ml q - sepharose fast - flow column ( amersham pharmacia , buckinghamshire , uk ) at 5 ml / in the flow - through was directly applied to a 20 ml nickel chelate column ( amersham pharmacia , buckinghamshire , uk ) previously charged with 0 . 1 m niso 4 . the nickel chelate column was washed with 5 column volumes of lysis buffer . the column was then step - eluted with lysis buffer containing 50 mm imidazole . this elution fraction was directly applied to a 2 litre g - 25 superfine desalting column ( amersham pharmacia , buckinghamshire , uk ) equilibrated in sp - sepharose buffer a ( 25 mm bis - tris ( ph 6 . 5 ), 50 mm nacl , 1 mm dtt and 2 μm e - 64 ). the protein was eluted in this buffer at 50 ml / in . the eluted fraction was then loaded onto a 20 ml sp - sepharose high - performance column ( amersham pharmacia , buckinghamshire , uk ) at a flow - rate of 5 mls / min . the flow - through was collected and dialysed overnight at 4 ° c . in heparin buffer a ( 25 mm bis - tris ( ph 6 . 5 ), 1 mm dtt and 2 μm e - 64 ). dialysis volume equalled 50 times the protein sample volume and the dialysis tubing used was 10 kda snakeskin ™ ( pierce , cheshire , uk ). the dialysed sample was then loaded onto a 20 ml heparin sepharose column ( amersham pharmacia , buckinghamshire , uk ), equilibrated in heparin buffer a . the column was eluted using a 10 column volume linear gradient with heparin buffer a containing 300 mm nacl at a flow - rate of 3 ml / min . fractions containing pde5 catalytic domain ( 534 - 875 ) were pooled and concentrated to 2 mg / ml using centrifugal protein concentrators ( vivascience , gloucestershire , uk ) and loaded at 1 . 5 m / min onto a superdex - 200 prep grade 26 / 60 column pre - equilibrated with 50 mm bis - tris ( ph 6 . 8 ), 500 mm nacl , 1 mm dtt and 2 μm e - 64 . the eluted fractions were analysed on tris - glycine sds page gels . pellet from the fermentation was resuspended into 5 mls lysis buffer per gram wet cell weight and mechanically broken using a continuous cell disrupter ( constant systems , warwickshire , uk ) at a pressure of 20 kpsi . the lysis buffer consisted of 50 mm bis - tris ( ph 6 . 8 ), 10 mm imidazole , 10 % glycerol , 50 mm sodium chloride and 3 mm β - mercaptoethanol ( β - me ) containing edta - free complete protease inhibitor cocktail tablets ( roche , east sussex , uk ). the lysate was chilled and centrifuged at 13000 g for 30 min to remove cell debris then passed through a 0 . 2 μm filter . all purifications were subsequently carried out using fplc purification system ( amersham pharmacia , buckinghamshire , uk ). the supernatant was applied to a 20 ml nickel chelate column ( amersham pharmacia , buckinghamshire , uk ) previously charged with 0 . 1 m niso 4 . the nickel chelate column was washed with 10 column volumes of buffer a ( lysis buffer with complete protease inhibitor tablets omitted ) followed by 10 column volumes of buffer a containing 50 mm imidazole . the column was then eluted with a gradient of 100 - 500 mm imidazole in buffer a . these eluted fractions were analysed using tris - glycine sds - page and stored overnight at 4 ° c . fractions containing pde5 catalytic domain were concentrated to 1 . 5 mg / ml using centriprep 10 kda molecular weight cut - off centrifugal concentrators ( amicon bioseparations , maine , usa ) at 3 , 000 rpm , 4 ° c . half of the concentrated fraction was then loaded onto a 320 ml sephacryl s300hr column ( amersham pharmacia , buckinghamshire , uk ) pre - equilibrated in 50 mm bis - tris ph 6 . 8 , 10 % glycerol , 50 mm nacl and 1 mm dl - dithiothreitol ( dtt ) at a flow - rate of 2 ml / min . the eluted fractions were analysed using tris - glycine sds - page and those containing pde5 catalytic domain were stored at − 80 ° c . pellet from both the e . coli and baculovirus fermentation was resuspended into 10 mls lysis buffer per gram wet cell weight and mechanically broken using a continuous cell disrupter ( constant systems , warwickshire , uk ) at a pressure of 20 kpsi . the lysis buffer consisted of 50 mm tris hcl ( ph 7 . 