US 20140O37547A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0037547 A1 DOstmann et al. (43) Pub. Date: Feb. 6, 2014

(54) NOVEL PEPTIDIC ACTIVATORS OF TYPE I Publication Classification CGMP DEPENDENT PROTEIN KINASES AND USES THEREOF (51) Int. Cl. C07K I4/00 (2006.01) (71) Applicants: Wolfgang Dostmann, Shelburne, VT CI2O I/48 (2006.01) (US); Brent W. Osborne, Ridgefield, NJ G06F 9/16 (2006.01) (US); Thomas M. Moon, Burlington, A6II 45/00 (2006.01) VT (US) (52) U.S. Cl. CPC ...... C07K 14/00 (2013.01); A61K 45/00 (72) Inventors: Wolfgang Dostmann, Shelburne, VT (2013.01); C12O 1/485 (2013.01); G06F 19/16 (US); Brent W. Osborne, Ridgefield, NJ (2013.01) (US); Thomas M. Moon, Burlington, USPC ...... 424/9.2: 514/262.1: 514/283: 514/263.4: VT (US) 514/267: 514/392: 514/334: 514/412: 514/250; (73) Assignee: The University of Vermont and State 514/470; 514/149; 514/21.3; 435/15; 530/300; Agricultural College, Burlington, VT 703/11 (US) (21) Appl. No.: 13/801.235 (57) ABSTRACT (22) Filed: Mar 13, 2013 The present invention relates to c(GMP protein kinase (PKG) and regulatory domains and methods of use thereof. The Related U.S. Application Data structural determination of PKG domains is also described. (60) Provisional application No. 61/610,325, filed on Mar. cGMP independent PKG activators and uses thereof are also 13, 2012. described.

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NOVEL PEPTIDIC ACTIVATORS OF TYPE I structural basis for cooperativity in PKG isoforms. The inven CGMP DEPENDENT PROTEIN KNASES AND tion includes compounds which competitively and allosteri USES THEREOF cally activate PKG and modulate vasodilation in vascular Smooth muscle. The use of these compounds, referred to RELATED APPLICATIONS herein as cCMP independent PKG activators as competitive disruptors of the regulatory/catalytic domain interaction pro 0001. This application claims priority under 35 U.S.C. vides a significant advance in a number of therapeutic areas. S119(e) to U.S. Provisional Application Ser. No. 61/610,325, The compounds are an entirely new class of PKG activators. entitled “cGMP DEPENDENT PROTEIN KINASE filed 0007. In some aspects the invention is a method for acti on Mar. 13, 2012, which is herein incorporated by reference vating PKG, by contacting a cell with a coMP independent in its entirety. PKG activator in an effective amount to activate PKG in the FEDERALLY SPONSORED RESEARCH cell. 0008. In other aspects the invention is a method of treating 0002 This invention was made with Government support a PKG deficient condition in a subject, comprising adminis under NIH grant number HL068891. Accordingly, the Gov tering to the Subject a therapeutically effective amount of a ernment has certain rights in this invention. cGMP independent PKG activator. The PKG deficient con dition is selected from the group consisting of cardiovascular FIELD OF THE INVENTION disorders, hypoxia, spinal cord injury, and stroke in some 0003. The present invention relates to activators of type I embodiments. isoforms of c(GMP dependent protein kinase (PKG) and 0009. The subject may also be administered another com methods ofuse and synthesis thereof. Methods for expression pound in the methods of the invention. In some embodiments and structural determination of regulatory domains of PKG the other compound is coMP. In other embodiments the other are also described. compound is a coMP dependent PKG activator, such as a PDE inhibitor or a NO-donor. In some embodiments the PDE BACKGROUND OF THE INVENTION inhibitor is selected from the group consisting of sildenafil citrate, vinpocetine, EHNA, anagrelide, enoXimone, mil 0004 PKG serves as an integral component of second rinone, mesembrine, rolipram, ibudilast, piclaimilast, luteolin, messenger signaling. In Smooth muscle, the PKG isoforms IC. drotaverine, roflumilast, tadalafil. Vardenafil. udenafil, avana and IB integrate the nitric oxide (NO) and natriuretic peptide fil, and papaverine. In other embodiments the NO-donor is mediated signal transduction pathways by modulating intra selected from the group consisting of isosorbide dinitrite, cellular Ca", cell contractility, and ultimately blood flow Diazeniumdiolates, NONOates, S-Nitrosothiols, NO hybrid (Hofmann et al., 2009; Francis et al., 1999; Pfeifer et al., drugs, and Zeolites. 1999). Understanding the regulatory mechanism of the PKG (0010. The cQMP independent PKG activator may be any holoenzyme is central to guiding pharmacological discover type of molecule. In some embodiments it is a peptide, and ies relevant to prevention and treatment of vascular diseases. optionally any of the peptides disclosed herein. An exemplary Direct pharmaceutical targeting of PKG IC. however, has peptide of the invention is DVSNKAYEDAEAKAKYEAE remained unsuccessful, largely due to the enzyme’s complex, AAFFANLKLSD (SEQID NO. 4). In other embodiments the multi-domain architecture. cGMP independent PKG activator is a small molecule. 0011. In other aspects the invention is a composition com SUMMARY OF THE INVENTION prising: a coMP independent PKG activator and a carrier. 0005. The present invention relates to PKG, regulatory The coMP independent PKG activator may be a peptide, such domains of PKG, methods of use and synthesis thereof, and as DVSNKAYEDAEAKAKYEAEAAFFANLKLSD (SEQ therapeutic and diagnostic reagents for modulating PKG. To ID NO. 4). Alternatively the coMP independent PKG activa better understand the molecular details of PKG regulation, we tor may be a small molecule. solved the first crystal structure of the intact regulatory 0012. A crystal comprising a regulatory domain of PKG is domain of PKGIC: 78-355 (PDBID: 3SHR). This structure provided according to other aspects of the invention. The exposed a unique helical domain, that we termed the Switch regulatory domain of PKG in the crystal comprises the amino helix (SW). The SW stabilizes an unexpected dimer interface acid sequence of SEQID NO: 1, wherein the crystal has unit between protomers called the knob/nest. The SW locus is cell dimensions of a=180.2 Angstroms, b=66.0 Angstroms, important for maintaining the structural integrity of PHG IC. and c=81.6 Angstroms, with a unit cell variability of 5% in all and serves as a key domain in PKG holoenzyme function and dimensions. In some embodiments the PKG regulatory acts as a modulator of the molecular forces that govern PHG domain of the crystal has a three-dimensional structure com IC. activation. prising two c(GMP binding domains, A and B, a hinge region 0006. The structure of the PKG regulatory domain identi and a Switch helix domain. fied according to the invention challenges the classic parallel 0013. A crystalline composition is provided in other dimer view of PKG holoenzyme assembly (FIG. 1). The aspects of the invention. The crystalline composition has a novel SW interface offers a dynamic view of trans-asymmet regulatory domain of PKG, wherein the PKG regulatory ric protomers and its function in the activation of PKG. The domain of the crystal has a three-dimensional structure com isoform-specific interprotomer coupling of PKG is mediated prising two c(GMP binding domains, A and B, a hinge region by SW and is required for native kinase activity. We identified and a Switch helix domain. a set of residues within the Switch helix, termed the knob 0014. The invention in other aspects is a regulatory that specifically interacts with a hydrophobic nest mainly domain of PKG in crystalline form comprising the amino acid provided by the coMP B-site. The knob/nest interaction is sequence 78-355 and having the structural coordinates as allosterically linked to the coMP B-site and provides the deposited in Protein Data Bank with accession code 3SHR. US 2014/0037547 A1 Feb. 6, 2014

0015. In yet other aspects, the invention is a crystallized element (SW) and the formation of a hydrophobic pocket PKG or portion thereof, wherein said crystallized PKG or (nest) by the coMP B-domain (bottom). A synthetic deriva portion thereof is crystallized under appropriate conditions tive of SW activates PKG in vitro and in vivo (upper left). such that the three dimensional structure of the PKG can be 0023 FIG. 2: A view of the pharmacophore derived from determined. the SW knob forming specific interactions with the nest in the 0016 A method of selecting or designing a compound that cGMP B-site. interacts with PKG and modulates PKG activity is also pro (0024 FIG. 3: A) Activation of PHG IC. is attenuated by vided according to aspects of the invention. The method com varying the C-terminus of SW-derived peptides in the pres prises the step of assessing the stereochemical complemen ence of 100 nM cQMP. Truncation of the knob residues tarity between the compound and a topographic region of abolishes peptide-based activation of PKG. B) A Schlid plot PKG, wherein the topographic region of the PKG is charac of the Rp-PET mediated inhibition of activation of PHGIC by terized by at least a portion of the amino acids positioned at SW:329-358. INSET: Secondary analysis of the concentra atomic coordinates as deposited in Protein Data Bank with tion-dependency of Rp-PET on the activation. Rp-PET accession code 3SHR wherein amino acid sequences of appears to sterically inhibits closure of the PBC, thus attenu switch helix domain thereofare set forth as SEQID NO: 2. In some embodiments the topographic region of PKG is the ating the activation PKG by the SW. switch helix domain defined by amino acids: 350-354. In (0025 FIG. 4: SW:329-358 activates single KCa channels other embodiments the method further involves testing the in inside-out membrane patches excised from cerebral artery compound in vitro or in vivo for its capacity to modulate the myocytes. A) Original records of single KCa channels show activity of PKG. ing the activating effects of c(GMP (left) and SW:329-358 0017. In some aspects the invention is an isolated peptide (right) in the presence of PHG Ia and ATP B) K channel comprising XXXX-XXX-Xs (SEQID NO: 8), wherein open-probability (NP) is significantly increased by c0MP each X is an amino acid, and wherein the peptide binds to a or SW:329-358 vs. PKG alone. C). A sequence-scrambled nest region or PKG. In some embodiments the Xs have the derivative of SW:329-358 does not increase K channel following values: X is Ala, Gly, Leu, Ile, Val, Cys, Ser, Thr, activity. or Pro; X is Ala, Gly, Leu, Ile, Val, Cys, Ser. Thr, or Pro: X (0026 FIG. 5: PKGIC.: Domain Organization and General is Phe, Tyr, or Trp; X is Phe, Tyr, or Trp; Xs is Ala, Gly, Leu, Architecture of PKG7 (A) D/D: docking and dimeriza Ile, Val, Cys, Ser. Thr, or Pro; X is ASn, Asp, Glu, Gln, Ser, or tion domain: AI: autoinhibitory domain; coMP-A/B: cQMP Thr; X, is Leu, Ile, Val, or Ala, and Xs is Lys Arg, Glu, His, binding sites A and B; SW: switch helix. Bars at the N termi Asp, Gln, Ser. Thr, or Tyr. nus indicate site of parallel homodimerization. (B) Overall 0018. In other embodiments the Xs have the following fold of PKG 78. values: X is Ala, Gly, Leu, Ile, or Val; X is Ala, Gly, Leu, Ile, (0027 FIG. 6: Topology of the coMP-Binding Domains or Val; X is Phe, Tyr, or Trp; X is Phe, Tyr, or Trp; Xs is Ala, (A) Overall fold of the tandem coMP binding domains shown Gly, Leu, Ile, or Val; X is ASn, Asp, or Glu, X, is Leu, Ile, Val, as cartoon representation with secondary structural elements or Ala, and Xs is Lys or Arg. labeled (switch helix not shown). (B) Overlay of c(GMP-A 0019. In other embodiments the peptide comprises (dark gray) with cGMP-B (white). RMSD=1.214 for Camol XX-FFANLXs (SEQID NO: 10), such that XX, and Xs ecules. PBC from coMP-A is labeled as PBC, C7-C are any amino acid, optionally Ala of Lys. disulfide bond is circled and labeled. 0020. An isolated peptide comprising, consisting of or (0028 FIG. 7 Features of the A-Domain (A) Sequence consisting essentially of FFANL (SEQID NO.9) is provided alignment for PBCs from PKA and PHGIC isoforms. Threo according to other aspects of the invention. nine residues in the 13 position of PKG PBCs are to high 0021. Each of the limitations of the invention can encom lighted by a box. (B) Composite omit map generated for the pass various embodiments of the invention. It is, therefore, cNT binding sites of both A-domains contoured to 1.5 s. anticipated that each of the limitations of the invention syn-cAMP is modeled into the positive density. (C) Specific involving any one element or combinations of elements can interactions between the sugar-phosphate moiety of cAMP be included in each aspect of the invention. This invention is and residues from the PBC. The 2'-hydroxyl hydrogen bonds not limited in its application to the details of construction and with Glu', the equatorial oxygen of the phosphate group the arrangement of components set forth in the following interacts with Arg'', and the apical oxygen contacts both the description or illustrated in the drawings. The invention is side-chain hydroxyl and backbone amino groups of Thr' '. capable of other embodiments and of being practiced or of The backbone carbonyl of Leu' bridges communication being carried out in various ways. Also, the phraseology and from the guanidinium group of Arg'' through the B2-63 loop terminology used herein is for the purpose of description and to Arg' in the B helix. (D) A hydrophobic face accommo should not be regarded as limiting. The use of “including.” dates the solvent-exposed nucleobase. (E) Schematic of cnT “comprising.” or “having.” “containing”, “involving, and binding to the A-domain of PKG IC” with cGMP mod variations thereof herein, is meant to encompass the items eled in place of the observed cAMP. Specific hydrogen bond listed thereafter and equivalents thereofas well as additional contacts are shown as dotted lines. Interactions coming from items. backbone amide (D) and carbonyl (o) are highlighted, whereas side-chain interactions are shown from the center of BRIEF DESCRIPTION OF DRAWINGS the circled residue. Hydrophobic interactions with the cMT 0022 FIG. 1: Outline of the specific application for the are shown as boxed residues. activation of c(GMP-dependent protein kinase given by the 0029 FIG. 8 Details of the Cys' '7-Cys' Disulfide Bond invention. Our new structure of PKG identified a hitherto (A) The disulfide bridge in the A-domain between Cys' and unknown interaction between protomers (upper right). This Cys' covalently links the A helix to the B helix. 2F-F interaction, called the knob?nest, is provided by the helical electron density contoured to 1.6 s is shown for the loop US 2014/0037547 A1 Feb. 6, 2014

