This application claims the benefit of U.S. Provisional Application No. 61/210,594, filed Mar. 20, 2009, which is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
- Top of Page
The instant application contains an ASCII “txt” compliant sequence listing submitted via EFS-WEB on Mar. 19, 2010, which serves as both the computer readable form (CRF) and the paper copy required by 37 C.F.R. Section 1.821(c) and 1.821(e), and is hereby incorporated by reference in its entirety. The name of the “txt” file created on Mar. 18, 2010, is: A-1455-WO-PCT-SeqList031810-482 ST25.txt, and is 348 kb in size.
Throughout this application various publications are referenced within parentheses or brackets. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
BACKGROUND OF THE INVENTION
- Top of Page
1. Field of the Invention
The present invention is related to the biochemical arts, in particular to therapeutic peptides and conjugates.
2. Discussion of the Related Art
Ion channels are a diverse group of molecules that permit the exchange of small inorganic ions across membranes. All cells require ion channels for function, but this is especially so for excitable cells such as those present in the nervous system and the heart. The electrical signals orchestrated by ion channels control the thinking brain, the beating heart and the contracting muscle. Ion channels play a role in regulating cell volume, and they control a wide variety of signaling processes.
The ion channel family includes Na+, K+, and Ca2+ cation and C1-anion channels. Collectively, ion channels are distinguished as either ligand-gated or voltage-gated. Ligand-gated channels include both extracellular and intracellular ligand-gated channels. The extracellular ligand-gated channels include the nicotinic acetylcholine receptor (nAChR), the serotonin (5-hydroxytryptamine, 5-HT) receptors, the glycine and γ-butyric acid receptors (GABA) and the glutamate-activated channels including kanate, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartate receptors (NMDA) receptors. (Harte and Ouzounis (2002), FEBS Lett. 514: 129-34). Intracellular ligand gated channels include those activated by cyclic nucleotides (e.g. cAMP, cGMP), Ca2+ and G-proteins. (Harte and Ouzounis (2002), FEBS Lett. 514: 129-34). The voltage-gated ion channels are categorized by their selectivity for inorganic ion species, including sodium, potassium, calcium and chloride ion channels. (Harte and Ouzounis (2002), FEBS Lett. 514: 129-34).
A unified nomenclature for classification of voltage-gated channels was recently presented. (Catterall et al. (2000), Pharmacol. Rev. 55: 573-4; Gutman et al. (2000), Pharmacol. Rev. 55, 583-6; Catterall et al. (2000) Pharmacol. Rev. 55: 579-81; Catterall et al. (2000), Pharmacol. Rev. 55: 575-8; Hofmann et al. (2000), Pharmacol. Rev. 55: 587-9; Clapham et al. (2000), Pharmacol Rev. 55: 591-6; Chandy (1991), Nature 352: 26; Goldin et al. (2000), Neuron 28: 365-8; Ertel et al. (2000), Neuron 25: 533-5). The K+ channels constitute the largest and best characterized family of ion channels described to date. Potassium channels are subdivided into three general groups: the 6 transmembrane (6™) K+ channels, the 2™-2™/leak K+ channels and the 2™/Kir inward rectifying channels. (Tang et al. (2004), Ann. Rev. Physiol. 66, 131-159). These three groups are further subdivided into families based on sequence similarity. The voltage-gated K+ channels, including (Kv1-6, Kv8-9), EAG (POTASSIUM CHANNEL, VOLTAGE-GATED, SUBFAMILY H, MEMBER 1), KQT (Potassium voltage-gated channel subfamily KQT member 1), and Slo (BKCa; POTASSIUM CHANNEL, CALCIUM-ACTIVATED, LARGE CONDUCTANCE, SUBFAMILY M, ALPHA MEMBER 1), are family members of the 6™ group. The 2™-2™ group comprises TWIK (POTASSIUM CHANNEL, SUBFAMILY K, MEMBER 1), TREK (POTASSIUM CHANNEL, SUBFAMILY K, MEMBER 2), TASK (POTASSIUM CHANNEL, SUBFAMILY K, MEMBER 3), TRAAK (POTASSIUM CHANNEL, SUBFAMILY K, MEMBER 4), and THIK (POTASSIUM CHANNEL, SUBFAMILY K, MEMBER 13, also known as TANDEM PORE DOMAIN HALOTHANE-INHIBITED POTASSIUM CHANNEL), whereas the 2™/Kir group consists of Kir1-7. Two additional classes of ion channels include the inward rectifier potassium (IRK) and ATP-gated purinergic (P2X) channels. (Harte and Ouzounis (2002), FEBS Lett. 514: 129-34).
