DE NOVO PROTEIN DECOYS OF ANGIOTENSIN-CONVERTING ENZYME 2 (ACE2) CROSS-REFERENCE TO RELATED APPLICATIONS [001] This application claims the benefit of priority of US Provisional Application No. 63/006,463, filed April 7, 2020; US Provisional Application No.63/028,401, filed May 21, 2020; US Provisional Application No.62/705,150, filed June 13, 2020; US Provisional Application No.63/055,051, filed July 22, 2020; US Provisional Application No. 63/060,489, filed August 3, 2020; US Provisional Application No.63/094,179, filed October 20, 2020; and US Provisional Application No.63/145,352, filed February 3, 2021; each of which is incorporated by reference herein in its entirety for any purpose. FIELD [002] The present invention is related to de novo protein decoys of ACE2. BACKGROUND [003] Viruses often exploit cell surface-associated proteins to enter and infect host cells. Neutralizing antibodies can inhibit this process by binding to the surface of the virus, impeding its interaction with the target cell’s surface protein(s) and/or preventing viral- associated conformational changes necessary for infection. Vaccination has, therefore, been a broadly useful tool to combat many viral diseases. RNA-based viruses are a frequent exception to this strategy. Neutralizing antibodies bind to viral proteins in a fundamentally different fashion than how the virus interacts with its cell target. Therefore, RNA viruses with high mutational rates often exploit such structural discrepancy to escape the immune system by remodeling the shape of their receptor-binding proteins to evade neutralizing antibodies while retaining the interaction with their target receptor(s). [004] Coronaviruses are large, enveloped, positive-stranded RNA viruses. The genome is packed inside a helical capsid formed by the nucleocapsid protein and is further surrounded by the viral envelope. At least three structural proteins are associated with the viral envelope, including the envelope-anchored spike protein. Coronaviruses recognize a variety of receptors, and binding by the spike protein mediates coronavirus entry into host cells via those receptors. Coronaviruses are believed to enter host cells via a two-step process. First, the spike protein binds to a receptor on the host cell surface through its S1 subunit and then fuses the viral and host membranes through its S2 subunit. Both the viral attachment step and membrane fusion process are mediated by recognition of the host receptor by the spike protein. The alphacoronavirus HCoV-NL63 and the betacoronavirus SARS-CoV both
recognize the receptor zinc peptidase angiotensin-converting enzyme 2 (ACE2). Recently, it has been discovered that ACE2 is also the functional receptor for 2019-nCov, a new SARS- like coronavirus that emerged from Wuhan, China in 2019, also referred to as SARS-CoV- 2. [005] SARS-CoV-2 is a highly contagious virus that causes coronavirus disease 2019 (COVID-19). Since its emergence in December 2019, SARS-CoV-2 has caused millions of cases of COVID19 and has become a global pandemic. While the majority of subjects are asymptomatic or have mild disease, a number of subjects will develop either severe disease with dyspnea or hypoxia or critical disease with symptoms of respiratory failure requiring positive pressure ventilation, shock, or multi-organ failure. A need exists for identifying agents that can prevent viruses, such as coronaviruses, from invading host cells. The present invention meets this and other needs. BRIEF DESCRIPTION OF THE DRAWINGS [006] Figure 1 – Figure 1 is a schematic showing inhibition of viral entry into cells by a de novo designed ACE2 protein decoy. SARS-CoV and SARS-CoV-2 viruses enter cells by first binding to the ACE2 receptor on the surface of human cells via the RBD domain of the spike protein (left). The de novo designed ACE2 decoy binds to the RBD domain in the same manner as natural ACE2 but, unlike ACE2, has no biological function. The decoys effectively sequester the virus from binding the native ACE2 receptors and prevent viral entry into cells while keeping native ACE2 function levels intact (right). [007] Figure 2A-F – Figure 2A-F demonstrate by yeast surface display that the de novo ACE2 protein decoy CTC-445 binds to SARS-CoV-2 Spike/RBD Protein. Figure 2A-B show a positive (ACE-2) control and Figure 2C-D show a negative control (human IL-21R). Figure 2E-F show CTC-445. [008] Figure 3A-D - Figure 3A-D demonstrate that control human ACE2 (3A-B) and de novo protein decoy CTC-445 (3C-D) bind to SARS-CoV-2 Spike/RBD Protein and compete with soluble ACE2 for binding to the Spike/RBD protein. [009] Figure 4 – Figure 4 shows the kinetics of binding of purified ACE2 protein decoy CTC-445 via an Octet biolayer interferometry (BLI) binding assay. [0010] Figure 5A-H – Figure 5A-H shows the kinetics of binding of select purified ACE2 protein decoys via Octet BLI binding assays. [0011] Figure 6A-H – Figure 6A-H shows the kinetics of binding of select purified ACE2 protein decoys via Octet BLI binding assays.