preceding the N helix and the disulfide bond. (B) Side view of as a critical site of dimer communication in PKG biology. the hydrophobic sheath surrounding the disulfide bond. These results offer new structural insight into the mechanism 0030 FIG.9 Comparative Overlay of Helical Subdomains of allosteric PKG to activation. The coordinates of the struc for PKG and PKA CNB Domains (A) Structural overlay of ture have been deposited in the Protein Data Bank with acces PKG A-domain (dark gray) with PKAA-domain (light gray) sion code 3SHR and are hereby incorporated by reference in from the unliganded, holoenzyme structure (left, 20CS) and their entirety. The crystal structure of the invention is useful the cAMP-bound form (right, 1 RGS). (B) Structural overlay for elucidating the mechanisms of action of PKG as well as of PKG B-domain (dark gray) with PKA B-domain (light for identifying novel therapeutics that may be used for treat gray) from the unliganded, holoenzyme structure (left, ing a variety of diseases. 2OCS) and the cAMP-bound form (right, 1 RGS). 0036. The greatest structural distinction between the two 0031 FIG. 10 Dimeric View of PKG7 Protomers (A) major cyclic nucleotide regulated protein kinases, PKA and Ribbon diagram of two PKG7 protomers as observed in PKG, is the fact that PKG maintains both its regulatory and the asymmetric unit. (B) “Front” and Top' views of surface catalytic elements on the same polypeptide chain (Gill et al., representation of the PKG dimer to illustrate the cross 1977), while PKA is divided into subunits (Gill and Garren, ing of Switch helices. 1971). To better understand how these molecular differences 0032 FIG. 11 Structural Details of the Switch Helix-Me contribute to the unique characteristics of each kinase, we diated Dimer Interface (A)“Top' view of switch helices (SW) crystallized the central portion of the regulatory domain of and their assemblage with the neighboring B-domain: hydro PKG IC. While prokaryotic expression of PKG constructs phobic knobs at the end of the SW, residues that comprise a containing the catalytic domain yields inactive, misfolded hydrophobic nest in the B-domain. (B) Details of the knob protein that is sequestered to inclusion bodies (Feil et al., nest assembly that mediates PKG7 dimerization. The 1993), previous attempts to crystallize full-length PKG from switch helix (lightest gray), and helical features from the mammalian systems have been unsuccessful as well. This is neighboring B-domain (darkest gray) are represented as rib in part due to conformational heterogeneity Stemming from bons. (C) Kinetic Analysis of WT PKGIC. (O) and SW knob mixed phosphorylation states at the N terminus and a pro mutants FSA, FSA, LSA (), NA (()), and FSA, teolytically exposed hinge at Arg’” (Aitken et al., 1984; Heil F351A N353A, L35A (0). et al., 1987; Scholten et al., 2007). We identified according to 0033 FIG. 12 DxMS for PKG7. (A) Secondary struc the invention that exclusion of both the N-terminal D/D tural features from the crystal structure of PKG Io.7 are domain, as well as the C-terminal catalytic domain gave rise positioned above sequence alignment for PKGIC, IB and II. to a stable, monomeric fragment that is readily expressed in Cys117 and Cys 195 (boxed) form a disulfide bond in the Escherichia coli. Multiple regulatory fragments were A-domain. Individual residues that comprise the hydrophobic designed to encompass both tandem coMP-binding domains nest in the B domain are highlighted and switch helix knob (PKG7-?, pKG7' and PKG7). The standard method residues are identified. DXMS results are presented under the for purification of recombinant PKG utilizes cyclic nucle sequence alignment. Percent deuteration was measured at otide affinity chromatography which saturates the allosteric four time points (3 s, 30 s. 300 s. 3000s). (B) Analysis of binding sites through an elution with high concentrations of DXMS from switch helix peptides. Percent deuteration as a cNT (Dostmann et al., 1999). To prevent this artificial expo function of time for D-E7 (O), D7-E7 () and F sure of PKG protein to cnTs, PKG constructs were engi K(A) is shown at the left with a summary of the initial rate neered with N-terminal hexa-histidine tags and purified using of deuterium exchange from 3-30 seconds at the right. standard immobilized metal affinity chromatography 0034 FIG. 13 Native PAGE analysis of PKG7. (A) 5 (IMAC) methods. Three PKG regulatory domain constructs ug of purified PKG7 in solution exhibited two distinct (PKG7, PKG7', and PKG7) were prepared. populations consistent with monomeric and dimeric forms of Despite high yielding, stable expression of all three con the protein (lane 2). (B) PKG 7 taken from crystals dis structs, only PKG yielded diffraction-quality crystals. Solved in Buffer A (see methods) migrated corresponding to The crystal structure is described below in detail in the the dimer. Each gel was 9.5% acrylamide lacking SDS. Gels Examples section. were run at constant voltage (200V) on ice for 2 hours. 0037 PKGI serves as the main intracellular receptor for cGMP and is a key branch points of the nitric oxide (NO) and DETAILED DESCRIPTION natriuretic peptide mediated signaling pathways. Most drugs, 0035. The coMP-dependent protein kinase (PKG) serves known to increase cQMP such as nitrates and inhibitors of as an integral component of second messenger signaling in a phosphodiesterases, are mediating their effects through PKG number of biological contexts including cell differentiation, (Bryan et al., 2009; Kots et al., 2011; Schlossmann et al., memory, and vasodilation. PKG is homodimeric and large 2005). These kinases modulate learning and memory, renin conformational changes accompany c0MP binding. How release, intestinal Secretion, platelet aggregation as well as ever, the structure of PKG and the molecular mechanisms vascular, gastrointestinal, bladder and penile Smooth muscle associated with protomer communication following c(GMP relaxation (Kemp-Harper et al., 2009; Sandner et al., 2009: induced activation were unknown. According to the invention Walteret al., 2009: Kleppish et al., 2009). The structure of the a 2.5 A crystal structure of a regulatory domain construct (aa PKG regulatory domain identified according to the invention, 78-355) containing both cGMP binding sites of PKGIC. was uncovered a unique, previously unrecognized helical domain developed. A distinct and segregated architecture with an (SW) that is important in kinase assembly and functionality. extended central helix separates the two coMP binding The presence of the SW directly contradicts prior models that domains. Additionally, a previously uncharacterized helical focus solely on the N-terminus as the only site of dimeriza domain (switch helix) promotes the formation of a hydropho tion. bic interface between protomers. Mutational disruption of 0038 Based on these findings we developed an alternative this interaction in full-length PKG implicates the switch helix view of PKG holoenzyme activation, which helps resolve US 2014/0037547 A1 Feb. 6, 2014

important unanswered questions regarding the role of dimer Waals, dipole and H-bond interactions are woven into a ization in the cyclic-GMP dependent protein kinases, in par concise network typically observed in drug-receptor sites. ticular, and, the role of symmetry in allosteric enzyme regu Further, the native affinity between knob and nest residues is lation, in general. The discovery of the SW and the location of driven by hydrophobic interactions (F350, F351, L354) that its binding site has prompted the development of SW-derived convey binding affinity as well as hydrogen-bond interactions PKG activators. These molecules have been shown to be (N353, T220) to that are known to contribute to binding functionally relevant by targeting PKG to elevate the open specificity (FIG. 2). The nest is accessible, yet sufficiently probability of large-conductance potassium channels (K1. deep to fully engulf Small ligands. These features prompted 1) in a similar fashion to that of c(GMP. Therefore, SW us to synthesize derivatives of the knob in an effort to ascer derivatives may provide a new platform for targeting the tain if molecules capable of mimicking the knob?nest inter activity of PKG isoforms independent of the classic nitric action could competitively displace the native SW domain oxide and natriuretic peptide driven pathways. and what consequences that would have on the function of 0039 All our experimental protocols have employed PKG. refined expression techniques that allow us to control the 0044 Starting with the 29-mer peptide (329-358), cover exposure to cyclic nucleotides and the degree of auto-phos ing the full length of the switch helix, an array of peptide phorylation in our recombinant preparations of PHGIC. (dele derivatives were synthesized to study the contributions from tion fragments and native enzyme). As a result, we solved the different segments of the SW helix peptide towards the func first crystal structure of any large fragment of PKG. This tional phenotype of PKG 1 C. Circular dichroism spectros structure of PHG IC. (78–356) contains the majority of the copy confirmed that the 29-mer SW peptide adopted a helical regulatory domain and the novel SW domain, which forms an secondary structure. Reversible binding of SW:329-358 to intermolecular boundary between the regulatory and the cata wild-type PHG Ia was confirmed by surface-plasmon-reso lytic domains. The experimental conditions that lead to Solv nance, while a sequence-scrambled control peptide demon ing the structure have precipitated additional advances in strated no binding. More importantly, SW: 329-358 dose obtaining crystals from other PKG constructs. dependently activated PHG Ia (K-500 uM) as determined by 0040. This interchain communication between PKG pro P-incorporation assay (FIG. 3). The knob/nest interaction tomers is Surprisingly concise, showing the hallmarks of a occurs at a site adjacent to the B-site that is associated with drug/receptor architecture, which we termed knob/nest inter cGMP binding. Priming the kinase with 100 nM coMP action (Shown in FIG. 2). Peptidic fragments of the SW resulted in a 20-fold shift in the SW329-358 mediated acti domain not only bind competitively to native PKG IC, but vation profile of PKG 1c (K-40 uM), strongly reiterates the also activate the enzyme through an allosteric mechanism that synergistic cross-talk between the coiMP-B site and the knob? involves the coMP-binding site B. SW peptides present the nest. Under coMP priming conditions, removal of the C-ter first class of Small non-cyclic nucleotide molecules capable minal residues LSD was sufficient to markedly decrease the of activating PKG. peptides activation potential (SW:329-355 K-500 uM). 0041. We also discovered that a shortened sequence con Additional removal of the knob residues (SW:329-349) ren taining only the knob residues is sufficient to activate PKG dered a completely inactive SW-peptide derivative (FIG. 3). (SW:348-355AAFFANLK). This confirms that the knob and To further test if the knob is not only necessary but also its defined Stereochemical orientation is the essential phar sufficient to activate PKG, we synthesized an 8-mer peptide macophore for this new target site within PKG necessary to that consisted only of the minimal knob motif (SW:348-355 activate PKG. As shown in FIG. 2, the pharmacophore is AAFFANLK). This knob-derived peptide did in fact activate provided by the contiguous arrangement of Phe350, Phe351, PKG (K-500 uM) albeit weakly, corresponding to 30% of and Leu354. This hydrophobic pocket provides approxi activation associated with cGMP. The activity of this short mately 20 kcal/mol of van der Waals interaction energy. In peptide is Surprising. To activate the kinase, this non-helical addition, the pharmacophore is Supplemented by a hydrogen 8-mer peptide first has to adopt the active helical conforma bond provided by ASn353 that lends specificity to the activa tion to activate PKG. However, this finding provides critical tor sequence (FIGS. 11A and 11B). support in favor that the confined knob/nest interaction site 0042. The maintenance and modulation of smooth muscle may serve as a novel target site for non-cyclic nucleotide tone is a persistent topic of investigation due to the pervasive driven activation of PKG. nature of cardiovascular disease in aging populations (Roger 0045. As outlined above, we have observed that SW:329 et al., 2011). The coMP-dependent protein kinase lies at the 358 activates PHG IC. in vitro and acts synergistically with heart of the regulation of Smooth muscle and acts as the cGMP to modulate activation. The isolation of the SW inde gatekeeper for nitric oxide and natriuretic peptide pathways pendent from the kinase allows us to discretely probe the nest (Kuhn, 2004; Bian et al., 2007; McDonald et al., 1995). in future studies using native and non-native amino acids. Therefore, it comes as a Surprise that the past thirty years have Moreover, the use of the SW: 329-358 allows us to answer yielded little definitive information regarding the structure more fundamental questions about the nature of the regula and function of this essential signaling node. The solution of tory and catalytic domains and the communication between the crystal structure of the largest fragment of PHG IC. has the B-site and the nest. Cyclic nucleotide analogs have been advanced the understanding of type I isoforms of PKG to used in previous investigations to competitively inhibit PKA include its reliance on the SW domain. and PKG (Dostmann et al., 1990; Dostmann and Taylor, 0043. The knob?nest interactions display several critical 1991; Ogreid et al., 1994: Dostmann, 1995). In particular, features of a typical ligand/drug-receptor binding site. The (RP)-8-Br-PET-cGMPS (Rp-PET) is a commercially-avail surface area provided by the nest is stereo-chemically well able analog that has a higher affinity for the PHG IC. regula defined and Suitable for accepting Small organic molecules as tory domain binding sites than ccjMP (Butt et al., 1995). The supplied by the native knob residues FFANL (MW=610 Da) structure of this inhibitor contains a B-phenyl-1,N-etheno that are also sterochemically defined (FIG. 2). The van-der group attached to the pyrimidine ring. We hypothesized pre US 2014/0037547 A1 Feb. 6, 2014

viously, based upon structural observations, that the presence 0051. In some embodiments the coMP independent PKG of this phenyl group in the syn orientation effectively blocks activator is a peptide of 15-30 amino acids in length. Thus, the PBC closure and reduces the potency of the SW:329-358 in a invention includes peptides and peptide mimetics that bind to dose-dependent manner. We have now found that when the the nest region. These peptides maybe structurally similar to Rp-PET is titrated into reactions measuring activation of the knob. For instance, an isolated peptide comprising PHGIC by SW:329-358, we observed a linear response of the XXX-XXXX,Xs (SEQID NO: 8), wherein each X is an activation constant to the amount of Rp-PET titrated (FIG.3). amino acid, and wherein the peptide binds to a nest region These results fully support our hypothesis that the knob is according to the invention. X refers to any amino acid, natu influenced by changes in the PBC, associated with the coMP rally occurring or modified. In some instances the peptide binding event. comprises FFANL (SEQ ID NO. 9). In some embodiments 0046. Once we had supporting evidence that molecules the Xs referred to the in SEQ ID NO: 8 have the following carrying the knob-type ligand conformation were capable of values: activating PKG we set out to determine if this novel mecha 0052 X is Ala, Gly, Leu, Ile, Val, Cys, Ser. Thr, or Pro nism of kinase activation was functionally relevant. The high 0053 X is Ala, Gly, Leu, Ile, Val, Cys, Ser, Thr, or Pro conductance calcium-activated potassium channel K1.1 is 0054 X is Phe, Tyr, or Trp a well-established intracellular target of PHG IC. in vascular 0055 X is Phe, Tyr, or Trp smooth muscle (VSM) cells. Using inside-out patch clamp 0056 X is Ala, Gly, Leu, Ile, Val, Cys, Ser, Thr, or Pro configurations from freshly dissociated VSM cells from rat 0057 X is Asn, Asp, Glu, Gln, Ser, or Thr posterior cerebral arteries, we measured the changes of open 0058 X, is Leu, Ile, Val, or Ala probability (NPo) of single KCa1.1 channels in response to 0059 X is Lys Arg, Glu, His, Asp, Gln, Ser, Thr, or Tyr PKG stimulation in membrane patches excised from freshly 0060. In other embodiments the Xs referred to the in SEQ dissociated vascular smooth muscle cells (FIG. 4). While ID NO: 8 have the following values: patches reconstituted with recombinant PKG, or PKG plus 0061 X is Ala, Gly, Leu, Ile, or Val scrambled SW329-358 peptide showed no significant 0062 X is Ala, Gly, Leu, Ile, or Val increase in NPo over non-PKG controls, SW329-358 peptide 0063 X is Phe, Tyr, or Trp increased NPo, similar to c(GMP 0064 X is Phe, Tyr, or Trp 0047 Direct communication between regulatory and cata 0065 X is Ala, Gly, Leu, Ile, or Val lytic elements is not without precedent in AGC kinases. In 006.6 X is Asn., Asp, or Glu, PKA, there are at least four major sites of contact between the 0067 X, is Leu, Ile, Val, or Ala R and C subunits, with CNB domain A providing the largest 0068 X is Lys or Arg docking surface for the C subunit (Boettcher et al., 2011; Kim 0069. The peptide preferably is XXFFANLXs (SEQ ID et al., 2007). Likewise, in PKC BII, the conserved to NH) NO: 10). Such that XX, and Xs are any amino acid and may motif in the catalytic domain is clamped by the diacylglyc be Ala of Lys. erol-binding C1 B domain until fully activated (Leonard et al., 0070 The minimal peptide length for binding the nest is 5 2011). While these interactions highlight the diversity of and preferably 8 amino acids. However, there can be over interdomain complementarity among AGC kinase family hanging amino acids on either side of the core structure. For members, they also demonstrate clear commonalities in their some well-studied peptides, it is known that additional over mechanisms of regulation. hanging amino acids on both the Nand C terminican augment 0048. This crystal structure provides the first atomic view binding. Thus the peptide may be 9 amino acids in length or of PKG and provides a new platform for understanding the it may be longer. For instance, the peptide may have addi allosteric regulation of the holoenzyme complex. The overall tional amino acids at the N and/or C terminus. The amino fold of PKG is surprising, in that a previously unchar acids at either terminus may be anywhere between 1 and 100 acterized allosteric interface promotes a novel means of com amino acids. In some embodiments the peptide includes 1-50, munication between PKG protomers wherein the cata 1-20, 1-15, 1-10, 1-5 or any integer range there between. lytic domain can be tethered between inactive and active When the peptide is referred to as “N-AAFFANLK-C” (SEQ states. The crossing of Switch helices between protomers ID NO: 7) the Cand—N refer to the terminus of the peptide (FIG. 10B) suggests that the catalytic domain from one pro and thus the peptide is only 8 amino acids in length. However tomer is regulated in part by the regulatory domain of the the 8 amino acid peptide may be linked to other non-peptide other protomer. moieties at either the —C or —N terminus or internally. 0071 Examples of specific peptides that are useful as a 0049 Based on the structures described herein, a class of cGMP independent PKG to activator include compounds having therapeutic activity has been discovered. DVSNKAYEDAEAKAKYEAEAAFFANLKLSD (SEQ ID These compounds, referred to herein as cCMP independent NO. 4); DVSNKAYEDAEAKAKYEAEAAFFANLK (SEQ PKG activators, are able to activate PKG in the absence of ID NO. 5): DVSNKAYEDAEAKAKYEAEAAFF (SEQ ID cGMP. The coMP independent PKG activators include pep NO. 6); and AAFFANLK (SEQID NO. 7). Peptides encom tides, nucleic acids, and Small molecules. In some embodi passing conservative substitutions of SEQ ID NOS.4-7 are ments acci MP independent PKG activator does not include also encompassed within the invention. Other peptides can BR4979 or BR498O. easily be identified by the skilled artisan using computer 0050. In certain embodiments, the coMP independent modeling and/or peptide screening. Such as peptide library PKG activators are peptides, antibodies or antigen-binding screens using the information provided herein. fragments that bind to the PKG activation domain. The pep 0072 Guidance as to appropriate amino acid substitutions tides, antibodies or fragments thereof may be selected for the that do not affect biological activity of the polypeptide of ability to bind the activation domain of PKG in the absence of interest may be found in the model of Dayhoffetal. (1978) in cGMP. Atlas of Protein Sequence and Structure (Natl. Biomed. Res. US 2014/0037547 A1 Feb. 6, 2014