Toxin peptides produced by a variety of organisms have evolved to target ion channels. Snakes, scorpions, spiders, bees, snails and sea anemone are a few examples of organisms that produce venom that can serve as a rich source of small bioactive toxin peptides or “toxins” that potently and selectively target ion channels and receptors. In most cases, these toxin peptides have evolved as potent antagonists or inhibitors of ion channels, by binding to the channel pore and physically blocking the ion conduction pathway. In some other cases, as with some of the tarantula toxin peptides, the peptide is found to antagonize channel function by binding to a region outside the pore (e.g., the voltage sensor domain).
Native toxin peptides are usually between about 20 and about 80 amino acids in length, contain 2-5 disulfide linkages and form a very compact structure. Toxin peptides (e.g., from the venom of scorpions, sea anemones and cone snails) have been isolated and characterized for their impact on ion channels. Such peptides appear to have evolved from a relatively small number of structural frameworks that are particularly well suited to addressing the critical issues of potency, stability, and selectivity. (See, e.g., Dauplais et al., On the convergent evolution of animal toxins: conservation of a diad of functional residues in potassium channel-blocking toxins with unrelated structures, J. Biol. Chem. 272(7):4302-09 (1997); Alessandri-Haber et al., Mapping the functional anatomy of BgK on Kv1.1, Kv1.2, and Kv1.3, J. Biol. Chem. 274(50):35653-61 (1999)). The majority of scorpion and Conus toxin peptides, for example, contain 10-40 amino acids and up to five disulfide bonds, forming extremely compact and constrained structure (microproteins) often resistant to proteolysis. The conotoxin and scorpion toxin peptides can be divided into a number of superfamilies based on their disulfide connections and peptide folds. The solution structure of many of these has been determined by Nuclear Magnetic Resonance (NMR) spectroscopy, illustrating their compact structure and verifying conservation of their family folding patterns. (E.g., Tudor et al., Ionisation behaviour and solution properties of the potassium-channel blocker ShK toxin, Eur. J. Biochem. 251(1-2):133-41 (1998); Pennington et al., Role of disulfide bonds in the structure and potassium channel blocking activity of ShK toxin, Biochem. 38(44): 14549-58 (1999); Jaravine et al., Three-dimensional structure of toxin OSK1 from Orthochirus scrobiculosus scorpion venom, Biochem. 36(6):1223-32 (1997); del Rio-Portillo et al.; NMR solution structure of Cn12, a novel peptide from the Mexican scorpion Centruroides noxius with a typical beta-toxin sequence but with alpha-like physiological activity, Eur. J. Biochem. 271(12): 2504-16 (2004); Prochnicka-Chalufour et al., Solution structure of discrepin, a new K+-channel blocking peptide from the alpha-KTx15 subfamily, Biochem. 45(6):1795-1804 (2006)). Conserved disulfide structures can also reflect the individual pharmacological activity of the toxin family. (Nicke et al. (2004), Eur. J. Biochem. 271: 2305-19, Table 1; Adams (1999), Drug Develop. Res.46: 219-34). For example, α-conotoxins have well-defined four cysteine/two disulfide loop structures (Loughnan, 2004) and inhibit nicotinic acetylcholine receptors. In contrast, ω-conotoxins have six cysteine/three disulfide loop consensus structures (Nielsen, 2000) and block calcium channels. Structural subsets of toxins have evolved to inhibit either voltage-gated or calcium-activated potassium channels.