5 ), 100 mm nacl , 1 mm dtt containing edta - free protease inhibitor cocktail tablets ( roche , east sussex , uk ) and 10 μm e - 64 . the lysate was chilled and centrifuged at 14000 g for 45 min to remove cell debris . all purifications were subsequently carried out using an akta explorer purification system ( amersham pharmacia , buckinghamshire , uk ). the supernatant was applied to a 50 ml q - sepharose fast - flow column ( amersham pharmacia , buckinghamshire , uk ) at 5 ml / min with the flow - through collected . the flow - through sample was then applied at 50 ml / min to a 2 litre g - 25 superfine desalting column ( amersham pharmacia , buckinghamshire , uk ) pre - equilibrated in blue sepharose buffer a ( 50 mm bis - tris ( ph 6 . 4 ), 50 mm nacl , 2 mm edta , 2 mm egta and 1 mm dtt ). the protein fraction was eluted in blue sepharose buffer a . the next column step was carried out in series , loading the sample initially onto a 20 ml sp - sepharose high performance column ( amersham pharmacia , buckinghamshire , uk ) then flow - through from this directly onto a 10 ml blue sepharose fast - flow column ( amersham pharmacia , buckinghamshire , uk ) at a flow - rate of 2 ml / min . once loading was complete , the sp - sepharose column was taken out of line and the blue sepharose column washed with 5 column volumes of blue sepharose buffer a . the column was washed with blue sepharose buffer a containing 1 m nacl until the absorbance 280 nm reached baseline and then washed with 5 column volumes of blue sepharose buffer a . pde5 * protein was step - eluted using blue sepharose buffer containing 20 mm cgmp ( na - salt ) ( sigma , dorset , uk ). fractions were assayed on tris - glycine sds gels ( invitrogen , gronigen , the netherlands ) and pooled accordingly . these fractions were concentrated to 2 . 5 mg / ml using centrifugal concentrators ( vivascience , gloucestershire , uk ) and loaded at 1 . 5 m / min onto a superdex - 200 prep grade 26 / 60 column pre - equilibrated with 50 mm bis - tris ( ph 6 . 8 ), 500 mm nacl , 1 mm dtt and 2 μm e - 64 . the eluted fractions were analysed on tris - glycine sds page gels . pde5 fractions from the final gel filtration column were thawed from − 80 ° c . and protein concentration measured . the solution was concentrated to 5 . 8 mg / ml using a centriprep 10 kda molecular weight cut - off centrifugal concentrator ( amicon bioseparations , maine , usa ) at 3 , 000 rpm , 20 ° c . then transferred to a centricon 10 kda molecular weight cut - off centrifugal concentrator ( amicon bioseparations , maine , usa ) and concentrated to 12 . 8 mg / ml at 4 , 000 rpm , 20 ° c . the protein solution was diluted to 10 mg / ml using ultrafiltrate from the final stage of concentration and frozen at − 80 ° c . prior to crystallisation , the protein solution was thawed and centrifuged for 2 min at 14 , 000 rpm in an eppendorf centrifuge . hanging drop vapour diffusion crystallisation trials were set up at 20 ° c . drops comprised of 2 μl reservoir buffer mixed with 2 μl protein solution were suspended on siliconised cover slips over 950 μl reservoir solutions containing 50 mm hepes ph 7 . 6 , 1 . 1m monobasic sodium phosphate and 1 . 1m monobasic potassium phosphate ( all from sigma , dorset , uk ). both crystallisation plates and reservoir solutions were chilled to 4 ° c . before set up . completed plates were placed in a 4 ° c . cold room . rod shaped crystals , up to 400 μm in largest dimension , grew from precipitate after 1 - 2 weeks . crystals were transferred gradually at 4 ° c ., via solutions of increasing glycerol concentration , to a solution containing 0 . 1 m hepes ph 7 . 6 , 2 . 