Found. Washington, D.C.), and Betts and Russell, Bioinfor bifunctional cross-linker); certain N-Succinimide esters (e.g., matics for Geneticists, chapter 14, 2003, herein incorporated discuccinimyidyl Suberate, dithiobis(Succinimidyl propi by reference. Conservative Substitutions, such as exchanging onate), and soluble bis-Sulfonic acid and salt thereof (see, one amino acid with another having similar properties, may e.g., Pierce Chemicals, Rockford, Ill.: Sigma-Aldrich Corp., be preferred. Examples of conservative substitutions include, St. Louis, Mo.). but are not limited to, Gly, Ala; Val. Ile-Leu, Asp.Glu; Lys.Arg; 0080 Preferably, a bifunctional cross-linker molecule is a Asn.Gln; and Phe,Trp.Tyr or alternatively within the follow heterobifunctional linker molecule, meaning that the linker ing classifications: hydrophobic (Ala, Ile, Pro, Val, Phe, Trp, has at least two different reactive sites, each of which can be Tyr), hydrophobic aliphatic side chains (Ala, Ile, Pro, Val), separately linked to a peptide or other molecule. Use of such hydrophobic aromatic side chains (Phe, Trp, Tyr), polar heterobifunctional linkers permits chemically separate and amino acids (ASn (negative), Glu (negative), Lys (positive), stepwise addition (vectorial conjunction) of each of the reac Arg (positive), His (neutral), Asp (neutral), Gln (neutral), Ser tive sites to a selected peptide sequence. Heterobifunctional (neutral), Thr (neutral), Tyr (neutral)), and Small amino acids linker molecules to useful in the invention include, without (Ala, Cys, Gly, Pro, Ser. Thr). limitation, m-maleimidobenzoyl-N-hydroxysuccinimide 0073. The peptide has a minimum length of 9 amino acids. ester (see, Green et al., Cell, 28: 477-487 (1982); Palkeret al., In some embodiments it has a length of 9-20 amino acids. The Proc. Natl. Acad. Sci (USA), 84: 2479-2483 (1987)); m-ma peptide may be cyclic or non-cyclic. In some embodiments leimido-benzoylsulfosuccinimide ester; Y-maleimidobutyric the peptide is PEGylated. In other aspects the invention is an acid N-hydroxysuccinimide ester; and N-succinimidyl 3-(2- isolated peptide comprising SEQID NO. 7. pyridyl-dithio)propionate (see, e.g., Carlos et al., Biochem.J., 0074 The amino acids may be naturally occurring amino 173: 723-737 (1978); Sigma-Aldrich Corp., St. Louis, Mo.). acids as well as non-naturally occurring amino acids. Natu I0081. The carboxyl terminal amino acid residue of the rally occurring amino acids are generally C-amino acids peptides described herein may also be modified to block or because the amino group is attached to the first carbon atom reduce the reactivity of the free terminal carboxylic acid after the COOH group. Conventionally a peptide is numbered group, e.g., to prevent formation of esters, peptide bonds, and from the N terminal to C terminal end. other reactions. Such blocking groups include forming an 0075. A composition of a peptide of the invention and a amide of the carboxylic acid group. Other carboxylic acid carrier is provided in other aspects. In some embodiments the groups that may be present in polypeptide may also be carrier is a liposome, such as a stealth liposome. In other blocked, again provided such blocking does not elicit an embodiments the carrier is a particle, for instance, a nanopar undesired immune reaction or significantly alter the capacity ticle or a low density to particle. In other embodiments the of the peptide to specifically function. carrier is a transmucosal absorption enhancer. I0082. The peptide for instance, may be linked to a PEG 0076. The peptides may also be linked to other molecules. molecule. Such a molecule is referred to as a PEGylated The two or more molecules may be linked directly to one peptide. The peptides useful herein are isolated peptides. another (e.g., via a peptide bond); linked via a linker mol I0083. In further embodiments, the peptide is an antibody ecule, which may or may not be a peptide; or linked indirectly or antigen biding fragment thereof against the activation to one another by linkage to a common carrier molecule, for domain of PKG. An antibody or antigen-binding fragment instance. thereof is selected from the group consisting of IgG1, IgG2. 0077. Thus, linker molecules (“linkers') may optionally IgG3, IgG4, IgM, IgA1, IgA2, IgASec, Ig|D, IgE or has immu be used to link the peptide to another molecule. Linkers may noglobulin constant and/or variable domain of IgG1, IgG2. be peptides, which consist of one to multiple amino acids, or IgG3, IgG4, IgM, IgA1, IgA2, Ig|D or IgE. In other embodi non-peptide molecules. Examples of peptide linker mol ments, the antibody is a bispecific or multispecific antibody. ecules useful in the invention include glycine-rich peptide In still other embodiments, the antibody is a recombinant linkers (see, e.g., U.S. Pat. No. 5,908.626), wherein more than antibody, a polyclonal antibody, a monoclonal antibody, a half of the amino acid residues are glycine. Preferably, such humanized antibody or a chimeric antibody, or a mixture of glycine-rich peptide linkers consist of about 20 or fewer these. In particularly preferred embodiments, the antibody is amino acids. a human antibody, e.g., a monoclonal antibody, polyclonal 0078 Linker molecules may also include non-peptide or antibody or a mixture of monoclonal and polyclonal antibod partial peptide molecules. For instance the peptide may be ies. In still other embodiments, the antibody is a bispecific or linked to other molecules using well known cross-linking multispecific antibody. Preferred antigen-binding fragments molecules such as glutaraldehyde or EDC (Pierce, Rockford, include a Fab fragment, a F(ab') fragment, and a Ffragment Ill.). Bifunctional cross-linking molecules are linker mol CDR3. Antibodies can be generated by injecting an animal, ecules that possess two distinct reactive sites. For example, preferably a rabbit or goat or mouse, with the PKG activation one of the reactive sites of a bifunctional linker molecule may domain. be reacted with a functional group on a peptide to form a I0084. In order to prepare polyclonal antibodies, fusion covalent linkage and the other reactive site may be reacted proteins containing the complete or defined fragments of the with a functional group on another molecule to form a cova PKG activation domain protein can be synthesized in bacteria lent linkage. General methods for cross-linking molecules by expression of corresponding DNA sequences in a Suitable have been reviewed (see, e.g., Means and Feeney, Bioconju cloning vehicle. The protein can then be purified, coupled to gate Chem., 1:2-12 (1990)). a carrier protein and mixed with Freund's adjuvant (to help 0079 Homobifunctional cross-linker molecules have two stimulate the antigenic response by the rabbits) and injected reactive sites which are chemically the same. Examples of into rabbits or other laboratory animals. Alternatively, protein homobifunctional cross-linker molecules include, without can be isolated from cultured cells expressing the protein. limitation, glutaraldehyde: N,N'-bis(3-maleimido-propio Following booster injections at bi-weekly intervals, the rab nyl-2-hydroxy-1,3-propanediol (a Sulfhydryl-specific homo bits or other laboratory animals are then bled and the sera US 2014/0037547 A1 Feb. 6, 2014 isolated. The sera can be used directly or purified prior to use, evaluating the potential of a selected ligand to associate with by various methods including affinity chromatography, Pro any of the molecules or molecular complexes set forth above. tein A-Sepharose, Antigen Sepharose, Anti-mouse-Ig This method includes the steps of: (a) employing computa Sepharose. The sera can then be used to probe protein extracts tional means, for example, Such as a programmable computer run on a polyacrylamide gel to identify the PKG activation including the appropriate Software known in the art or as domain. Alternatively, synthetic peptides can be made to the disclosed herein, to perform a fitting operation between the antigenic portions of the protein and used to inoculate the potential therapeutic compound and the activation domain of animals. PKG and (b) analyzing the results of the fitting operation to 0085. To produce monoclonal PKG activation domain quantify the association between the potential therapeutic antibodies, mice are injected multiple times, the mice spleens compound and the activation domain of PKG. Several meth are removed and resuspended in a phosphate buffered saline ods can be used to Screen potential therapeutic compounds for (PBS). The spleen cells serve as a source of lymphocytes, the ability to associate with the activation domain of PKG. Some of which are producing antibody of the appropriate Selected potential therapeutic compounds may be positioned specificity. These are then fused with a permanently growing in a variety of orientations associating with the activation myeloma partner cell, and the products of the fusion are domain of PKG. This may be accomplished using software plated into a number of tissue culture wells in the presence of such as QUANTA (Molecular Simulations, Inc., San Diego, a selective agent such as HAT. The wells are then screened to Calif., USA.) and SYBYL (TRIPOS, St. Louis, Mo., USA), identify those containing cells making useful antibody by followed by energy minimization and molecular dynamics ELISA. These are then freshly plated. After a period of with standard molecular mechanics force fields, such as growth, these wells are again screened to identify antibody CHARMM (Molecular Simulations, Inc., San Diego, Calif., producing cells. Several cloning procedures are carried out USA) and AMBER (P. A. Kollman, University of California until over 90% of the wells contain single clones which are at San Francisco, San Francisco, Calif., USA). positive for antibody production. From this procedure a stable I0089 Any of the biological or biochemical functions line of clones is established which produce the antibody. The mediated by the activation domain of PKG can be used as an monoclonal antibody can then be purified by affinity chroma endpoint assay to identify an agent that modulates PKG activ tography using Protein A Sepharose, ion-exchange chroma ity (a putative therapeutic compound). The assays may tography, as well as variations and combinations of these include all of the biochemical or biochemical/biological techniques. events described herein, in the references-cited herein, incor I0086 For antibodies to be used in therapy in humans, they porated by reference for these endpoint assay targets, and preferably are humanized. Humanization of antibodies other functions known to those of ordinary skill in the art or involves replacing native mouse sequences with human that can be readily identified. Compounds can be identified sequences as to lower the chance of an immune response once through cellular assayS. Cellular assays may involve, for the therapeutic antibody is introduced into humans. instance, expressing the activation domain of PKG in cells 0087 Computational techniques can be used to screen, and testing a variety of compounds for their ability to bind to identify, select, and design compounds capable of binding to the expressed peptide. The assay may be performed with the activation domain of PKG and functioning as cCMP labeled compounds, facilitating identification of the com independent PKG activators. In particular, computational pound that binds. In another embodiment a biological readout techniques can be used to identify or design ligands, such as can be used to identify a putative therapeutic compound. agonists and/or antagonists, that associate with the activation Biological assays will allow for the identification of both domain of PKG. Once identified and screened for biological agonists and antagonists or inhibitors. Competition binding activity, these agonists, antagonists, and combinations assays may also be used to discover compounds that interact thereof, may be used therapeutically, for example, to increase with the activation domain of PKG (e.g. binding partners PKG activity. Data stored in a machine-readable storage and/or ligands). Thus, a compound is exposed to the activa medium that is capable of displaying a graphical three-di tion domain of PKG under conditions that allow the com mensional representation of the structure of the potential pound to bind or to otherwise interact with the polypeptide. A therapeutic compound or a structurally homologous mol peptide orantibody or fragment thereofagainst the activation ecule or molecular complex, as identified herein, or portions domain of PKG may be added to the mixture. If the test thereof may thus be advantageously used for drug discovery. compound interacts with the activation domain of PKG, it The structure coordinates of the potential therapeutic com decreases the amount of peptide or antibody that can bind to pounds are used to generate a three-dimensional image that the activation domain of PKG. To perform cell free drug can be computationally fit to the three-dimensional image of screening assays, it is sometimes desirable to immobilize the the activation domain of PKG. The three-dimensional activation domain of PKG, or its target molecule to facilitate molecular structure encoded by the data in the data storage separation of complexes from uncomplexed forms of one or medium can then be computationally evaluated for its ability both of the proteins, as well as to accommodate automation of to associate with the potential therapeutic compound. When the assay. Agents that modulate the activation domain of PKG the molecular structures encoded by the data is displayed in a can be identified using one or more of the above assays, alone graphical three-dimensional representation on a computer or in combination. It is generally preferable to use a cell screen, the protein structure can also be visually inspected for based or cell free system first and then confirm activity in an potential association with the potential therapeutic com animal or other model system. Such model systems are well pound. known in the art and can readily be employed in this context. 0088. One embodiment of the method of drug design 0090. To unlock the therapeutic potential of the activation involves evaluating the potential association of a candidate domain of PKG, the structure of the activation domain of therapeutic compound with the activation domain of PKG. PKG has been elucidated. The invention presented herein The method of drug design thus includes computationally discloses the materials and methods that were used to deter US 2014/0037547 A1 Feb. 6, 2014