Due to their potent and relatively selective blockade of specific ion channels, toxin peptides have been used for many years as tools to investigate the pharmacology of ion channels. Other than excitable cells and tissues such as those present in heart, muscle and brain, ion channels are also important to non-excitable cells such as immune cells. Accordingly, the potential therapeutic utility of toxin peptides has been considered for treating various immune disorders, in particular by inhibition of potassium channels such as Kv1.3 and IKCa1 since these channels indirectly control calcium signaling pathway in lymphocytes. (E.g., Kem et al., ShK toxin compositions and methods of use, U.S. Pat. No. 6,077,680; Lebrun et al., Neuropeptides originating in scorpion, U.S. Pat. No. 6,689,749; Beeton et al., Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases, Molec. Pharmacol. 67(4):1369-81 (2005); Possani Postay et al., VM23 and VM24, two scorpion peptides that block human T-lymphocyte potassium channels (subtype kv1.3) with high selectivity and decrease the in vivo DTH-responses in rats, WO 2008/139243; Mouhat et al., K+ channel types targeted by synthetic OSK1, a toxin from Orthochirus scrobiculosus scorpion venom, Biochem. J. 385:95-104 (2005); Mouhat et al., Pharmacological profiling of Orthochirus scrobiculosus toxin 1 analogs with a trimmed N-terminal domain, Molec. Pharmacol. 69:354-62 (2006); Mouhat et al., OsK1 derivatives, WO 2006/002850 A2; B. S. Jensen et al. The Ca2+-activated K+ Channel of Intermediate Conductance: A Molecular Target for Novel Treatments?, Current Drug Targets 2:401-422 (2001); Rauer et al., Structure-guided Transformation of Charybdotoxin Yields an Analog That Selectively Targets Ca2+-activated over Voltage-gated K+ Channels, J. Biol. Chem. 275: 1201-1208 (2000); Castle et al., Maurotoxin: A Potent Inhibitor of Intermediate Conductance Ca2+-Activated Potassium Channels, Molecular Pharmacol. 63: 409-418 (2003); Chandy et al., K+ channels as targets for specific Immunomodulation, Trends in Pharmacol. Sciences 25: 280-289 (2004); Lewis & Garcia, Therapeutic Potential of Venom Peptides, Nat. Rev. Drug Discov. 2: 790-802 (2003); Han et al., Structural basis of a potent peptide inhibitor designed for Kv1.3 channel, a therapeutic target of autoimmune disease, J. Biol. Chem. 283(27):19058-65 (2008)].
Calcium mobilization in lymphocytes is known to be a critical pathway in activation of inflammatory responses [M. W. Winslow et al. (2003) Current Opinion Immunol. 15, 299]. Compared to other cells, T cells show a unique sensitivity to increased levels of intracellular calcium and ion channels both directly and indirectly control this process. Inositol triphosphate (IP3) is the natural second messenger which activates the calcium signaling pathway. IP3 is produced following ligand-induced activation of the T cell receptor (TCR) and upon binding to its intracellular receptor (a channel) causes unloading of intracellular calcium stores. The endoplasmic reticulum provides one key calcium store. Thapsigargin, an inhibitor of the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA), also causes unloading of intracellular stores and activation of the calcium signaling pathway in lymphocytes. Therefore, thapsigargin can be used as a specific stimulus of the calcium signaling pathway in T cells. The unloading of intracellular calcium stores in T cells is known to cause activation of a calcium channel on the cell surface which allows for influx of calcium from outside the cell. This store operated calcium channel (SOCC) on T cells is referred to as “CRAC” (calcium release activated channel) and sustained influx of calcium through this channel is known to be critical for full T cell activation [S. Feske et al. (2005) J. Exp. Med. 202, 651 and N. Venkatesh et al. (2004) PNAS 101, 8969]. For many years it has been appreciated that in order to maintain continued calcium influx into T cells, the cell membrane must remain in a hyperpolarized condition through efflux of potassium ions. In T cells, potassium efflux is accomplished by the voltage-gated potassium channel Kv1.3 and the calcium-activated potassium channel IKCa1 [K. G. Chandy et al. (2004) TIPS 25, 280]. These potassium channels therefore indirectly control the calcium signaling pathway, by allowing for the necessary potassium efflux that allows for a sustained influx of calcium through CRAC.