3m monobasic sodium phosphate and 20 % glycerol as a cryoprotectant . samples were then flash - frozen prior to x - ray data collection . the pde5 fractions from the final gel filtration column were pooled ( total volume of 25 ml ) and the protein concentration was assayed ( 0 . 2 mg / ml ). the protein solution was supplemented with 10 μm e - 64 and 1 mg / ml leupeptin ( sigma , dorset , uk ). the solution was concentrated to 3 mg / ml using a centriprep 10 kda molecular weight cut - off centrifugal concentrator ( amicon bioseparations , maine , usa ) at 3 , 000 rpm , 4 ° c . a three - fold molar equivalent of sildenafil ( 10 mg / ml aqueous stock solution ) was added to the protein solution , which was then further concentrated to 8 mg / ml . a further one - molar equivalent of sildenafil was added to this solution , which was concentrated to 10 mg / ml . prior to crystallisation , the protein solution was centrifuged for 5 min at 14 , 000 rpm in an eppendorf centrifuge . hanging drop vapour diffusion crystallisation trials were set up at 20 ° c . drops comprised of 2 μl reservoir buffer mixed with 2 μl protein solution were suspended on siliconised cover slips over 900 μl reservoir solutions containing 0 . 1 m tris ph 8 . 0 , 50 mm ammonium phosphate , ph 7 . 0 , 16 - 26 % peg2kmme ( all from sigma , dorset , uk ). block shaped crystals , up to 300 μm in largest dimension , grew from precipitate after 2 - 5 days . crystals were transferred to a solution containing 0 . 1 m tris , ph 8 . 0 , 250 mm nacl , 10 % glycerol and 12 - 22 % peg2kmme as a cryoprotectant . samples were then flash - frozen prior to x - ray data collection . the pde5 * fractions from the final gel filtration column were pooled ( total volume of 25 mls ) and the protein concentration was assayed ( 0 . 2 mg / ml ). the protein solution was supplemented with 10 μm e - 64 and 1 mg / ml leupeptin ( sigma , dorset , uk ). the solution was concentrated to 10 mg / ml using a centriprep 10 kda molecular weight cut - off centrifugal concentrator ( amicon bioseparations , maine , usa ) at 3 , 000 rpm , 4 ° c . prior to crystallisation , the protein solution was centrifuged for 5 min at 14 , 000 rpm in an eppendorf centrifuge . hanging drop vapour diffusion crystallisation trials were set up at 4 ° c . drops comprised of 2 μl reservoir buffer mixed with 2 μl protein solution were suspended on cover slips over 900 μl - reservoir solutions containing 0 . 2m sodium acetate , 0 . 1 m tris hydrochloride ph 8 . 5 , 30 % w / v polyethylene glycol 8000 ( the solution is component number 22 in the crystal screen ® from hampton research , california , usa .) plate - shaped crystals , up to 700 μm in largest dimension , grew after 1 - 2 days . crystals were transferred to a solution containing 0 . 16m sodium acetate , 80 mm tris hydrochloride ph 8 . 5 , 24 % w / v polyethylene glycol 8000 and 10 % glycerol and then frozen during x - ray data collection . purified pde5 * protein was supplemented with 10 μm e - 64 and 1 mg / ml leupeptin ( sigma , dorset , uk ). following the exact method described above for wild - type pde5 catalytic domain , a complex of pde5 * with sildenafil was made , and concentrated to a final protein concentration of 10 mg / ml . hanging drop vapour diffusion crystallisation trials were set up at 4 ° c . drops comprised of 2 μl reservoir buffer mixed with 2 μl protein solution were suspended on siliconised cover slips over 900 μl reservoir solutions containing 0 . 1 m tris ph 7 . 4 , 50 mm ammonium phosphate , ph 7 . 5 , 30 - 24 % peg2kmme ( sigma , dorset , uk ). thin plate crystals grew after 2 - 5 days , the largest of these , up to 600 μm in the largest dimension , from the 28 % peg2kmme conditions . crystals were transferred to a solution containing 0 . 1 m tris ph 7 . 