mine the structure and mechanism of function of the activa has been extensively described in numerous publications, tion domain of PKG. The invention also encompasses com including standard biochemistry text books, such as “Bio positions and methods of use of native activation domains of chemistry’ by Geoffrey Zubay, Addison-Wesley Publishing PKG as well as modified activation domains of PKG having Co., 1986 edition, which describes conservative and non at least one deletion or Substitution from a native activation conservative Substitutions and properties of amino acids domain of PKG, for instance, when it is desirable to compete which lead to their definition as polar, non-polar or acidic. with a naturally occurring PKG. As used herein, a native 0.095 The modified activation domains of PKG having at activation domain of PKG is a naturally occurring form of least one substitution, deletion or insertion have, in some PKG including two coMP binding domains, a hinge region, embodiments, a native conformation. A native conformation and a switch helix. The native activation domain of PKG may as used herein refers to a tertiary structure that is similar to the have the sequence of any naturally occurring PKG, but pref tertiary structure of native activation domain of PKG. The erably has the sequence of a human PKG. tertiary structure of modified or native activation domains of 0091. The activation domain of PKG may be an isolated PKG can be assessed using structural analysis such as crys peptide. An isolated peptide or molecule is a molecule that is tallography or by functional analysis, such as binding and/or substantially pure and is free of other substances with which activity assays and NMR spectroscopy. it is ordinarily found in nature or in vivo systems to an extent practical and appropriate for its intended use. In particular, 0096 Crystallographic data can be obtained by perform the molecular species are sufficiently pure and are sufficiently ing crystallographic analysis on crystals of the activation free from other biological constituents of host cells so as to be domain of PKG using for instance, the methods described in useful in, for example, producing pharmaceutical prepara the Examples. Alternatively crystals can be grown by various tions or sequencing if the molecular species is a nucleic acid, methods, such as, for example, sitting or hanging drop vapor peptide, or polysaccharide. Because an isolated molecular diffusion. In general, crystallization can be performed at a species of the invention may be admixed with a pharmaceu temperature of from about 4°C. to about 60°C. The activation tically-acceptable carrier in a pharmaceutical preparation or domain of PKG can be crystallized from a solution including be mixed with some of the components with which it is NaCl, MgCl, Tris buffer and polyethylene glycol (PEG). The associated in nature, the molecular species may comprise Solution can include a precipitant, such as ammonium sulfate. only a small percentage by weight of the preparation. The Structural data describing a crystal can be obtained, for molecular species is nonetheless Substantially pure in that it example, by X-ray diffraction. X-ray diffraction data for the has been substantially separated from the substances with crystals can be collected by a variety of means in order to which it may be associated in living systems. obtainstructural coordinates. Suitable X-ray sources include 0092. The activation domain of PKG may be in the context rotating anode and synchrotron sources (e.g., NSLS, of, or separate from, the full length PKG or portions thereof. Brookhaven, N.Y.). The X-ray diffraction data can be used to If the activation domain of PKG is in the context of the PKG construct an electron density map of the activation domain of it may form a full length or partial PKG. The full length or PKG. Creation of an electron density map typically involve partial PKG may include the native sequence or may include using information regarding the phase of the insertions, deletions or substitutions. When the peptide con 0097. X-ray scatter. Methods for calculating phase from sists of the activation domain of PKG it is not found in the X-ray diffraction data, include, without limitation, multi context of a partial or full length PKG. wavelength anomalous dispersion (MAD), multiple isomor 0093 Modified activation domains of PKG having at least phous replacement (MIR), multiple isomorphous replace one Substitution, deletion or insertion are also useful accord ment with anomalous scattering (MIRAS), reciprocal space ing to the invention. As used herein, a “conservative amino Solvent flattening, molecular replacement, and single isomor acid substitution' or “conservative substitution” refers to an phous replacement with anomalous scattering (SIRAS), or a amino acid Substitution in which the Substituted amino acid combination thereof. These methods generate phase informa residue is of similar charge as the replaced residue and is of tion by making isomorphous structural modifications to the similar or smaller size than the replaced residue. Conservative native protein, such as by including a heavy atom or changing Substitutions of amino acids include Substitutions made the scattering strength of a heavy atom already present, and amongst amino acids within the following groups: (a) the then measuring the diffraction amplitudes for the native pro Small non-polar amino acids, A, M. I. L., and V; (b) the Small tein and each of the modified cases. If the position of the polar amino acids, G. S. T and C; (c) the amidoamino acids, additional heavy atom or the change in its scattering strength Q and N; (d) the aromatic amino acids, F, Y and W: (e) the is known, then the phase of each diffracted X-ray can be basic amino acids, K. Rand II; and (f) the acidic amino acids, determined by solving a set of simultaneous phase equations. E and D. Substitutions which are charge neutral and which The location of heavy atom sites can be identified using a replace a residue with a smaller residue may also be consid computer program, such as SHELXS, (Sheldrick, Institut ered "conservative substitutions' even if the residues are in Anorg. Chemie, Gottingen, Germany) or Sharp (Global Phas different groups (e.g., replacement of phenylalanine with the ing, Cambridge, UK) and diffraction data can be processed smaller isoleucine). The term “conservative amino acid sub using computer programs such as MOSFLM, SCALA, stitution” also refers to the use of amino acid analogs or SOLOMON, (“The CCP4 Suite: Programs for Protein Crys variants. tallography.” 1997, Acta Crystallogr. Sect. D, 54, 905-921; 0094 Methods for making amino acid substitutions, addi deLa Fortelle et al. 1997, Meth. Enzym. 276, 472-494) and tions or deletions are well known in the art. The terms “con HKL2000 (HKL Research, Charlottesville, Va.). Upon deter servative substitution”, “non-conservative substitutions', mination of the phase, an electron density map of the complex “non-polar amino acids”, “polar amino acids, and "acidic can be constructed. amino acids are all used consistently with the prior art ter 0098. The electron density map can subsequently be used minology. Each of these terms is well-known in the art and to derive a representation of a polypeptide, a complex, or a US 2014/0037547 A1 Feb. 6, 2014 fragment of a polypeptide or complex by fitting a three salt, buffering agents, preservatives, compatible carriers, dimensional model of a polypeptide or complex into the adjuvants, and optionally other therapeutic ingredients. electron density map. 0099. The conformation of the activation domain of PKG 0105 For use in therapy, an effective amount of the coMP may also be assessed by whether the activation domain of independent PKG activator can be administered to a subject PKG is able to bind to compounds that the native activation by any mode that delivers the coMP independent PKG acti domain of PKG binds to. The binding of the activation vator to the desired surface. Administering the pharmaceuti domain of PKG to c(GMP may be determined according to cal composition of the present invention may be accom standard procedures. plished by any means known to the skilled artisan. Preferred 0100 Thus the compounds of the invention are useful for routes of administration include but are not limited to oral, treating Subjects having a PKG deficient condition as well as parenteral, intramuscular, intranasal, Sublingual, intratra other disorders described herein. A “subject' shall mean a cheal, inhalation, ocular, vaginal, and rectal. human or vertebrate mammal including but not limited to a 0106 For oral administration, the coMP independent dog, cat, horse, cow, pig, sheep, goat, or primate, e.g., mon PKG activator can be formulated readily by combining the key. active compound(s) with pharmaceutically acceptable carri 0101 Thus, in some aspects the invention is a method of ers well known in the art. Such carriers enable the compounds treating a PKG deficient condition in a subject. The method of the invention to be formulated as tablets, pills, dragees, involves administering to the Subject a therapeutically effec capsules, liquids, gels, syrups, slurries, Suspensions and the tive amount of a coMP independent PKG activator. A PKG like, for oral ingestion by a subject to be treated. Pharmaceu deficient condition includes but is not limited to cardiovascu tical preparations for oral use can be obtained as Solid excipi lar disorders, hypoxia, spinal cord injury, and stroke. ent, optionally grinding a resulting mixture, and processing 0102 The compounds of the invention are useful in effec the mixture of granules, after adding Suitable auxiliaries, if tive amounts. The term effective amount refers to the amount desired, to obtain tablets or dragee cores. Suitable excipients necessary or sufficient to realize a desired biologic effect. are, in particular, fillers such as Sugars, including lactose, Combined with the teachings provided herein, by choosing Sucrose, mannitol, or Sorbitol; cellulose preparations such as, among the various active compounds and weighing factors for example, maize starch, wheat starch, rice starch, potato Such as potency, relative bioavailability, patient body weight, starch, gelatin, gum tragacanth, methyl cellulose, hydrox severity of adverse side-effects and preferred mode of admin ypropylmethyl-cellulose, Sodium carboxymethylcellulose, istration, an effective prophylactic or therapeutic treatment and/or polyvinylpyrrollidone (PVP). If desired, disintegrating regimen can be planned which does not cause Substantial agents may be added. Such as the cross-linked polyvinyl toxicity and yet is effective to treat the particular subject. The pyrrolidone, agar, or alginic acid or a salt thereof Such as effective amount for any particular application can vary Sodium alginate. Optionally the oral formulations may also be depending on Such factors as the disease or condition being formulated in saline or buffers, i.e. EDTA for neutralizing treated, the particular cGMP independent PKG activator internal acid conditions or may be administered without any being administered, the size of the subject, or the severity of carriers. the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular 0107 Also specifically contemplated are oral dosage cGMP independent PKG activator and/or other therapeutic forms of the above component or components. The compo agent without necessitating undue experimentation. It is pre nent or components may be chemically modified so that oral ferred generally that a maximum dose be used, that is, the delivery of the derivative is efficacious. Generally, the chemi highest safe dose according to some medical judgment. Mul cal modification contemplated is the attachment of at least tiple doses per day may be contemplated to achieve appropri one moiety to the component molecule itself, where said ate systemic levels of compounds. Appropriate system levels moiety permits (a) inhibition of proteolysis; and (b) uptake can be determined by, for example, measurement of the into the blood stream from the stomach or intestine. Also patient’s peak or sustained plasma level of the drug. “Dose” desired is the increase in overall stability of the component or and “dosage' are used interchangeably herein. components and increase in circulation time in the body. 0103 Generally, daily oral doses of active compounds will Examples of Such moieties include: polyethylene glycol, be from about 0.01 milligrams/kg per day to 1000 milligrams/ copolymers of ethylene glycol and propylene glycol, car kg per day. It is expected that oral doses in the range of 0.5 to boxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl 50 milligrams/kg, in one or several administrations per day, pyrrolidone and polyproline. Abuchowski and Davis, 1981, will yield the desired results. Dosage may be adjusted appro “Soluble Polymer-Enzyme Adducts In: Enzymes as Drugs, priately to achieve desired drug levels, local or systemic, Hocenberg and Roberts, eds., Wiley-Interscience, New York, depending upon the mode of administration. For example, it N.Y., pp. 367-383: Newmark, et al., 1982, J. Appl. Biochem. is expected that intravenous administration would be from an 4:185-189. Other polymers that could be used are poly-1,3- order to several orders of magnitude lower dose per day. In the dioxolane and poly-1,3,6-tioxocane. Preferred for pharma event that the response in a subject is insufficient at Such ceutical usage, as indicated above, are polyethylene glycol doses, even higher doses (or effective higher doses by a dif moieties. ferent, more localized delivery route) may be employed to the 0.108 For the component the location of release may be the extent that patient tolerance permits. Multiple doses per day stomach, the Small intestine (the duodenum, the jejunum, or are contemplated to achieve appropriate systemic levels of the ileum), or the large intestine. One skilled in the art has compounds. available formulations which will not dissolve in the stomach, 0104. The formulations of the invention are administered yet will release the material in the duodenum or elsewhere in in pharmaceutically acceptable solutions, which may rou the intestine. Preferably, the release will avoid the deleterious tinely contain pharmaceutically acceptable concentrations of effects of the stomach environment, either by protection of US 2014/0037547 A1 Feb. 6, 2014

the coMP independent PKG activator or by release of the include but are not limited to; Stearic acid including its mag biologically active material beyond the stomach environ nesium and calcium salts, polytetrafluoroethylene (PTFE), ment, Such as in the intestine. liquid paraffin, vegetable oils and waxes. Soluble lubricants 0109 To ensure full gastric resistance a coating imperme may also be used such as sodium lauryl Sulfate, magnesium able to at least pH 5.0 is essential. Examples of the more lauryl Sulfate, polyethylene glycol of various molecular common inertingredients that are used as enteric coatings are weights, Carbowax 4000 and 6000. cellulose acetate trimellitate (CAT), hydroxypropylmethyl 0117 Glidants that might improve the flow properties of cellulosephthalate (HPMCP), HPMCP50, HPMCP 55, poly the drug during formulation and to aid rearrangement during vinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, compression might be added. The glidants may include cellulose acetate phthalate (CAP), Eudragit L., Eudragit S, starch, talc, pyrogenic silica and hydrated silicoaluminate. and Shellac. These coatings may be used as mixed films. 0118. To aid dissolution of the therapeutic into the aque 0110. A coating or mixture of coatings can also be used on ous environment a surfactant might be added as a wetting tablets, which are not intended for protection against the agent. Surfactants may include anionic detergents such as stomach. This can include Sugar coatings, or coatings which Sodium lauryl Sulfate, dioctyl sodium SulfoSuccinate and dio make the tablet easier to Swallow. Capsules may consist of a ctyl Sodium sulfonate. Cationic detergents might be used and hard shell (such as gelatin) for delivery of dry therapeutic i.e. could include benzalkonium chloride or benzethomium chlo powder; for liquid forms, a softgelatin shell may be used. The ride. The list of potential non-ionic detergents that could be shell material of cachets could be thick starch or other edible included in the formulation as Surfactants are lauromacrogol paper. For pills, lozenges, molded tablets or tablet triturates, 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated moist massing techniques can be used. castor oil 10, 50 and 60, glycerol monostearate, polysorbate 0111. The therapeutic can be included in the formulation 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose as fine multi-particulates in the form of granules or pellets of and carboxymethyl cellulose. These surfactants could be particle size about 1 mm The formulation of the material for present in the formulation of the coMP independent PKG capsule administration could also be as a powder, lightly activator either alone or as a mixture in different ratios. compressed plugs or even as tablets. The therapeutic could be 0119 Pharmaceutical preparations which can be used prepared by compression. orally include push-fit capsules made of gelatin, as well as 0112 Colorants and flavoring agents may all be included. soft, sealed capsules made of gelatin and a plasticizer, Such as For example, the coMP independent PKG activator may be glycerol or Sorbitol. The push-fit capsules can contain the formulated (such as by liposome or microsphere encapsula active ingredients in admixture with filler such as lactose, tion) and then further contained within an edible product, binders such as starches, and/or lubricants such as talc or Such as a refrigerated beverage containing colorants and fla magnesium Stearate and, optionally, stabilizers. In soft cap Voring agents. Sules, the active compounds may be dissolved or Suspended in 0113. One may dilute or increase the volume of the thera Suitable liquids, such as fatty oils, liquid paraffin, or liquid peutic with an inert material. These diluents could include polyethylene glycols. In addition, stabilizers may be added. carbohydrates, especially mannitol, a-lactose, anhydrous lac Microspheres formulated for oral administration may also be tose, cellulose, Sucrose, modified dextrans and starch. Certain used. Such microspheres have been well defined in the art. All inorganic salts may be also be used as fillers including cal formulations for oral administration should be in dosages cium triphosphate, magnesium carbonate and Sodium chlo Suitable for Such administration. ride. Some commercially available diluents are Fast-Flo, I0120 For buccal administration, the compositions may Emdex, STA-RX 1500, Emcompress and Avicell. take the form of tablets or lozenges formulated in conven 0114 Disintegrants may be included in the formulation of tional manner. the therapeutic into a solid dosage form. Materials used as I0121 For administration by inhalation, the compounds for disintegrates include but are not limited to starch, including use according to the present invention may be conveniently the commercial disintegrant based on starch, Explotab. delivered in the form of an aerosol spray presentation from Sodium starch glycolate, Amberlite, sodium carboxymethyl pressurized packs or a nebulizer, with the use of a suitable cellulose, ultramylopectin, Sodium alginate, gelatin, orange propellant, e.g., dichlorodifluoromethane, trichlorofluo peel, acid carboxymethyl cellulose, natural sponge and ben romethane, dichlorotetrafluoroethane, carbon dioxide or tonite may all be used. Another form of the disintegrants are other suitable gas. In the case of a pressurized aerosol the the insoluble cationic exchange resins. Powdered gums may dosage unit may be determined by providing a valve to deliver be used as disintegrants and as binders and these can include a metered amount. Capsules and cartridges of e.g. gelatin for powdered gums such as agar, Karaya or tragacanth. Alginic use in an inhaler or insufflator may be formulated containing acid and its Sodium salt are also useful as disintegrants. a powder mix of the compound and a suitable powder base 0115 Binders may be used to hold the therapeutic agent Such as lactose or starch. together to form a hard tablet and include materials from 0.122 Also contemplated herein is pulmonary delivery of natural products such as acacia, tragacanth, starch and gela the coMP independent PKG activator. The coMP indepen tin. Others include methyl cellulose (MC), ethyl cellulose dent PKG activator is delivered to the lungs of a mammal (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrroli while inhaling and traverses across the lung epithelial lining done (PVP) and hydroxypropylmethyl cellulose (HPMC) to the blood stream. Other reports of inhaled molecules could both be used in alcoholic solutions to granulate the include Adjei et al., 1990, Pharmaceutical Research, 7:565 therapeutic. 569; Adjei et al., 1990, International Journal of Pharmaceu 0116. An anti-frictional agent may be included in the for tics, 63:135-144 (leuprolide acetate); Braquet et al., 1989, mulation of the therapeutic to prevent sticking during the Journal of Cardiovascular Pharmacology, 13(suppl. 5): 143 formulation process. Lubricants may be used as a layer 146 (endothelin-1); Hubbard et al., 1989, Annals of Internal between the therapeutic and the die wall, and these can Medicine, Vol. III, pp. 206-212 (a1-antitrypsin); Smith et al., US 2014/0037547 A1 Feb. 6, 2014