Sustained increases in intracellular calcium activate a variety of pathways in T cells, including those leading to activation of NFAT (Nuclear Factor of Activated T cells), NF-kB (NUCLEAR FACTOR OF KAPPA LIGHT CHAIN GENE ENHANCER IN B CELLS) and AP-1 [ACTIVATOR PROTEIN 1; Quintana-A (2005) Pflugers Arch.—Eur. J. Physiol. 450, 1]. These events lead to various T cell responses including alteration of cell size and membrane organization, activation of cell surface effector molecules, cytokine production and proliferation. Several calcium sensing molecules transmit the calcium signal and orchestrate the cellular response. Calmodulin is one molecule that binds calcium, but many others have been identified (M. J. Berridge et al. (2003) Nat. Rev. Mol. Cell. Biol. 4,517). The calcium-calmodulin dependent phosphatase calcineurin is activated upon sustained increases in intracellular calcium and dephosphorylates cytosolic NFAT. Dephosphorylated NFAT quickly translocates to the nucleus and is widely accepted as a critical transcription factor for T cell activation (F. Macian (2005) Nat. Rev. Immunol. 5, 472 and N. Venkatesh et al. (2004) PNAS 101, 8969). Inhibitors of calcineurin, such as cyclosporin A (Neoral, S and Immune) and FK506 (Tacrolimus) are a main stay for treatment of severe immune disorders such as those resulting in rejection following solid organ transplant (I. M. Gonzalez-Pinto et al. (2005) Transplant. Proc. 37, 1713 and D. R. J. Kuypers (2005) Transplant International 18, 140). Neoral has been approved for the treatment of transplant rejection, severe rheumatoid arthritis (D. E. Yocum et al. (2000) Rheumatol. 39, 156) and severe psoriasis (J. Koo (1998) British J. Dermatol. 139, 88). Preclinical and clinical data has also been provided suggesting calcineurin inhibitors may have utility in treatment of inflammatory bowel disease (IBD; Baumgart D C (2006) Am. J. Gastroenterol. Mar. 30; Epub ahead of print), multiple sclerosis (Ann. Neurol. (1990) 27, 591) and asthma (S. Rohatagi et al. (2000) J. Clin. Pharmacol. 40, 1211). Lupus represents another disorder that may benefit from agents blocking activation of helper T cells. Despite the importance of calcineurin in regulating NFAT in T cells, calcineurin is also expressed in other tissues (e.g. kidney) and cyclosporine A & FK506 have a narrow safety margin due to mechanism based toxicity. Renal toxicity and hypertension are common side effects that have limited the promise of cyclosporine & FK506. Due to concerns regarding toxicity, calcineurin inhibitors are used mostly to treat only severe immune disease (Bissonnette-R et al. (2006) J. Am. Acad. Dermatol. 54, 472). Kv1.3 inhibitors offer a safer alternative to calcineurin inhibitors for the treatment of immune disorders. This is because Kv1.3 also operates to control the calcium signaling pathway in T cells, but does so through a distinct mechanism to that of calcineurin inhibitors, and evidence on Kv1.3 expression and function show that Kv1.3 has a more restricted role in T cell biology relative to calcineurin, which functions also in a variety of non-lymphoid cells and tissues.
Calcium mobilization in immune cells also activates production of the cytokines interleukin 2 (IL-2) and interferon gamma (designated interchangeably herein as IFNg, IFN-g or IFN-γ) which are critical mediators of inflammation. IL-2 induces a variety of biological responses ranging from expansion and differentiation of CD4+ and CD8+ T cells, to enhancement of proliferation and antibody secretion by B cells, to activation of NK cells [S. L. Gaffen & K. D. Liu (2004) Cytokine 28, 109]. Secretion of IL-2 occurs quickly following T cell activation and T cells represent the predominant source of this cytokine Shortly following activation, the high affinity IL-2 receptor (IL2-R) is upregulated on T cells endowing them with an ability to proliferate in response to IL-2. T cells, NK cells, B cells and professional antigen presenting cells (APCs) can all secrete IFNg upon activation. T cells represent the principle source of IFNg production in mediating adaptive immune responses, whereas natural killer (NK) cells & APCs are likely an important source during host defense against infection [K. Schroder et al. (2004) J. Leukoc. Biol. 75, 163]. IFNg, originally called macrophage-activating factor, upregulates antigen processing and presentation by monocytes, macrophages and dendritic cells. IFNg mediates a diverse array of biological activities in many cell types [U. Boehm et al. (1997) Annu Rev. Immunol. 15, 749] including growth & differentiation, enhancement of NK cell activity and regulation of B cell immunoglobulin production and class switching.