4 , 250 mm nacl , 10 % glycerol and 26 - 20 % peg2kmme as a cryoprotectant . samples were then flash - frozen prior to x - ray data collection . the structure of recombinant human pde5 was solved by multiple wavelength anomalous dispersion ( mad ) using four wavelengths at the zinc l lll edge . native x - ray diffraction data were collected with an mar ccd at station bm14 at the esrf , grenoble , france . all data were processed using the hkl package ( otwinowski & amp ; minor , 1997 ). data collection statistics are summarised in table 1a . the crystals belong to space group p6 2 with unit cell dimensions a = 94 . 921 å , b = 94 . 921 å , c = 81 . 850 å , a = β = 90 ° γ = 120 °. they contain 1 molecule per asymmetric unit ( m w = 39 , 654 . 71 da ) and have a calculated solvent content of 43 . 23 % ( v m = 2 . 18 ; matthews , 1968 ). anomalous heavy atom sites were located using solve ( terwilliger & amp ; berendzen , 1997 ). refinement of the heavy atom parameters and phase calculation was performed with sharp ( de la fortelle & amp ; bricogne , 1997 ). phases were improved by 100 cycles of solvent flattening with solomon ( abrahams & amp ; leslie , 1996 ). the resulting map was of good quality and used to trace about 70 % of the structure using quanta ( quanta98 , 1998 , version 98 . 1111 ; molecular simulations inc ., san diego , calif . 92121 - 3752 , usa ). the model was refined against a set of native structure factors ( f p - calc ) derived with sharp from a combination of experimental native ( f p ) and derivative ( f ph ) structure factors . refinement was carried out in the resolution range 30 - 2 . 5 å using xplor ( brunger et al ., 1998 ). partial structure factors from a flat bulk - solvent model and anisotropic b - factor correction were supplied throughout the refinement . the r - factor for the current model is 0 . 260 ( free r - factor , 5 % of data , 0 . 319 ) for all data in the resolution range 30 - 2 . 5 å . the refinement statistics are summarised in table 2a . the current model contains 296 out of 342 amino acid residues calculated on the basis of the construct and is well defined in most regions of the polypeptide chain . no interpretable electron density is observed for residues : 534 , 657 - 673 , 790 - 804 and 863 - 875 . analysis of the structure using procheck ( laskowski , et al ., 1993 ) shows 12 residues from the four molecules in the asymmetric unit are in disallowed regions . data collection , structure determination and refinement of wild - type pde5 with sildenafil the structure of recombinant human pde5 was solved by multiple wavelength anomalous dispersion ( mad ) using three wavelengths at the zinc l lll edge . native x - ray diffraction data were collected with an mar ccd at station bm14 at the esrf , grenoble , france . all data were processed using the hkl package ( otwinowski & amp ; minor , 1997 ). data collection statistics are summarised in table 1b . the crystals belong to space group p2 1 2 1 2 1 with unit cell dimensions a = 94 . 179 å , b = 103 . 645 å , c = 141 . 942 å , α = β = γ = 90 °. they contain 4 molecules per asymmetric unit ( m w = 39 , 654 . 71 da ) and have a calculated solvent content of 43 . 23 % ( v m = 2 . 18 ; matthews , 1968 ). anomalous heavy atom sites were located using solve ( terwilliger & amp ; berendzen , 1997 ) and confirmed with snb ( smith et al ., 1998 ). refinement of the heavy atom parameters and phase calculation was performed with sharp ( de la fortelle & amp ; bricogne , 1997 ). phases were improved by 100 cycles of solvent flattening with solomon ( abrahams & amp ; leslie , 1996 ). the resulting map was of good quality and used to trace about 80 % of the structure using quanta ( quanta98 , 1998 , version 98 . 1111 ; molecular simulations inc ., san diego , calif . 92121 - 3752 , usa ). the model was refined against a set of native structure factors ( f p - calc ) derived with sharp from a combination of experimental native ( f p ) and derivative ( f ph ) structure factors . refinement was carried out in the resolution range 30 - 2 . 