1989, J. Clin. Invest. 84:1145-1146 (a-1-proteinase); Oswein forms as Suspensions, solutions or emulsions in oily or aque et al., 1990, Aerosolization of Proteins’. Proceedings of ous vehicles, and may contain formulatory agents such as Symposium on Respiratory Drug Delivery II, Keystone, Suspending, stabilizing and/or dispersing agents. Colorado, March, (recombinant human growth hormone); 0.130 Pharmaceutical formulations for parenteral admin Debs et al., 1988, J. Immunol. 140:3482-3488 (interferon-g istration include aqueous solutions of the active compounds and tumor necrosis factor alpha) and Platz et al., U.S. Pat. No. in water-soluble form. Additionally, suspensions of to the 5.284,656 (granulocyte colony stimulating factor). A method active compounds may be prepared as appropriate oily injec and composition for pulmonary delivery of drugs for sys tion suspensions. Suitable lipophilic solvents or vehicles temic effect is described in U.S. Pat. No. 5,451,569, issued include fatty oils such as sesame oil, or synthetic fatty acid Sep. 19, 1995 to Wong et al. esters, such as ethyl oleate or triglycerides, or liposomes. 0123 Contemplated for use in the practice of this inven Aqueous injection Suspensions may contain Substances tion are a wide range of mechanical devices designed for which increase the viscosity of the Suspension, such as pulmonary delivery of therapeutic products, including but not sodium carboxymethyl cellulose, sorbitol, or dextran. limited to nebulizers, metered dose inhalers, and powder Optionally, the Suspension may also contain Suitable stabiliz inhalers, all of which are familiar to those skilled in the art. ers or agents which increase the Solubility of the compounds 0.124. Some specific examples of commercially available to allow for the preparation of highly concentrated Solutions. devices suitable for the practice of this invention are the I0131 Alternatively, the active compounds may be in pow Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. der form for constitution with a suitable vehicle, e.g., sterile Louis, Mo.; the Acorn II nebulizer, manufactured by Mar pyrogen-free water, before use. quest Medical Products, Englewood, Colo.; the Ventolin 0.132. The compounds may also be formulated in rectal or metered dose inhaler, manufactured by Glaxo Inc., Research vaginal compositions such as Suppositories or retention Triangle Park, N.C.; and the Spinhaler powder inhaler, manu enemas, e.g., containing conventional Suppository bases Such factured by Fisons Corp., Bedford, Mass. as cocoa butter or other glycerides. 0.125 All such devices require the use of formulations I0133. In addition to the formulations described previously, suitable for the dispensing of c(3MP independent PKG acti the compounds may also be formulated as a depot prepara vator. Typically, each formulation is specific to the type of tion. Such long acting formulations may be formulated with device employed and may involve the use of an appropriate Suitable polymeric or hydrophobic materials (for example as propellant material, in addition to to the usual diluents, adju an emulsion in an acceptable oil) or ion exchange resins, or as vants and/or carriers useful in therapy. Also, the use of lipo sparingly soluble derivatives, for example, as a sparingly Somes, microcapsules or microspheres, inclusion complexes, soluble salt. or other types of carriers is contemplated. 0.134. The pharmaceutical compositions also may com 0126 Nasal delivery of a pharmaceutical composition of prise Suitable Solid or gel phase carriers or excipients. the present invention is also contemplated. Nasal delivery Examples of Such carriers or excipients include but are not allows the passage of a pharmaceutical composition of the limited to calcium carbonate, calcium phosphate, various present invention to the blood stream directly after adminis Sugars, starches, cellulose derivatives, gelatin, and polymers tering the therapeutic product to the nose, without the neces Such as polyethylene glycols. sity for deposition of the product in the lung. Formulations for 0.135 Suitable liquid or solid pharmaceutical preparation nasal delivery include those with dextran or cyclodextran. forms are, for example, aqueous or saline Solutions for inha 0127. For nasal administration, a useful device is a small, lation, microencapsulated, encochleated, coated onto micro hard bottle to which a metered dose sprayer is attached. In one scopic gold particles, contained in liposomes, nebulized, embodiment, the metered dose is delivered by drawing the aerosols, pellets for implantation into the skin, or dried onto a pharmaceutical composition of the present invention Solution sharp object to be scratched into the skin. The pharmaceutical into a chamber of defined volume, which chamber has an compositions also include granules, powders, tablets, coated aperture dimensioned to aerosolize and aerosol formulation tablets, (micro)capsules, suppositories, syrups, emulsions, by forming a spray when a liquid in the chamber is com Suspensions, creams, drops or preparations with protracted pressed. The chamber is compressed to administer the phar release of active compounds, in whose preparation excipients maceutical composition of the present invention. In a specific and additives and/or auxiliaries Such as disintegrants, binders, embodiment, the chamber is a piston arrangement. Such coating agents, Swelling agents, lubricants, flavorings, Sweet devices are commercially available. eners or solubilizers are customarily used as described above. 0128. Alternatively, a plastic squeeze bottle with an aper The pharmaceutical compositions are Suitable for use in a ture or opening dimensioned to aerosolize an aerosol formu variety of drug delivery systems. For a brief review of meth lation by forming a spray when Squeezed is used. The opening ods for drug delivery, see Langer, 1990, Science 249, 1527 is usually found in the top of the bottle, and the top is gener 1533, which is incorporated herein by reference. ally tapered to partially fit in the nasal passages for efficient I0136. The cQMP independent PKG activator and option administration of the aerosol formulation. Preferably, the ally other therapeutics may be administered perse (neat) or in nasal inhaler will provide a metered amount of the aerosol the form of a pharmaceutically acceptable salt. When used in formulation, for administration of a measured dose of the medicine the salts should be pharmaceutically acceptable, but drug. non-pharmaceutically acceptable salts may conveniently be 0129. The compounds, when it is desirable to deliver them used to prepare pharmaceutically acceptable salts thereof. systemically, may be formulated for parenteral administra Such salts include, but are not limited to, those prepared from tion by injection, e.g., by bolus injection or continuous infu the following acids: hydrochloric, hydrobromic, Sulphuric, Sion. Formulations for injection may be presented in unit nitric, phosphoric, maleic, acetic, Salicylic, p-toluene Sul dosage form, e.g., in ampoules or in multi-dose containers, phonic, tartaric, citric, methane Sulphonic, formic, malonic, with an added preservative. The compositions may take Such Succinic, naphthalene-2-Sulphonic, and benzene Sulphonic. US 2014/0037547 A1 Feb. 6, 2014

Also, such salts can be prepared as alkaline metal or alkaline 0.141. The therapeutic agent(s) may be contained in con earth salts, such as Sodium, potassium or calcium salts of the trolled release systems. The term “controlled release' is carboxylic acid group. intended to refer to any drug-containing formulation in which 0.137 Suitable buffering agents include: acetic acid and a the manner and profile of drug release from the formulation salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid are controlled. This refers to immediate as well as non-im and a salt (0.5-2.5% w/v); and phosphoric acid and a salt mediate release formulations, with non-immediate release (0.8-2% w/v). Suitable preservatives include benzalkonium formulations including but not limited to Sustained release chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); and delayed release formulations. The term “sustained parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% release' (also referred to as “extended release') is used in its w/v). conventional sense to refer to a drug formulation that provides 0.138. The pharmaceutical compositions of the invention for gradual release of a drug over an extended period of time, contain an effective amount of a coMP independent PKG and that preferably, although not necessarily, results in Sub activator and optionally therapeutic agents included in a phar stantially constant blood levels of a drug over an extended maceutically-acceptable carrier. The term pharmaceutically time period. The term “delayed release' is used in its conven acceptable carrier means one or more compatible solid or tional sense to refer to a drug formulation in which there is a liquid filler, diluents or encapsulating Substances which are time delay between administration of the formulation and the suitable for administration to a human or other vertebrate release of the drug there from. “Delayed release' may or may animal. The term carrier denotes an organic or inorganic not involve gradual release of drug over an extended period of ingredient, natural or synthetic, with which the active ingre time, and thus may or may not be “sustained release.” dient is combined to facilitate the application. The compo nents of the pharmaceutical compositions also are capable of 0142. Use of a long-term sustained release implant may be being commingled with the compounds of the present inven particularly suitable for treatment of chronic conditions. tion, and with each other, in a manner Such that there is no "Long-term' release, as used herein, means that the implantis interaction which would substantially impair the desired constructed and arranged to deliver therapeutic levels of the pharmaceutical efficiency. active ingredient for at least 7 days, and preferably 30-60 0.139. The therapeutic agent(s), including specifically but days. Long-term Sustained release implants are well-known not limited to the coMP independent PKG activator, may be to those of ordinary skill in the art and include some of the provided in particles. Particles as used herein means nano or release systems described above. microparticles (or in some instances larger) which can consist 0143. The coMP independent PKG activator can be com in whole or in part of the coMP independent PKG activator or bined with other therapeutic agents. The coMP independent the other therapeutic agent(s) as described herein. The par PKG activator and other therapeutic agent may be adminis ticles may contain the therapeutic agent(s) in a core Sur tered simultaneously or sequentially. When the other thera rounded by a coating, including, but not limited to, an enteric peutic agents are administered simultaneously they can be coating. The therapeutic agent(s) also may be dispersed administered in the same or separate formulations, but are throughout the particles. The therapeutic agent(s) also may be administered at the same time. The other therapeutic agents adsorbed into the particles. The particles may be of any order are administered sequentially with one another and with release kinetics, including Zero order release, first order cGMP independent PKG activator, when the administration release, second order release, delayed release, Sustained of the other therapeutic agents and the coMP independent release, immediate release, and any combination thereof, etc. The particle may include, in addition to the therapeutic agent PKG activator is temporally separated. The separation in time (s), any of those materials routinely used in the art of phar between the administration of these compounds may be a macy and medicine, including, but not limited to, erodible, matter of minutes or it may be longer. nonerodible, biodegradable, or nonbiodegradable material or 0144. Thus, the coMP independent PKG activator may combinations thereof. The particles may be microcapsules also be administered in conjunction with an another therapy, which contain the coMP independent PKG activator in a such as a coMP dependent PKG activator. A ccjMP depen Solution or in a semi-solid State. The particles may be of dent PKG activator is a compound that can activate PKG virtually any shape. when coMP is bound to PKG. These compounds include for 0140. Both non-biodegradable and biodegradable poly instance PDE inhibitors and NO-donors. The PDE inhibitor meric materials can be used in the manufacture of particles for may be, for example, sildenafil citrate, vinpocetine, EHNA, delivering the therapeutic agent(s). Such polymers may be anagrelide, enoXimone, milrinone, mesembrine, rolipram, natural or synthetic polymers. The polymer is selected based ibudilast, piclamilast, luteolin, drotaverine, roflumilast, tad on the period of time over which release is desired. Bioadhe alafil. Vardenafil. udenafil, avanafil, or papaverine. The NO sive polymers of particular interest include bioerodible donor may be, for example, isosorbide dinitrite, DiaZenium hydrogels described by Sawhney et. al., 1993, Macromol diolates, NONOates, S-Nitrosothiols, NO hybrid drugs, or ecules 26, 581-587, the teachings of which are incorporated Zeolites. 4. The compounds of the invention may also be herein. These include polyhyaluronic acids, casein, gelatin, administered with cGMP to the subject. glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, 0145 The present invention is further illustrated by the poly(methyl methacrylates), poly(ethyl methacrylates), poly following Examples, which in no way should be construed as (butylmethacrylate), poly(isobutyl methacrylate), poly(hexy further limiting. The entire contents of all of the references lmethacrylate), poly(isodecyl methacrylate), poly(lauryl (including literature references, issued patents, published methacrylate), poly(phenyl methacrylate), poly(methyl acry patent applications, and co-pending patent applications) cited late), poly(isopropyl acrylate), poly(isobutyl acrylate), and throughout this application are hereby expressly incorporated poly(octadecyl acrylate). by reference. US 2014/0037547 A1 Feb. 6, 2014

EXAMPLES 1.6MGuPHCl, 0.8% formic acid. In addition, nondeuterated samples were prepared by incubating the protein in buffered 0146 Materials and Methods 0147 Bacterial Protein Expression and Purification: DNA HO. Fully deuterated samples were prepared by incubating for bovine PHG Io, encoding amino acids Q7-K was the protein in D20 buffer containing 1% formic acid overnight amplified by PCR using the primers 50-CGGGATCCATG at room temperature. The samples were frozen on dry ice and CAGGCATTCCGGAAGTTC-30 (sense, SEQID NO.1) and stored at -80° C. until analysis by mass spectrometry. 50-GGAATTCCTACTACTTCAGGTTGGCGAAG-30 (an Samples were manually thawed on wet ice and immediately tisense, SEQID NO. 2). The PCR product was digested with analyzed by LC-MS. Procedures for pepsin digestion for BamHI and EcoRI and ligated into pRSETA (Invitrogen) in DXMS have been described previously (Burns-Hamuro et al., frame with the N-terminal 6x-His tag. The plasmid was trans 2005; Hamuro et al., 2004: Pantazatos et al., 2004; Spraggon formed into E. coli BL21 and 5 ml starter cultures in LB were et al., 2004). Briefly, the samples were passed through an allowed to grow to OD600–0.6 at 37 C, 300 RPM in the immobilized pepsin column and the protease-generated pep presence of 50 mg/ml Ampicillin. One liter of Overnight tides were collected on a C18HPLC column The peptides Express (Novagen) media was inoculated 1:1000 in 4 liter were eluted from the C18 column and the effluent was baffled flasks with the mid-log starter prep and grown at 25° directed to a Thermo Finnigan LCQ electrospray ion trap type C., 300 RPM for 16-24 hr. Bacteria were harvested by cen mass spectrometer with data acquisition in either MS1 profile trifugation at 5K RPM for 10 min at 4°C., and pellets were mode or data dependent MS2 mode. The pepsin-generated stored at -80°C. Pellets were resuspended in 5 ml/gram of 50 peptides from the MS/MS data sets were identified using mM MES (pH 6.8), 100 mM NaCl, 5 mM MgCl2, 3 mM SEQUEST (Thermo Finnigan Inc.), followed by analysis TCEP, 5% glycerol (Buffer A) plus protease inhibitors. Cells using customized DXMS data reduction software (Siena were lysed at 1200 psi using a French pressure cell. Homo Analytics Inc., Modesto, Calif.). Corrections for back genates were clarified via centrifugation at 15K RPM for 90 exchange were made through measurement of loss of deute min at 4°C. and recombinant PKGIC was purified from rium from fully deuterated samples. Deuterium incorporation the Supernatants on a Profinia protein purification system for each peptide was calculated using the methods of Zhang (Biorad) using the native IMAC protocol. Eluted protein was and Smith (1993): subjected to three rounds of dialysis in 2 liters of buffer A at 4° C. A secondary purification step was performed on an AKTAprime FPLC system by passing Ni2+-purified protein deuteration level(%) = over a HiLoad 16/60 Superdex 75 (GE Healthcare) gel filtra ar) or Nix100 tion column and collecting 1 ml fractions in Buffer A. Protein homogeneity was assessed via SDS-PAGE and Coomassie where m(P), mCN), and mCF) are the centroid value of the staining. Desired fractions were pooled and concentrated partially deuterated, nondeuterated, and fully deuterated pep using 10K MWCO centrifugal concentrators (Sartorius). tide, respectively. The experiments were performed twice, Typical yields were 100 mg purified protein per liter of media. and the reported results are the average of the two experi 0148 Crystallization, Data Collection, and Model Refine mentS. ment: Sparse matrix kits from Hampton Research were used 0150 Cyclic NucleotideAnalysis: To confirm the copuri to screen initial crystal growth conditions. Crystals used for fication of cAMP purified PKG7 was incubated on ice in data collection were grown via hanging drop vapor diffusion a 1:1 ratio of acetonitrile. The extract was clarified via cen in 2.2 M (NH)SO 100 mM Tris (pH 8.0), 0.2% MPD at a trifugation at 4° C. and the Supernatant was Subjected to protein concentration of 25-35 mg/ml at 20°C. Crystals were UV-spectroscopy. The extract had a single peak with almax cryoprotected for 10-30 minin 2 MLiSO 100 mM Tris (pH of 258 nm and lacked the characteristic cQMP shoulder. The 8.0), 0.2% MPD and flash frozen in liquid N2. 2.5A diffrac same extract was subjected to HPLC analysis using a Merck tion data were collected at beamline 8.2.1 at the Advanced Hitachi HPLC system comprising a L-2130 pump, a L-2400 Light Source, Lawrence Berkeley National Laboratory. UV detector, a L-2350 column oven, and a L-2200 autosam HKL2000 was used for data processing and scaling (Minor, pler, and data were processed with EZChrom Elite evaluation 1997). Phases were generated with the program Phaser (Mc software (3.2.1). Isocratic runs were performed at 30°C. on a Coy et al., 2007) using the PKA regulatory domain (PDB ID RP-18 reversed phase silica column (250 3 4 mm, ODS 1RGS) as a search model. Crystals grew in the C2 space group A.YMC) with 2.5% isopropanol and 25 mM triethylammo with two molecules per asymmetric unit. The PKG Io." nium formate buffer (pH 6.9) with a flow of 1 ml/min at 255 model was manually built into electron density using the programs TURBO-FRODO (Roussel and Cambillau, 1991) 0151. Mammalian Protein Expression: WT PHG Io. was and Coot (Emsley et al., 2010). CNS (Brunger et al., 1998) cloned into pcDNA 3.1 using BamHI and EcoRI. Mutations was used for structure refinement. Publication-quality images were made using QuickChange site-directed mutagenesis were generated using Pymol (The PyMOL Molecular Graph (Agilent Technologies, Santa Clara, Calif.) per manufactur ics System, Version 1.2r3pre, Schrodinger, LLC). Interface er's recommendations. HEK293 cells grown in 10 cm dishes and assembly measurements of symmetry mates were calcu were transfected for 5 hr using Metafectene (Biontex, San lated using the protein interfaces, Surfaces, and assemblies Diego, Calif.) at a ratio of 20 mg DNA per 60 ml lipid. Sixty service PISA at European Bioinformatics Institute (Krissinel hours after transfection cells were harvested by scraping into and Henrick, 2007). PBS, 2 mM benzamidine-HCl, 200 mM EDTA. Protein con 0149 Deuterium on Exchange: All deuterium exchange centration was determined by the Bradford method and reactions were performed on ice, in a 4° C. cold room. stocks were stored in 50% glycerol at -20°C. Exchange reactions were initiated by adding 6 ml buffered 0152 Kinetic Analysis: Determination of activation con DO to 2 ml purified PKGIC” at 7 mg/ml. At the appro stants was performed using a Y--P1-ATP transfer assay as priate time points exchange was quenched by adding 12 ml previously reported (Ruth et al., 1991; Tegge et al., 1995). US 2014/0037547 A1 Feb. 6, 2014