CD40L (TUMOR NECROSIS FACTOR LIGAND SUPERFAMILY, MEMBER 5) is another cytokine expressed on activated T cells following calcium mobilization and upon binding to its receptor on B cells provides critical help allowing for B cell germinal center formation, B cell differentiation and antibody isotype switching. CD40L-mediated activation of CD40 (B CELL-ASSOCIATED MOLECULE CD40; Also known as TUMOR NECROSIS FACTOR RECEPTOR SUPERFAMILY, MEMBER 5) on B cells can induce profound differentiation and clonal expansion of immunoglobulin (Ig) producing B cells [S. Quezada et al. (2004) Annu Rev. Immunol. 22, 307]. The CD40 receptor can also be found on dendritic cells and CD40L signaling can mediate dendritic cell activation and differentiation as well. The antigen presenting capacity of B cells and dendritic cells is promoted by CD40L binding, further illustrating the broad role of this cytokine in adaptive immunity. Given the essential role of CD40 signaling to B cell biology, neutralizing antibodies to CD40L have been examined in preclinical and clinical studies for utility in treatment of systemic lupus erythematosis (SLE),—a disorder characterized by deposition of antibody complexes in tissues, inflammation and organ damage [J. Yazdany and J Davis (2004) Lupus 13, 377].
Small molecule inhibitors of Kv1.3 and IKCa1 potassium channels and the major calcium entry channel in T cells, CRAC, have also been developed to treat immune disorders (A. Schmitz et al. (2005) Molecul. Pharmacol. 68, 1254; K. G. Chandy et al. (2004) TIPS 25, 280; H. Wulff et al. (2001) J. Biol. Chem. 276, 32040; C. Zitt et al. (2004) J. Biol. Chem. 279, 12427), but obtaining small molecules with selectivity toward some of these targets has been difficult.
The identification of selective and potent peptide Kv1.3 inhibitors with prolonged in vivo activity has been a long standing challenge. Production of toxin peptides is a complex process in venomous organisms, and is an even more complex process synthetically. Due to their conserved disulfide structures and need for efficient oxidative refolding, toxin peptides present challenges to synthesis. Although toxin peptides have been used for years as highly selective pharmacological inhibitors of ion channels, the high cost of synthesis and refolding of the toxin peptides and their short half-life in vivo have impeded the pursuit of these peptides as a therapeutic modality.
Much of the focus has been given to analogs of the 35-residue ShK peptide, which has a short in vivo half-life of about 30 minutes, and which is a potent inhibitor of both the Kv1.3 ion channel and the Kv1.1 ion channel, a potassium channel expressed in the human nervous system. (E.g., Harvey et al., A three-residue, continuous binding epitope peptidomimetic of ShK toxin as a Kv1.3 inhibitor, Bioorganic & Medicinal Chem. Lett. 15:3193-96 (2005); Lanigan et al., Designed peptide analogues of the potassium channel blocker ShK toxin, Biochem. 40:15528-37 (2001)). Position 22 of ShK has been identified as a key residue which confers Kv1.3 selectivity, and ShK binding to Kv1.3 is sensitive to substitution at Lys9 and Arg11. For example, [Dap22]ShK (SEQ ID NO:317; also known as ShK-Dap22) is a picomolar range inhibitor of Kv1.3 with a reported 35-fold selectivity for murine Kv1.3 over murine Kv1.1. (See, e.g., Kem et al., ShK toxin compositions and methods of use, U.S. Pat. No. 6,077,680). [Dap22]ShK is reported to display about 20-fold selectivity for human K(v)1.3 over K(v)1.1, when measured by the whole-cell voltage clamp method but not in equilibrium binding assays (Middleton R E, et al., Substitution of a single residue in Stichodactyla helianthus peptide, ShK-Dap22, reveals a novel pharmacological profile. Biochemistry. 2003 Nov. 25 42(46):13698-707). The ShK-Dap22 molecule was reported to have similar potency to native ShK and to provide potent blockade of Kv1.3 with an IC50 of about 23 μM as measured by whole cell patch clamp electrophysiology. (Kalman et al., ShK-Dap22, a potent Kv1.3-specific immunosuppressive polypeptide, J. Biol. Chem. 273(49):32697-707 (1998)).