2 å using cnx ( brunger et al ., 1998 ) with the “ mlhl ” maximum likelihood target function . partial structure factors from a flat bulk - solvent model and anisotropic b - factor correction were supplied throughout the refinement . the r - factor for the current model is 0 . 235 ( free r - factor , 5 % of data , 0 . 28 ) for all data in the resolution range 30 - 2 . 2 å . the refinement statistics are summarised in table 2b . the current model contains 1261 out of 1364 amino acid residues calculated on the basis of the construct and is well defined in most regions of the polypeptide chain . no interpretable electron density is observed for residues : molecule a 534 - 536 , 665 - 681 and 863 - 875 ; molecule d 534 , 667 - 681 and 865 - 875 ; molecule b 534 - 536 , 667 and 865 - 875 and molecule c 534 - 536 , 663 - 678 and 863 - 875 . analysis of the structure using procheck ( laskowski , et al ., 1993 ) shows only four residues from the four molecules in the asymmetric unit are in disallowed regions . the structure of the baculovirus engineered pde5 * was solved by molecular replacement ( mr ) using the pde5 * coordinates obtained from the complex with sildenafil ( see example 15 ). x - ray diffraction data were collected with an raxisiv image plate detector on an in - house ru200hb rotating anode ( rigaku ), with blue osmic mirrors ( msc ). all data were processed using the hkl package ( otwinowski & amp ; minor , 1997 ). data collection statistics are summarised in table 1a . the crystals belong to the monoclinic space group p2 1 , with unit cell dimensions a = 54 . 983 å , b = 77 . 153 å , c = 80 . 660 å , α = γ = 90 ° β = 101 . 311 °. they contain 2 molecules per asymmetric unit ( m w = 37 , 562 da ) and have a calculated solvent content of 45 . 1 % ( v m = 2 . 26 ; matthews , 1968 ). molecular replacement was performed using amore ( ccp4 ). the resulting map was of good quality and the structure was refitted using quanta . refinement was carried out in the resolution range 30 - 1 . 6 å using cnx ( brünger et al ., 1998 ) with the “ mlf ” maximum likelihood target function . partial structure factors from a flat bulk - solvent model and anisotropic b - factor correction were supplied throughout the refinement . the r - factor for the current model is 0 . 301 ( free r - factor , 5 % of data , 0 . 326 ) for all data in the resolution range 30 - 1 . 6 å . the refinement statistics are summarised in table 2a . the current model contains 323 residues per molecule , 537 - 858 ( residue glu 681a has been numbered to maintain pde5 numbering scheme ). no interpretable electron density is observed for residues : 534 , 535 and 536 in molecules a or b . analysis of the structure using procheck ( laskowski , et al ., 1993 ) shows only two residues from the two molecules in the asymmetric unit are in disallowed regions . the structure of the baculovirus engineered pde5 * was solved by molecular replacement ( mr ) using a combined model of wild - type pde5 with the structure for the second sub - domain from pde4 as a search model . x - ray diffraction data were collected with an raxisiv image plate detector on an in - house ru200hb rotating anode ( rigaku ), with blue osmic mirrors ( msc ). all data were processed using the hkl package ( otwinowski & amp ; minor , 1997 ). data collection statistics are summarised in table 1b . the crystals belong to the monoclinic space group p2 1 , with unit cell dimensions a = 54 . 93 å , b = 77 . 77 å , c = 82 . 05 å , α = γ = 90 ° β = 100 . 955 °. they contain 2 molecules per asymmetric unit ( m w = 37 , 562 . 41 da ) and have a calculated solvent content of 45 . 87 % ( v m = 2 . 