Briefly, 0.5 mg of wild-type or mutant protein from PKG ture of PKG 7. FIG. 12 shows a DXMS for PKG 7. The transfected HEK293 cells was incubated in buffer with vari secondary structural features from the crystal structure of ous concentration of c(3MP in the presence of Ig-32P1-ATP PKG IC” are positioned above sequence alignment for and the PKG-specific peptide substrate TOAKRKKSLAMA PKGIC, If and II. Cys117 and to Cys195 form a disulfide (SEQID NO3) (Dostmann et al., 1999). Aliquots, spotted on bond in the A-domain. Residues that comprise the hydropho P81 Whatman paper were subjected to scintillation counting. bic nest in the B domain are highlighted and switch helix knob All experiments were performed in the presence of the PKA residues are highlighted within the box. DXMS results are specific inhibitor, PKI' (70 nM) to suppress endogenous presented under the sequence alignment. Percent deuteration PKA activity. Mock-transfected cells did not show any activ was measured at four time points (3 s. 30s, 300 s. 3000 s). 1ty. Percent deuteration as a function of time for D’-E7 (O), D7-E7 () and F-K (A) is shown at the left with a Example 1 summary of the initial rate of deuterium exchange from 3-30 seconds at the right. Overall Fold of PHG Io. 7 (O155 Both cGMP-binding sites exhibit classic features of 0153. Crystals grew in the C2 space group with two mol the conserved CNB fold (FIG. 6A). A rigid eight-stranded ecules per asymmetric unit. Superimposition of Ca atoms B-barrel sandwiches the phosphate binding cassette (PBC) from the two molecules sites gave an average root mean between 3 strands 6 and 7 which serves as a docking site for square deviation (rmsd) of 0.882. Initial phases were attained cNTs. The B-barrel itself is flanked at the N terminus by the by molecular replacement using the CNB-A domain of PKA CN helix, the 310 loop, and the CA helix (collectively termed RIa (PDB ID 1 RGS) as a search model. The structure was the N3A motif) and C-terminally by the OB/C.C helix. Sur refined to 2.5A with working and free-R factor values of 0.22 prisingly, the two CNB domains presented in this structure and 0.28, respectively as shown in Table 1. Each protein have been captured in different conformational states as is molecule in the asymmetric unit contained 278 amino acids, evident when comparing helical elements between A- and 1 cAMP molecule, and 2 phosphates. B-domains (FIG. 6B). Superimposition of the two c(3MP binding yields an rmsd of 1.124. While the B-barrels super TABLE 1. impose well, there is variance in the helical subdomains. FIG. 6 shows the topology of the coMP-Binding Domains. The Data Collection and Refinement Statistics overall fold of the tandem coiMP binding domains is shown as a cartoon representation with secondary structural elements PKG I,78-355 labeled (switch helix not shown). Data collection 0156 The two coMP-binding sites are separated by an Space group C2 elongated B/C helix at the end of the A-domain (FIG. 6A). Cell dimensions (A) This creates a dumbbell-like topology between the two CNB domains and resembles the conformation adopted by regula A. 18O2 tory subunits of PKA when bound to the catalytic subunits in B 66.O C 81.6 the holoenzyme conformation (Kim et al., 2007: Wu et al., B() 113.8 2007). A kink in the Chelix in the middle of this cleft is a No. of molecule per asymmetrical unit 2 likely point at which the two CNB domains undergo c(3MP Resolution (A) 2.50 mediated structural rearrangements. Residues in this kink are Rmerge 0.085 (0.52) Completeness (%) 97.9 (93.5) clearly solvent exposed (FIG. 5B; FIG. 12A) and proteolysis |sigma 18.7 (1.9) at Arg' readily occurs in the absence of coMP (Chu et al., No. reflections 32.166 1997: Scholten et al., 2007). Incubation with cGMP induces Refinement conformational changes that prevent Such proteolysis. Fur Resolution (A) SO.O-2SO thermore, c0MP binding increases solvent protection in this Rai? Ree (%) 22.1.27.7 region (Alverdi et al., 2008) further suggesting that the A-do No. of protein residues 556 main Chelix may be the center of to c(GMP induced confor No. of ligandsions 4 mational changes in the regulatory domain. No. of water molecules 2O7 Rmsd (O157. The extended conformation of PKG7 does not appear to allow direct communication between the A- and Bond lengths (A) O.OO8 B-domains. This suggests that the structural determinants Bond angles () 1.4 mediating cooperative binding of c(GMP are not contributed Ramachandran angles (%) by the core of the regulatory domain. In support of this find Most favored 83.3 ing, previous studies have demonstrated a loss of cooperativ Disallowed None ity upon deletion of regions outside the coMP-binding domains Values in parentheses are for highest-resolution shell. 0158 (Dostmannet al., 1996; Heilet al., 1987). Moreover, 0154) The overall fold presents both cGMP-binding in PKA holoenzyme structures of both type Ia (Kim et al., domains of the PHG IC. regulatory domain. c6MP-binding 2007) and IIa (Wu et al., 2007) the catalytic subunit docks to site A (residues 87-210) begins following a loop in the “hinge the cleft created by the extended topology of the CNB region.” for which we see clear backbone density, followed by domains in the regulatory subunits. Residue Gln18, just cGMP-binding site B (residues 211-327) and a new helical C-terminal to the AI domain in PKG, is positioned in the subdomain we have termed switch helix (SW, residues 328 center of the two coMP-binding sites (FIG. 5B), which is 355) (FIG. 5B; FIG. 12A, secondary structural elements). where the N terminus of PKG interacts with the catalytic core FIG. 5 shows the domain organization and general architec of the enzyme via the AI domain (Heil et al., 1987; Hofmann US 2014/0037547 A1 Feb. 6, 2014

et al., 2009). While the precise docking mechanism may indication that the chT occupying the A-domain was Syn differ in the PKG holoenzyme, the catalytic domain is likely oriented cAMP. HPLC analysis and UV-spectroscopy con to sit in this cleft and participate in crosstalk between the two firmed the presence of cAMP and the absence of c(GMP (see cGMP-binding sites similar to the architectural arrangements methods). observed in PKA. 0162 The primary interactions made with the cyclic nucleotide come from residues buried within the PBC. Spe Example 2 cific contacts are made by Glu'7, Arg'7, and Thr'77 (FIG. 7C). Glu'7 forms a hydrogen bond with the 2'OH of the The A-Domain Is cAMP Bound ribose and Arg''' docks the equatorial oxygen of the phos 0159. The docking of cNTs to a CNB domain is made phate. Thr'77 hydrogen bonds with the apical oxygen of the possible by the conserved and mobile motif of the PBC (FIG. phosphate group with both side-chain hydroxyland backbone 7A) (Diller et al., 2001; McKay et al., 1982: Su et al., 1995). amino groups. Modeling of syn-oriented coMP into the This short, 14 residue helix-turn contains residues that coor A-domain maintains these same contacts and shows that the dinate the ribose-phosphate moiety and allows for the pref 2-amino group is primed to interact with the Thr'77 side-chain erential binding of cAMP versus coMP. Invariantly, all CNB hydroxyl. The Glu and Arg contacts are similar to those used domains from cnT-regulated protein kinases have a Glu in the by the cAMP-PKA interaction and the previously predicted 3 position and an Arg in the 12 position, which hydrogen interactions with Thr'77 are confirmed (Shabb et al., 1991; bonds with the 2'OH and equatorial oxygen, respectively. Weber et al., 1989). A schematic summary of these specific Unique to PKG is a Thr or Ser in the 13 position of the PBC, interactions with cGMP modeled into the A-domain is pre which has been predicted to provide specificity for cGMP by sented in FIG. 7E. providing a hydrogenbond potential with both the 2-NH2 and (0163 As has been described for other CNB domainstruc apical oxygen of c(GMP (Shabb and Corbin, 1992; Weber et tures, cnT docking to the PBC is stabilized by a capping al., 1989). PKA contains an Ala in the 13-position and muta mechanism that sandwiches the nucleotide base between tion of this residue to a Thr produces a PKA mutant that no hydrophobic surface on the b-barrel, and a hydrophobic cap longer discriminates between cAMP and coMP (Shabb et al., that moves into place upon cNT-induced structural rearrange 1990). ments of the protein (Berman et al., 2005; Das et al., 2007). In (0160 FIG. 7 shows the features of the A-Domain. For the PKG 7 structure, the backside of the adenine ring is instance the sequence alignment for PBCs from PKA and flanked by an array of hydrophobic contacts with no obvious PHGIC isoforms is shown in 7A. Threonine residues in the 13 hydrogen bond donor or acceptor potentials (FIG. 7D). The position of PKG PBCs are highlighted by a box. A composite identity of the specific capping residue is not disclosed, how omit map generated for the cMT binding sites of both A-do ever, because the two CNB domains are in an extended, mains contoured to 1.5 s. syn-cAMP is modeled into the inactive conformation and capping occurs only after ligand positive density as shown in FIG. 7B. Specific interactions mediated conformational changes cause the two CNB between the sugar-phosphate moiety of cAMP and residues domains to form a more compact structure. from the PBC are shown in FIG.7C. The 2'-hydroxyl hydro 0164. A second shell of regulation is provided by the gen bonds with Glu'', the equatorial oxygen of the phos B2-33 loop. In PKA this loop stabilizes the PBC arginine and phate group interacts with Arg'', and the apical oxygen provides allosteric communication of binding events in the contacts both the side-chain hydroxyl and backbone amino PBC to the B helix. In the A-domain of PKGIC, Arg'7° is groups of Thr'77. The backbone carbonyl of Leu' bridges coordinated by a number of conserved residues from the communication from the guanidinium group of Arg176 B2-33 loop (FIG. 7C). A hydrophobic interaction is made through the B3-33 loop to Arg' in the B helix. A hydropho with Ile', and the backbone carbonyl and amide of Arg'' bic face accommodates the solvent-exposed nucleobase, as forms hydrogen bonds with the Gly' amide and Asp'' shown in 7D. A schematic of cNT binding to the A-domain of carbonyl, respectively. Furthermore, the guanidinium group PKGIC with cGMP modeled in place of the observed of Arg'' interacts with the carbonyl of Leu'. Communica cAMP is shown in 7E. Specific hydrogen bond contacts are tion between the B2-33 loop and the B helix occurs via shown as dotted lines. Interactions coming from backbone Arg'. The backbone amide of Arg' is bridged by the amide (D) and carbonyl (o) are highlighted, whereas side carbonyl of Ser'7. This is where PKG differs from PKA. The chain interactions are shown from the center of the circled equivalent to Ser'7 is Asp'' in the PKA RIaA-domain residue. Hydrophobic interactions with the cnT are shown as which hydrogen bonds not only with the backbone amide of boxed residues. the Argin the Bhelix, but also coordinates the guanidinium of (0161) To assess the occupancy of PBCs in the PKG 7 the PBC Arg. In doing so, a direct link from the PBC to the B structure, initial phases from the molecular replacement Solu helix hinge is made via the B2-?33 loop. In contrast, the PKG tion were used to generate a simulated annealing composite A-domain does not utilize this same direct allosteric mecha omit map. In order to minimize model bias, side chains for the nism. This may be a critical divergence in how these two invariant Glu, Arg, and Thr residues were omitted in each cNT-regulated protein kinases communicate binding events PBC. A strong positive peak was observed in the A-domain in their PBCs with the rest of the molecule. which resembled a cyclic-30.50-nucleotide monophosphate containing a purine moiety in the syn configuration (FIG. Example 3 7B), while the B-domain appeared cnT-free. While electron density at the 2'OH of the ribose and the 6 position of the A Cys'7-Cys' Disulfide Bond Locks A and B purine was evident, we could not attribute the 6 position Helices in the A-Domain density to being either a keto?enol or amino group. However, (0165. The presence of disulfide bonds in PHG IC. have there was no density extending from the 2 position where the been reported and an oxidation-induced mechanism of acti 2-amino group of c(3MP should reside. This was a clear Vation has been proposed as complementary mechanism to US 2014/0037547 A1 Feb. 6, 2014

cyclic nucleotide mediated regulation of kinase activity (Bur induced conformational changes bring the two CNB domains goyne et al., 2007; Landgrafet al., 1991). PHGIC contains 11 closer together, which is then stabilized by the association of cysteine residues (Takio et al., 1984), 5 of which have been a hydrophobic capping residue with the nucleotide base. Suggested to contribute to oxidation-induced activation. Although there is sequence and spatial variability as to the Cys', just C-terminal of the D/D domain forms an intermo identity of the cap that secures the nucleotide base to the lecular disulfidebond with Cys' from the opposing protomer B-barrel, this allosteric mechanism is present in all CNB in the holoenzyme assembly (Burgoyne et al., 2007). It has domain structures described to date. been suggested that H2O2-induced oxidation of PHGIC pro motes kinase activation via a bridging of these two cysteines. TABLE 2 Additionally, exposure of PHG IC. to divalent cations with positive redox potentials promotes enzyme activation via dis Summary of the Different Positions for Each Helical Component ulfide bond formation between Cys'7-Cys' and/or Cys'- of CNB Domains from PKA RIC. and the PKG7 Structure Cys' (Landgraf et al., 1991). It was unclear, however, CNB Domain State PBC N3A Motif B.C Helix whether Cys'7-Cys' or Cys'°-Cys' was exclusively PKARIC: Ca apo OPEN IN OUT responsible for the observed oxidation-induced activation. PKARIC: cAMP cNT bound CLOSED OUT IN Despite the observations that cysteine oxidation can lead to PKG-A Transition CLOSED IN OUT cGMP-independent activation of PKG IC, no molecular PKG-B Transition OPEN OUT OUT mechanism of activation has been proposed. (0166 The PKG 7 structure reveals a disulfide bridge between Cys'7 in the A helix and Cys' at the start of the B 0169. The two CNB domains presented here are in a helix (FIG. 8A). Residues immediately C-terminal of these mixed, hybrid configuration, with helical elements in both two cysteines are solvent-exposed (FIG. 12A), while a hydro apo and cnT bound orientations (Table 2). As only the high phobic sheath (Phe, Ile''', Ile', and Ile') surrounds the affinity A-domain is cnTbound, the A-domain PBC is closed, disulfide bridge and provides solvent protection (FIG.8B). In relative to that of the B-domain (FIG. 6B). However, the support of the oxidation-induced activation of PKG IC. pre Subsequent conformational changes inherent to CNB viously observed (Landgraf et al., 1991), the recruitment of domains are not observed. An overlay of the CNB domains Phe' to the hydrophobic sheath provides a hypothesis as to from PKG and PKA indicate that the overall fold is con how the enzyme might be activated in the absence of c(GMP. served, but positional differences in the helical subdomain The capping of Cys'7-Cys.195 by Phe serves to order the evince the hybrid nature of this PKG structure (FIGS. 9A and loop that precedes the N helix of the A-domain (FIG. 8A). As 9B). The B/C helix of the A-domain is clearly extended and the AI domain residesjust adjacent to Phe, autoinhibition of the N3A motif is In, representative of a CNB domain to in the the kinase may be relieved through a movement of this loop to apo, unliganded State rather than a chT-bound conformation the hydrophobic sheath. The reorganization of this region (Komev et al., 2008: FIG. 9A). Despite the A-domain PBC upon oxidation of Cys'-Cys' may disrupt the interaction being occupied by cAMP, allosteric communication of this between the AI and catalytic center thereby releasing the binding event with the rest of the domain is severed. Interest kinase from a state of autoinhibition. Furthermore, the Sulf ingly, the Cys'-Cys' disulfide bond provides a major hydryl group of Cys' sits on 38 of the B-domain and points structural determinant for this stable transition state. The inward toward the center of the B-barrel (not shown). It seems covalent bridging of the A helix to the B helix prevents the unlikely that Cys' is capable of forming a disulfide bridge B/C helix from closing upon cNT binding to the PBC. In turn, with Cys' from the activation loop in the catalytic domain. the N3A motif cannot move out from the b-barrel (FIG.9A, Our structure supports the Cys'-Cys' disulfide bond as arrow). This disulfide bond therefore uncouples communica being involved in the metal-induced activation of PKGIC. tion of allosteric events in the A-domain from being transmit (0167 FIG. 8 shows some details of the Cys'7-Cys' ted to the B-domain. Disulfide Bond. The disulfide bridge in the A-domain (0170 The B-domain of this PKG structure is similarly between Cys'7 and Cys' covalently links the A helix to the locked in a hybrid conformation. This CNB domain is cnT B helix. 2F-F electron density contoured to 1.6 s is shown free, thus the unbound PBC is Open and maintains an for the loop preceding the N helix and the disulfide bond. extended B/C helix (FIG. 9B). However, the N3A motif is also Out, an orientation reminiscent of a cMT-bound state Example 4 (Kornev et al., 2008). The B-domain N3A motif is stabilized in the Out position by a set of hydrophobic residues originat Mixed Configuration of the Two CNB Domains ing from the C-terminal end of the SW in the other molecule in the asymmetric unit. The two symmetry mates are related 0168 The cnT-dependent structural dynamics of CNB by noncrystallographic symmetry and this interaction pro domains are well established (Berman et al., 2005; Komev et motes a previously uncharacterized docking interface al., 2008; Rehmann et al., 2007). While the B-barrel does not between PHG IO protomers, which is described in detail appear to undergo significant conformational changes upon below. cNT binding, the C-helical subdomain moves. In and Out relative to the barrel (Table 2). Ligand association with the Example 5 PBC initiates these structural changes as several residues make specific interactions with the nucleotide and close the PKG 7Protomers Interact Via Their Switch PBC. This structural change is communicated through hydro Helices phobic residues to the N3A motif and B/C helix. As a result, an extended B/C helix forms a hinge and closes inward (0171 Remarkably, the PKG7 crystal structure forms a toward the B-barrel. This rearrangement is accompanied by symmetry related dimer through the formation of an interface an outward shift of the N3A motif. In PKA, these cNT between the SW and the opposing B-domain (FIGS. 10A and US 2014/0037547 A1 Feb. 6, 2014