Other ShK analogs with phosphotyrosine (“pY”), or other anionically charged chemical entities, or fluorescein modifications at the N-terminus have reportedly resulted in some improved selectivity for mKv1.3 over mKv1.1. An example includes Shk-L5, which involves a phosphotyrosine-AEEA modification at the N-terminus of the ShK peptide. (Beeton et al., Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases, Molec. Pharmacol. 67(4):1369-81 (2005); Chandy et al., Analogs of ShK toxin and their uses in selective inhibition of Kv1.3 potassium channels, WO 2006/042151 A2; Pennington et al., Engineering a stable and selective peptide blocker of the Kv1.3 channel in T lymphocytes, Molecular Pharmacology Fast Forward, published Jan. 2, 2009 as doi:10.1124/mol.108.052704 (2009)). AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid (also known as 8-Amino-3,6-Dioxaoctanoic Acid) and is used as a “linker” group in peptide chemistry in its N-Fmoc-protected form. In ShK-L5, this AEEA hydrophilic bifunctional linker is use as a very short bridge between a phosphotyrosine residue and the ShK peptide. However, from a biochemical perspective, the phosphotyrosine group is not metabolically stable, and the phospho group can be cleaved under physiological conditions. Beeton et al. (2005) indicated that ShK-L5 has an estimated circulating half-life of about 50 min in rats following subcutaneous or intravascular injection, which is comparable to that for the native ShK peptide. (See, e.g., Beeton et al., Selective blockade of T lymphocyte K+ channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis, Proc. Natl. Acad. Sci. USA 98(24):13942-47 (2001)). Thus phosphotyrosine-linked derivative of ShK with improved metabolic stability but which also retain high potency have been sought. (Chaurdran, Tet. Letters, 28, 4051-4054 (2007). More recently, M. W. Pennington et al. described ShK-192, which incorporates phosphonophenylalanine-AEEA- at the N-terminus instead of phosphotyrosine-AEEA. (Pennington et al., Engineering a stable and selective peptide blocker of the Kv1.3 channel in T lymphocytes, Molec. Pharmacol. 75(4):762-73 (2009)).
A desideratum provided by the present invention is compositions of matter including ShK peptide analogs with improved Kv1.3 inhibition activity, in vivo stability and/or selectivity, which may also be fused, or otherwise covalently conjugated to a vehicle.
- Top of Page
OF THE INVENTION
The present invention is directed to a composition of matter, which comprises an amino acid sequence of the following formula:
SEQ ID NO: 4
or a pharmaceutically acceptable salt thereof,
Xaa1Xaa2 is absent; or Xaa1 is absent and Xaa2 is Glu, Ser, Ala, or Thr; or Xaa1 is Arg or Ala and Xaa2 is Glu, Ser, Ala, or Thr;
Xaa4 is an alkyl, basic, or acidic amino acid residue;
Xaa6 is Thr, Tyr, Ala, or Leu;
Xaa7 is Leu, Ile, Ala, or Lys;
Xaa8 is Pro, Ala, Arg, Lys, 1-Nal, or Glu;
Xaa9 is Lys, Ala, Val or an acidic amino acid residue;
Xaa10 is Ser, Glu, Arg, or Ala;
Xaa11 is Arg, Glu; or Ala;
Xaa13 is Thr, Ala, Arg, Lys, 1-Nal, or Glu;
Xaa14 is Gln, Ala or an acidic amino acid residue;
Xaa15 is an alkyl or aromatic amino acid residue;
Xaa16 is a basic, alkyl, or aromatic amino acid residue, other than Ala, Gln, Glu or Arg;
Xaa18 is Ala or an acidic or basic amino acid residue;
Xaa19 is Thr, Ala or a basic amino acid residue;
Xaa20 is Ser, Ala, or a basic amino acid residue;
Xaa21 is an alkyl or aromatic amino acid residue, other than Ala or Met;
Xaa22 is Lys or Ala;
Xaa23 is Tyr or Ala;
Xaa24 is Arg, Lys, or Ala;
Xaa25 is Tyr, Leu, or Ala;
Xaa26 is Ser, Thr, Asn, Ala, or an aromatic amino acid residue;
Xaa27 is Leu, Ala, Asn, or an aromatic amino acid residue;
Xaa29 is 1-Nal, 2-Nal, Ala, or a basic amino acid residue;
Xaa30 is Ala or an acidic or basic amino acid residue;
Xaa31 is Thr, Ala, or an aromatic amino acid residue;