29 ; matthews , 1968 ). molecular replacement was performed using amore ( ccp4 ). the resulting map was of good quality and the structure was refitted using quanta . refinement was carried out in the resolution range 30 - 1 . 6 å using cnx ( brunger et al ., 1998 ) with the “ mlf ” maximum likelihood target function . partial structure factors from a flat bulk - solvent model and anisotropic b - factor correction were supplied throughout the refinement . the r - factor for the current model is 0 . 286 ( free r - factor , 5 % of data , 0 . 307 ) for all data in the resolution range 30 - 1 . 6 å . the refinement statistics are summarised in table 2b . the current model contains 323 residues per molecule , 537 - 858 ( residue glu 681a has been numbered to maintain pde5 numbering scheme ). no interpretable electron density is observed for residues : 534 , 535 and 536 in molecules a or b . analysis of the structure using procheck ( laskowski , et al ., 1993 ) shows only two residues from the two molecules in the asymmetric unit are in disallowed regions . lengthy table referenced here us20070015205a1 - 20070118 - t00001 please refer to the end of the specification for access instructions . lengthy table referenced here us20070015205a1 - 20070118 - t00002 please refer to the end of the specification for access instructions . lengthy table referenced here us20070015205a1 - 20070118 - t00003 please refer to the end of the specification for access instructions . lengthy table referenced here us20070015205a1 - 20070118 - t00004 please refer to the end of the specification for access instructions . lengthy table referenced here us20070015205a1 - 20070118 - t00005 please refer to the end of the specification for access instructions . lengthy table referenced here us20070015205a1 - 20070118 - t00006 please refer to the end of the specification for access instructions . lengthy table referenced here us20070015205a1 - 20070118 - t00007 please refer to the end of the specification for access instructions . lengthy table referenced here us20070015205a1 - 20070118 - t00008 please refer to the end of the specification for access instructions . abrahams , j . p . & amp ; leslie , a . 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( 1997 ) acta crystallogr . d 53 , 571 - 579 crystallogr . d 54 , 799 - 804 . abbreviations camp cyclic adenosine monophosphate cgmp cyclic guanosine monophosphate pde phosphodiesterase pgk protein kinase g mad multi - wavelength anomalous dispersion pcr polymerase chain reaction 2yt 16 g tryptone , 10 g yeast extract , 5 g nacl per litre solution tris tris [ hydroxymethyl ] amino - methane e - 64 epoxysuccinyl - 1 - leucylamido -( 4 - guanidino ) butane dtt dl - dithiothreitol β - me β - mercaptoethanol iptg β - d - isopropyl - thiogalactopyranoside edta ethylenediamine tetraacetic acid bis - tris bis [ 2 - hydroxyethyl ] imino - tris [ hydroxymethyl ] methane peg polyethylene glycol peg2kmme polyethylene glycol 2000 monomethyl ether peg4kmme polyethylene glycol 4000 monomethyl ether peg8kmme polyethylene glycol 8000 monomethyl ether rmsd root mean square deviation rpm revolutions per minute moi ( or moi ) multiplicity of infection sildenafil 5 -[ 2 - ethoxy - 5 -( 4 - methyl - 1 - piperazinylsulphonyl ) phenyl ]- 1 - methyl - 3 - n - propyl - 1 , 6 - dihydro - 7h - pyrazolo [ 4 , 3 - d ] pyrimidin - 7 - one , which is also known as 1 -[[ 3 -( 6 , 7 - dihydro - 1 - methyl - 7 - oxo - 3 - propyl - 1h - pyrazolo [ 4 , 3 - d ] pyrimidin - 5 - yl )- 4 - ethoxyphenyl ] sulphonyl ]- 4 - methylpiperazine ( see ep - a - 0463756 ) uk - 092 , 480 see “ sildenafil ” the patent application contains a lengthy table section . a copy of the table is available in electronic form from the uspto web site ( http :// seqdata . uspto . gov /? pagerequest = docdetail & amp ; docid = us20070015205a1 ) an electronic copy of the table will also be available from the uspto upon request and payment of the fee set forth in 37 cfr 1 . 19 ( b )( 3 ).