10B). It is well established that PKG assembles into parallel changes in KA, combined with the observed decrease in Hill homodimers, an assembly mediated by leucine Zipper motifs coefficient, illustrates the importance of the knob-nest inter at the immediate N termini. The structure presented here face in maintaining the kinetic fidelity of PKG IC. In the provides the first evidence for intermolecular communication context of full-length PKGIC, the SW seems to act as a tether at sites distal to the classical N-terminal D/D domain. The SW for the catalytic domain, disruption of which causes the extends from the B-domain and residues at the C terminus of kinase to be more easily activated. each SW interact with an open hydrophobic network in the B-domain of the opposing protomer (FIGS. 11A and 11B). TABLE 3 Hydrophobic “knobs' at the end of the SW (residues 350 354) stabilize the open N3A motif in the B-domain of the Summary of Kinetic Analysis from FIG. 7C neighboring protomer. Side chains from Phe, Phe, and Leu fill the void created by an extensive hydrophobic Mutation K (nM) IlH Fold Activation “nest' (FIGS. 11A and 11B). The eight residues that comprise WT (n = 12) 67 2 17 O.O9 11.0 F350A/F351A/L354A (n = 4) 7OS 1.6 0.06 12.3 the nest (Phe, Leu??", Leu?, Tro??, Gln, Phe, NA (n = 9) 571 17 O.O6 5.9 Ile324, Leu) are noncontiguous and recruited from the F350A/F35A/L35-A (n = 10) 15.3 - 17 O.80.09 3.1 entire B-domain (FIG. 12A). This knob-nest interface is fur Activation constants (K) and Hill coefficients (nt) are presented for WTPKG Ioand SW ther strengthened by ASn, the only polar residue at the end knob mutants. A statistically significant difference inK was noted for both the NA and of the SW, which forms a hydrogen bond with the backbone FAFANALA mutants (p<0.001) compared with WT PKG Io. carbonyl of Thr' in the 310 loop of the B-domain (FIGS. 11A and 11B). Formation of this dimeric assembly protects 2740 A 2 of surface area, and has a free energy (DG) of 15.8 References kcal/mol required for dissociation, an indication that this 0.174 Aitken, A., Hemmings, B. A., and Hofmann, F. arrangement is thermodynamically stable in Solution. Native (1984). Identification of the residues on cyclic GMP-depen PAGE analysis of PKG7 in solution displays a small dent protein kinase that are autophosphorylated in the pres population of dimeric protein (FIG. 13A). Similarly, the ence of cyclic AMP and cyclic GMP Biochim Biophys. Acta migration of crystalline PKG' is consistent with a dimer 790, 219-225. (FIG. 13B). (0175 Alverdi, V., Mazon, H., Versluis, C., Hemrika, W., 0172) Our initial DxMS studies on PKG7 offered fur Esposito, G., van den Heuvel, R., Scholten, A., and Heck, A. ther validation of the significance of this knob-nest interface. J. (2008). c6MP-binding prepares PKG for substrate binding Analysis of peptide fragments from the SW indicated that the by disclosing the C-terminal domain.J. Mol. Biol. 375, 1380 C-terminal residues containing the hydrophobic knobs and 1393. Asn had a slower rate of deuterium exchange compared (0176 Berman, H. M., Ten Eyck, L. F., Goodsell, D. S., with residues in the more solvent-exposed region of this helix. Haste, N.M., Kornev, A., and Taylor, S. S. (2005). The cAMP This finding suggested that the very C terminus of the SW is binding domain: an ancient signaling module. Proc. Natl. protected in Solution and that the knob-nest interaction may Acad. Sci. USA 102, 45-50. serve as the focal point of interchain communication between 0177 Bian, K. and F. Murad, Nitric oxide signaling in PKG protomers. Interestingly, the knob residues appear to be vascular biology. Journal of the American Society of Hyper unique to PKGI isoforms, as PKG II has a large amino acid tension: JASH, 2007. 1(1): p. 17-29. Boettcher, A.J., Wu, J., insertion at the site of the SW (see sequence alignment, FIG. Kim, C., Yang, J., Bruystens, J., Cheung, N., Pennypacker, J. 12A). However, hydrophobic residues at the equivalent nest K., Blumenthal, D.A., Kornev, A.P., and Taylor, S.S. (2011). positions are conserved in both PKGI and II isoforms. Realizing the allosteric potential of the tetrameric protein kinase ARIa holoenzyme. Structure 19, 265-276. Example 6 0.178 Brunger, A.T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P. Grosse-Kunstleve, R. W., Jiang, J. S. Kusze Mutational Disruption of the Knob-Nest Interface in wski, J., Nilges, M., Pannu, N. S., et al. (1998). Crystallog Full-Length PHG IC. Decreases the Activation raphy & NMR system: A new software suite for macromo Constant and Suggests a Tethering Mechanism for lecular structure determination. Acta Crystallogr. D Biol. the Catalytic Domain Crystallogr. 54,905-921. 0173 The functional relevance of the knob-nest interface (0179 Bryan, N.S., K. Bian, and F. Murad, Discovery of the was probed by Alanine Scanning mutagenesis of the SW knob nitric Oxide signaling pathway and targets for drug develop residues in full-length PHG IC. containing both the N-termi ment. Frontiers in bioscience: a journal and virtual library, nal D/D and the catalytic domains. Extracts from HEK293 2009. 14: p. 1-18. cells expressing wild-type and mutant PHG IC. were exam 0180 Burgoyne, J. R., Madhani, M., Cuello, F. Charles, ined for phosphoryl transfer activity. Removal of the specific R. L., Brennan, J. P. Schroder, E., Browning, D. D., and hydrogen bond provided by Asn (NA) resulted in a Eaton, P. (2007). Cysteine redox sensor in PKGIa enables significantly decreased activation constant (FIG. 11C and oxidant-induced activation. Science 317, 1393-1397. Table 3). The entire knob nest interface was disrupted by a 0181 Burns-Hamuro, L. L., Hamuro, Y. Kim, J. S., quadruple mutation wherein all hydrophobic knob residues in Sigala, P., Fayos, R., Stranz, D.D., Jennings, P. A., Taylor, S. addition to the specific Asn were substituted for alanine S., and Woods, V. L., Jr. (2005). Distinct interaction modes of (FA, FA, NA, LA). Neutralization of these inter an AKAP bound to two regulatory subunit isoforms of protein actions further reduced the activation constant greater than kinase A revealed by amide hydrogen/deuterium exchange. 4-fold (FIG. 11C and Table 3). Additionally, this mutant Protein Sci. 14, 2982-2992. displayed a loss of cooperativity (nH). While we were unable 0182. Butt, E., D. Pohler, H. G. Genieser, J. P. Huggins, to calculate absolute basal and Vmax values, the relative to and B. Bucher, Inhibition of cyclic GMP-dependent pro US 2014/0037547 A1 Feb. 6, 2014

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Role of the (New York: Academic Press). receptor in the mechanism of action of adenosine 30:50 0212 Ogreid, D. W. Dostmann, H. G. Genieser, P. cyclic monophosphate. Proc. Natl. Acad. Sci. USA 68, 786 Niemann, S. O. Doskeland, and B. Jastorff, (Rip)- and (Sp)- 790. 8-piperidino-adenosine 3',5'-(cyclic)thiophosphates dis (0195 Gill, G. N., Walton, G. M., and Sperry, P.J. (1977). criminate completely between site A and B of the regulatory Guanosine 30:50-monophosphate-dependent protein kinase subunits of cAMP-dependent protein kinase type I and II. from bovine lung. Subunit structure and characterization of European journal of biochemistry/FEBS, 1994. 221 (3): p. the purified enzyme. J. Biol. Chem. 252, 6443-6449. 1089-94. 0196. Hamuro, Y., Anand, G. S., Kim, J. S., Juliano, C., 0213 Osborne, B. W., et al., Crystal structure of c(GMP Stranz, D. D., Taylor, S. S., and to Woods, V. L., Jr. (2004). dependent protein kinase reveals novel site of interchain com Mapping intersubunit interactions of the regulatory Subunit munication. Structure, 2011, 19(9): p. 1317-27. (Rlalpha) in the type I holoenzyme of protein kinase A by 0214 Pantazatos, D., Kim, J. S., Klock, H. E., Stevens, R. amide hydrogen/deuterium exchange mass spectrometry C., Wilson, I. A., Lesley, S.A., and Woods, V. L., Jr. (2004). (DXMS). J. Mol. Biol. 340, 1185-1196. Rapid refinement of crystallographic protein construct defi (0197) Heil, W. G., Landgraf, W., and Hofmann, F. (1987). nition employing enhanced hydrogen/deuterium exchange A catalytically active fragment of c(GMP-dependent protein MS. Proc. Natl. Acad. Sci. USA 101,751–756. US 2014/0037547 A1 Feb. 6, 2014

0215 Pfeifer, A., Ruth, P., Dostmann, W., Sausbier, M., 0226 Su,Y., Dostmann, W. R., Herberg, F. W. Durick, K., Klatt, P., and Hofmann, F. (1999). Structure and function of Xuong, N.H., Ten Eyck, L., Taylor, S. S., and Varughese, K. cGMP-dependent protein kinases. Rev. Physiol. Biochem. I. (1995). Regulatory subunit of protein kinase A: structure of Pharmacol. 135, 105-149. deletion mutant with cAMP binding domains. Science 269, 0216 Rehmann, H. Wittinghofer, A., and Bos, J. L. 807-813. (2007). Capturing cyclic nucleotides in action: Snapshots 0227 Takio, K., Wade, R. D., Smith, S. B., Krebs, E. G., Walsh, K. A., and Titani, K. (1984). Guanosine cyclic 30.50 from crystallographic studies. Nat. Rev. Mol. Cell Biol. 8, phosphate dependent protein kinase, a chimeric protein 63-73. homologous with two separate protein families Biochemistry 0217 Roger, V. L., et al., Heart disease and stroke statis 23, 4207-4218. tics 2011 update: a report from the American Heart Asso 0228 Tegge, W., Frank, R., Hofmann, F., and Dostmann, ciation. Circulation, 2011. 123(4): p. e18-e209. W. R. (1995). Determination of cyclic nucleotide-dependent 0218. Roussel, A., and Cambillau, C. (1991). Silicon protein kinase Substrate specificity by the use of peptide Graphics, Inc (CA: Mountain View). libraries on cellulose paper. Biochemistry 34, 10569-10577. 0219 Sandner, P. D. Neuser, and E. Bischoff, Erectile 0229 Walter, U. and S. Gambaryan, c0MP and coMP dysfunction and lower urinary tract. Handbook of experi dependent protein kinase in platelets and blood cells. Hand mental pharmacology, 2009(191): p. 507-31. book of experimental pharmacology, 2009(191): p. 533-48. 0220 Schlossmann, J. and F. Hofmann, c0MP-dependent 0230 Weber, I.T., Shabb, J. B., and Corbin, J. D. (1989). protein kinases in drug discovery. Drug discovery today, Predicted structures of the coMP binding domains of the 2005. 10(9): p. 627-34. cGMP-dependent protein kinase: a key alanine/threonine dif 0221 Scholten, A., Fuss, H., Heck, A. J., and Dostmann, ference in evolutionary divergence of cAMP and coMP bind W. R. (2007). The hinge region operates as a stability switch ing sites. Biochemistry 28, 6.122-6127. in coiMP-dependent protein kinase I alpha. FEBS J. 274, 0231 Wu, J., Brown, S. H. von Daake, S., and Taylor, S. 2274-2286. S. (2007). PKA type Ilalpha holoenzyme reveals a combina 0222 Shabb, J. B., Buzzeo, B.D., Ng, L., and Corbin, J. D. torial strategy for isoform diversity. Science 318, 274–279. (1991). Mutating protein kinase cAMP-binding sites into 0232 Zhang, Z., and Smith, D. L. (1993). Determination cGMP-binding sites. Mechanism of c(GMP selectivity. J. of amide hydrogen exchange by mass spectrometry: a new Biol. Chem. 266, 24320-24326. tool for proteinstructure elucidation. Protein Sci. 2, 522-531. 0223 Shabb, J. B., and Corbin, J. D. (1992). Cyclic nucle 0233. The foregoing written specification is considered to otide-binding domains in proteins having diverse functions. J. be sufficient to enable one skilled in the art to practice the Biol. Chem. 267,5723-5726. invention. The present invention is not to be limited in scope 0224) Shabb, J. B., Ng, L., and Corbin, J. D. (1990). One by examples provided, since the examples are intended as a amino acid change produces a high affinity c0MP-binding single illustration of one aspect of the invention and other site in cAMP-dependent protein kinase. J. Biol. Chem. 265, functionally equivalent embodiments are within the scope of 16031-16034. the invention. Various modifications of the invention in addi 0225 Spraggon, G., Pantazatos, D., Klock, H. E., Wilson, tion to those shown and described herein will become appar I.A., Woods, V. L., Jr., and Lesley, S.A. (2004). On the use of ent to those skilled in the art from the foregoing description DXMS to produce more crystallizable proteins: structures of and fall within the scope of the appended claims. The advan the T. maritima proteins TM0160 and TM1171. Protein Sci. tages and objects of the invention are not necessarily encom 13,3187-3199. passed by each embodiment of the invention.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 41

<21 Os SEQ ID NO 1 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polynucleotide

<4 OOs SEQUENCE: 1

cgggat.c cat gcaggcatt C cqgaagttc 29

<21 Os SEQ ID NO 2 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polynucleotide

<4 OOs SEQUENCE: 2

ggaattic ct a ct acttic agg ttggcgaag 29 US 2014/0037547 A1 Feb. 6, 2014 20

- Continued

<210s, SEQ ID NO 3 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 3 Thr Glin Ala Lys Arg Llys Llys Ser Lieu Ala Met Ala 1. 5 1O

<210s, SEQ ID NO 4 &211s LENGTH: 30 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 4 Asp Wal Ser Asn Lys Ala Tyr Glu Asp Ala Glu Ala Lys Ala Lys Tyr 1. 5 1O 15 Glu Ala Glu Ala Ala Phe Phe Ala Asn Lieu Lys Lieu. Ser Asp 2O 25 3O

<210s, SEQ ID NO 5 &211s LENGTH: 27 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 5 Asp Wal Ser Asn Lys Ala Tyr Glu Asp Ala Glu Ala Lys Ala Lys Tyr 1. 5 1O 15 Glu Ala Glu Ala Ala Phe Phe Ala Asn Lieu Lys 2O 25

<210s, SEQ ID NO 6 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 6 Asp Wal Ser Asn Lys Ala Tyr Glu Asp Ala Glu Ala Lys Ala Lys Tyr 1. 5 1O 15

Glu Ala Glu Ala Ala 2O

<210s, SEQ ID NO 7 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OO > SEQUENCE: 7 Ala Ala Phe Phe Ala Asn Lieu Lys 1. 5

<210s, SEQ ID NO 8 US 2014/0037547 A1 Feb. 6, 2014 21

- Continued

&211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Xaa is A. G. L., I, W C S T or P 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Xaa is A. G. L., I, W C S T or P 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Xaa is F, Y or W 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Xaa is F, Y or W 22 Os. FEATURE <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Xaa is A. G. L., I, W C S T or P 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Xaa is N, DE, Q, S or T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Xaa is L., I, W or A 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Xaa is K. R. E. H. D. Q S T or Y <4 OOs, SEQUENCE: 8

Xaa Xala Xala Xala Xala Xala Xala Xala 1. 5

<210s, SEQ ID NO 9 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 9

Phe Phe Ala Asn Lieu. 1. 5

<210s, SEQ ID NO 10 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) ... (2) <223> OTHER INFORMATION: Xaa can be any amino acid 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Xaa can be any amino acid <4 OOs, SEQUENCE: 10

Xaa Xala Phe Phe Ala Asn Lieu. Xaa 1. 5 US 2014/0037547 A1 Feb. 6, 2014 22

- Continued

<210s, SEQ ID NO 11 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 11 Gly Glu Gly Gly Ser Phe Gly Glu Lieu Ala Lieu. Ile Tyr Gly Thr Pro 1. 5 1O 15 Arg Ala Ala Thr Val Lys Ala 2O

<210s, SEQ ID NO 12 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 12 Pro Ser Asp Tyr Phe Gly Glu Ile Ala Lieu. Ile Met Asn Arg Pro Arg 1. 5 1O 15

Ala Ala Thr Wal Wall Ala 2O

<210s, SEQ ID NO 13 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 13 Ser Glu Gly Gly Ser Phe Gly Glu Lieu Ala Lieu. Ile Tyr Gly Thr Pro 1. 5 1O 15 Arg Ala Ala Thr Val Lys Ala 2O

<210s, SEQ ID NO 14 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 14 Pro Ser Asp Tyr Phe Gly Glu Ile Ala Lieu. Lieu. Lieu. Asn Arg Pro Arg 1. 5 1O 15

Ala Ala Thr Wal Wall Ala 2O

<210s, SEQ ID NO 15 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 15 Asp Asn Arg Gly Ser Phe Gly Glu Lieu Ala Lieu Met Tyr Asn Thr Pro 1. 5 1O 15 US 2014/0037547 A1 Feb. 6, 2014 23

- Continued Arg Ala Ala Thir Ile Val Ala 2O

<210s, SEQ ID NO 16 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 16 Lys Gly Glin Tyr Phe Gly Glu Lieu Ala Lieu Val Thr Asn Llys Pro Arg 1. 5 1O 15 Ala Ala Ser Ala Tyr Ala 2O

<210s, SEQ ID NO 17 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 17 Asp Asn Arg Gly Ser Phe Gly Glu Lieu Ala Lieu Met Tyr Asn Thr Pro 1. 5 1O 15 Arg Ala Ala Thir Ile Thr Ala 2O

<210s, SEQ ID NO 18 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 18 Arg Gly Glin Tyr Phe Gly Glu Lieu Ala Lieu Val Thr Asn Llys Pro Arg 1. 5 1O 15

Ala Ala Ser Ala His Ala 2O

<210s, SEQ ID NO 19 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 19 Gly Pro Gly Llys Val Phe Gly Glu Lieu Ala Ile Leu Tyr Asn Cys Thr 1. 5 1O 15 Arg Thr Ala Thr Val Lys Thr 2O

<210s, SEQ ID NO 2 O &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 2O US 2014/0037547 A1 Feb. 6, 2014 24

- Continued Lys Gly Asp Trp Phe Gly Glu Lys Ala Lieu. Glin Gly Glu Asp Val Arg 1. 5 1O 15

Thir Ala Asn. Wall Ile Ala 2O

<210s, SEQ ID NO 21 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 21 Gly Pro Gly Llys Val Phe Gly Glu Lieu Ala Ile Leu Tyr Asn Cys Thr 1. 5 1O 15 Arg Thr Ala Thr Val Lys Thr 2O

<210s, SEQ ID NO 22 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 22 Lys Gly Asp Trp Phe Gly Glu Lys Ala Lieu. Glin Gly Glu Asp Val Arg 1. 5 1O 15

Thir Ala Asn. Wall Ile Ala 2O

<210s, SEQ ID NO 23 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 23 Gly Pro Gly Llys Val Phe Gly Glu Lieu Ala Ile Leu Tyr Asn Cys Thr 1. 5 1O 15 Arg Thr Ala Thr Val Lys Thr 2O

<210s, SEQ ID NO 24 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 24 Lys Gly Asp Trp Phe Gly Glu Lys Ala Lieu. Glin Gly Glu Asp Val Arg 1. 5 1O 15

Thir Ala Asn. Wall Ile Ala 2O

<210s, SEQ ID NO 25 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide US 2014/0037547 A1 Feb. 6, 2014 25

- Continued

<4 OOs, SEQUENCE: 25 Gly Pro Gly Llys Val Phe Gly Glu Lieu Ala Ile Leu Tyr Asn Cys Thr 1. 5 1O 15 Arg Thr Ala Thr Val Lys Thr 2O

<210s, SEQ ID NO 26 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 26 Lys Gly Asp Trp Phe Gly Glu Lys Ala Lieu. Glin Gly Glu Asp Val Arg 1. 5 1O 15

Thir Ala Asn. Wall Ile Ala 2O

<210s, SEQ ID NO 27 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 27 Gly Pro Gly Llys Val Phe Gly Glu Lieu Ala Ile Leu Tyr Asn Cys Thr 1. 5 1O 15 Arg Thr Ala Thr Val Arg Thr 2O

<210s, SEQ ID NO 28 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 28 Arg Gly Asp Ser Phe Gly Glu Lys Ala Lieu. Glin Gly Glu Asp Ile Arg 1. 5 1O 15

Thir Ala Asn. Wall Ile Ala 2O

<210s, SEQ ID NO 29 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 29 Ser Gly Ala Lys Val Lieu. Gly Glu Lieu Ala Ile Lieu. Tyr Asn. Cys Glin 1. 5 1O 15 Arg Thr Ala Thr Ile Thr Ala 2O

<210s, SEQ ID NO 3 O &211s LENGTH: 22 212. TYPE: PRT US 2014/0037547 A1 Feb. 6, 2014 26

- Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 30 Lys Gly Asp Phe Phe Gly Glu Lys Ala Lieu. Glin Gly Asp Asp Lieu. Arg 1. 5 1O 15 Thir Ala Asn. Ile Ile Cys 2O

<210s, SEQ ID NO 31 211 LENGTH: 77 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 31 Glin Ala Phe Arg Llys Phe Thr Lys Ser Glu Arg Ser Lys Asp Lieu. Ile 1. 5 1O 15 Lys Glu Ala Ile Lieu. Asp Asn Asp Phe Met Lys Asn Lieu. Glu Lieu. Ser 2O 25 3O Glin Ile Glin Glu Ile Val Asp Cys Met Tyr Pro Val Glu Tyr Gly Lys 35 4 O 45 Asp Ser Cys Ile Ile Lys Glu Gly Asp Val Gly Ser Lieu Val Tyr Val SO 55 6 O Met Glu Asp Gly Llys Val Glu Val Thir Lys Glu Gly Val 65 70 7s

<210s, SEQ ID NO 32 211 LENGTH: 77 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 32 Val Thr Lieu Pro Phe Tyr Pro Llys Ser Pro Glin Ser Lys Asp Lieu. Ile 1. 5 1O 15 Lys Glu Ala Ile Lieu. Asp Asn Asp Phe Met Lys Asn Lieu. Glu Lieu. Ser 2O 25 3O Glin Ile Glin Glu Ile Val Asp Cys Met Tyr Pro Val Glu Tyr Gly Lys 35 4 O 45 Asp Ser Cys Ile Ile Lys Glu Gly Asp Val Gly Ser Lieu Val Tyr Val SO 55 6 O Met Glu Asp Gly Llys Val Glu Val Thir Lys Glu Gly Val 65 70 7s

<210s, SEQ ID NO 33 &211s LENGTH: 79 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 33 Phe Ser Phe Glu Lys Ala Arg Val Arg Lys Asp Ser Ser Glu Lys Llys 1. 5 1O 15

Lieu. Ile Thr Asp Ala Lieu. Asn Lys Asn Glin Phe Lieu Lys Arg Lieu. Asp 2O 25 3O US 2014/0037547 A1 Feb. 6, 2014 27

- Continued

Pro Glin Glin Ile Lys Asp Met Val Glu. Cys Met Tyr Gly Arg Asn Tyr 35 4 O 45 Gln Glin Gly Ser Tyr Val Ile Lys Glin Gly Glu Pro Gly Asn His Ile SO 55 6 O Phe Val Lieu Ala Glu Gly Arg Lieu. Glu Val Phe Glin Gly Glu Lys 65 70 7s

<210s, SEQ ID NO 34 &211s LENGTH: 79 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 34 Llys Lieu. Cys Thir Met Gly Pro Gly Llys Val Phe Gly Glu Lieu Ala Ile 1. 5 1O 15 Lieu. Tyr Asn Cys Thr Arg Thr Ala Thr Val Lys Thr Lieu Val Asn Val 2O 25 3O Llys Lieu. Trp Ala Ile Asp Arg Glin Cys Phe Glin Thir Ile Met Met Arg 35 4 O 45 Thr Gly Lieu. Ile Llys His Thr Glu Tyr Met Glu Phe Leu Lys Ser Val SO 55 6 O Pro Thr Phe Glin Ser Leu Pro Glu Glu Ile Leu Ser Lys Lieu. Ala 65 70 7s

<210s, SEQ ID NO 35 &211s LENGTH: 79 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 35 Lieu. Leu Ser Ser Ile Pro Met Trp Thir Thr Phe Gly Glu Lieu. Ala Ile 1. 5 1O 15 Lieu. Tyr Asn. Cys Thr Arg Thr Ala Ser Wall Lys Ala Ile Thir Asn. Wall 2O 25 3O Llys Thir Trp Ala Lieu. Asp Arg Glu Val Phe Glin Asn. Ile Met Arg Arg 35 4 O 45 Thir Ala Glin Ala Arg Asp Glu Glu Tyr Arg Asn. Phe Lieu. Arg Ser Val SO 55 6 O Ser Lieu. Lieu Lys Asn Lieu Pro Glu Asp Llys Lieu. Thir Lys Ile Ile 65 70 7s

<210s, SEQ ID NO 36 211 LENGTH: 77 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 36 Asp Val Lieu. Glu Glu Thir His Tyr Glu Asin Gly Glu Tyr Ile Ile Arg 1. 5 1O 15 Glin Gly Ala Arg Asp Gly Asp Thr Phe Phe Ile Ile Ser Lys Gly Lys 2O 25 3O

Val Asn Val Thr Arg Glu Asp Ser Pro Asn. Glu Asp Pro Val Phe Lieu US 2014/0037547 A1 Feb. 6, 2014 28

- Continued

35 4 O 45 Arg Thr Lieu. Gly Lys Gly Asp Trp Phe Gly Glu Lys Ala Lieu. Glin Gly SO 55 6 O Glu Asp Val Arg Thr Ala Asn Val Ile Ala Ala Glu Ala 65 70 7s

<210s, SEQ ID NO 37 &211s LENGTH: 76 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OO > SEQUENCE: 37 Asp Val Lieu. Glu Glu Thir His Tyr Glu Asin Gly Glu Tyr Ile Ile Arg 1. 5 1O 15 Glin Gly Ala Arg Gly Asp Thr Phe Phe Ile Ile Ser Lys Gly Llys Val 2O 25 3O Asn Val Thir Arg Glu Asp Ser Pro Asn. Glu Asp Pro Val Phe Lieu. Arg 35 4 O 45 Thir Lieu. Gly Lys Gly Asp Trp Phe Gly Glu Lys Ala Lieu. Glin Gly Glu SO 55 6 O Asp Val Arg Thr Ala Asn Val Ile Ala Ala Glu Ala 65 70 7s

<210s, SEQ ID NO 38 211 LENGTH: 77 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 38 Asp Cys Lieu. Glu Val Glu Tyr Tyr Asp Llys Gly Asp Tyr Ile Ile Arg 1. 5 1O 15 Glu Gly Glu Glu Gly Ser Thr Phe Phe Ile Lieu Ala Lys Gly Llys Val 2O 25 3O Llys Val Thr Glin Ser Thr Glu Gly His Asp Gln Pro Gln Lieu. Ile Llys 35 4 O 45 Thir Lieu Gln Lys Gly Glu Tyr Phe Gly Glu Lys Ala Lieu. Ile Ser Asp SO 55 6 O Asp Val Arg Ser Ala Asn. Ile Ile Ala Glu Glu Asn Asp 65 70 7s

<210s, SEQ ID NO 39 &211s LENGTH: 46 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 39 Val Thr Cys Lieu Val Ile Asp Arg Asp Ser Phe Llys His Lieu. Ile Gly 1. 5 1O 15 Gly Lieu. Asp Asp Val Ser Asn Lys Ala Tyr Glu Asp Ala Glu Ala Lys 2O 25 3O Ala Lys Tyr Glu Ala Glu Ala Ala Phe Phe Ala Asn Lieu Lys 35 4 O 45 US 2014/0037547 A1 Feb. 6, 2014

- Continued

<210s, SEQ ID NO 4 O &211s LENGTH: 73 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 4 O Val Ala Cys Lieu Val Ile Asp Arg Glu Thir Phe Asn Glin Thr Val Gly 1. 5 1O 15 Thir Phe Asp Glu Lieu. Glin Llys Tyr Lieu. Glu Gly Tyr Val Ala Thr Lieu. 2O 25 3O Asn Arg Asp Asp Glu Lys Arg His Ala Lys Arg Ser Met Ser Ser Trip 35 4 O 45 Llys Lieu. Ser Lys Ala Lieu. Ser Lieu. Glu Met Ile Glin Lieu Lys Glu Lys SO 55 6 O Val Ala Arg Phe Ser Ser Thr Ser Pro 65 70

<210s, SEQ ID NO 41 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 41 Asp Wal Ser Asn Lys Ala Tyr Glu Asp Ala Glu Ala Lys Ala Lys Tyr 1. 5 1O 15

Glu Ala Glu Ala Ala Phe Phe 2O

What is claimed is: 10. The method of claim 2, wherein the coMP independent 1. (canceled) PKG activator is a peptide. 2. A method of treating a PKG deficient condition in a 11. The method of claim 10, wherein the peptide is: Subject, comprising administering to the Subject a therapeu tically effective amount of acci MP independent PKG activa tOr. DWSNKAYEDAEAKAKYEAEAAFFANLKLSD. (SEQ ID NO. 4) 3. The method of claim 2, wherein the PKG deficient 12. The method of claim 2, wherein the coMP independent condition is selected from the group consisting of cardiovas PKG activator is a small molecule. cular disorders, hypoxia, spinal cord injury, and stroke. 13. A composition comprising: acci MP independent PKG 4. The method of claim 2, further comprising administer ing c(GMP to the subject. activator and a carrier. 5. The method of claim 2, further comprising administer 14. The composition of claim 13, wherein the coMP inde ing acciMP dependent PKG activator to the subject. pendent PKG activator is a peptide. 6. The method of claim 5, wherein the coMP dependent 15. The composition of claim 14, wherein the peptide is: PKG activator is a PDE inhibitor. 7. The method of claim 6, wherein the PDE inhibitor is DWSNKAYEDAEAKAKYEAEAAFFANLKLSD. (SEQ ID NO. 4) selected from the group consisting of sildenafil citrate, Vin pocetine, EHNA, anagrelide, enoXimone, milrinone, mesem 16. The composition of claim 13, wherein the coMP inde brine, rolipram, ibudilast, piclamilast, luteolin, drotaverine, pendent PKG activator is a small molecule. roflumilast, tadalafil. Vardenafil. udenafil, avanafil, and 17-21. (canceled) papaverine. 22. A method of selecting or designing a compound that 8. The method of claim 5, wherein the acGMP dependent interacts with PKG and modulates PKG activity, the method PKG activator is a NO-donor. comprising the step of assessing the stereochemical comple 9. The method of claim 8, wherein the NO-donor is mentarity between the compound and a topographic region of selected from the group consisting of isosorbide dinitrite, PKG, wherein the topographic region of the PKG is charac Diazeniumdiolates, NONOates, S-Nitrosothiols, NO hybrid terized by at least a portion of the amino acids positioned at drugs, and Zeolites. atomic coordinates as deposited in Protein Data Bank with US 2014/0037547 A1 Feb. 6, 2014 30 accession code 3SHR wherein amino acid sequences of X, is Leu, Isl, Val, or Ala, and switch helix domain thereof are set forth as SEQID NO: 2. Xs is Lys Arg, Glu, His, Asp, Gln, Ser, Thr, or Tyr. 23. A method of claim 22, wherein the topographic region 27. The peptide of claim 25, wherein the Xs have the of PKG is the switch helix domain defined by amino acids: following values: 350-354. values: 24. The method of claim 22, further comprising testing the X is Ala, Gly, Leu, Ile, or Val compound in vitro or in vivo for its capacity to modulate the X is Ala, Gly, Leu, Ile, or Val activity of PKG. X is Phe, Tyr, or Trp 25. An isolated peptide comprising XXX-XXXX-Xs X is Phe, Tyr, or Trp (SEQ ID NO: 8), wherein each X is an amino acid, and wherein the peptide binds to a nest region or PKG. X is Ala, Gly, Leu, Ile, or Val 26. The peptide of claim 25, wherein the Xs have the X is ASn, Asp, or Glu, following values: X, is Leu, Isl, Val, or Ala, and X is Ala, Gly, Leu, Ile, Val, Cys, Ser. Thr, or Pro Xs is Lys or Arg. X is Ala, Gly, Leu, Ile, Val, Cys, Ser. Thr, or Pro 28. The peptide of claim 25, wherein the peptide comprises X is Phe, Tyr, or Trp XX-FFANLXs (SEQ ID NO: 10), such that XX, and Xs X is Phe, Tyr, or Trp are any amino acid, optionally Ala of Lys. X is Ala, Gly, Leu, Ile, Val, Cys, Ser. Thr, or Pro 29. (canceled) X is Asn, Asp, Glu, Gln, Ser, or Thr