[0012] Figure 7A-H – Figure 7A-H shows the kinetics of binding of select purified ACE2 protein decoys CTC-445 variants via Octet BLI binding assays. [0013] Figure 8A-C – Circular dichroism absorption at 222 nm of ACE2 protein decoys CTC-445 (A), CTC-445.2 (B) and CTC-445.2d (C). The insets show far UV wavelength spectra of the test articles at 20 °C, after heating to 95-99 °C (dashed) and after cooling the heated sample to 20 °C. The Y axes for 8B and 8C is the same as that for 8A. [0014] Figure 9A-C – Figure 9A-C shows the thermal recovery of ACE2 protein decoys CTC-445, CTC-445.2 and CTC-445.2d after repeated cycles of heating and cooling. The data shows that the designed proteins refold even after repeated thermal denaturation. [0015] Figure 10A-C – Figure 10A-C show the kinetics of binding of purified ACE2 protein decoys CTC-445 (A), CTC 445.2 (B) and CTC-445.2d (C) via Octet BLI binding assays. [0016] Figure 11 – Figure 11 provides a plot of the potency of select ACE2 protein decoys vs their molecular weight. IC
50 is measured by ELISA. [0017] Figure 12A-B – Figure 12A shows a neutralization assay performed using a non- replicative VSV pseudovirus carrying a luciferase reporter gene and expressing the spike protein of SARS-CoV-2 on its surface. Viral neutralization with ACE2 protein decoys CTC- 445.2 and CTC-445.2d was performed on HEK 293T cells overexpressing ACE2. The test proteins were pre-incubated with pseudovirus prior to incubation with cells. Samples were tested in duplicate utilizing 3-fold serial dilutions started at 20 μM (CTC-445.2) or 10 μM (CTC-445.2d). A cell viability assay (12B) was run in parallel. [0018] Figure 13 – Figure 13 shows bioavailability of ACE2 protein decoy CTC-445.2d in mice lung (top) and plasma (bottom) after intranasal administration. Protein concentration in lung lysates and blood plasma are quantified using Meso Scale Discovery platform. [0019] Figure 14 – Figure 14 shows ACE2 functional activity as measured by enzymatic release of a free fluorophore from Mca-APK(DNP) substrate. ACE2 inhibition was shown using DX600 peptide as a positive control. [0020] Figure 15A-B – Figure 15 shows the kinetics of binding of ACE2 protein decoys CTC-445.2 (A) and CTC-445.2d (B) to SARS-CoV-1 via Octet BLI binding assays. [0021] Figure 16A-E – Figure 16A-E show the shows the kinetics of binding of ACE2 protein decoy CTC-445.2 to SARS-CoV-2 RBD mutants via Octet binding assays. [0022] Figure 17 – Figure 17 provides the designed ACE2 protein decoy CTC-445 in complex with the SARS-CoV-2 spike protein RBD (surface representation). The graph on
the right on the cartoon representations shows biased forward folding simulations of designed sequence. The designed sequence was subjected to ab initio structure prediction using Rosetta. Each point in the plot represents an independent folding trajectory which was computed by Monte Carlo insertion of fragments from solved protein structures. Folding simulations were biased towards the designed conformation by using a small subset of fragments at each residue position with the lowest RMSD (9- and 3-mers) to the designed structure. Red (or black for B&W) dots are trajectories computed using the 5 fragments from Rosetta's vall structural database with lowest RMSD to the designs at each amino acid position. Brown (or gray) dots are trajectories computed using fragments from the design model itself plus the 8 lowest RMSD 3mers and 9mers. The funnel-shaped energy landscape suggests that the designed structure is the global energy minima and has a substantial energy gap with respect to alternative conformations. [0023] Figure 18A-B –Figure 18A-B shows a structural alignment of ACE2 protein decoy CTC-640 with a non-redundant database of known structures. Structural alignment was performed using MICAN in rewiring and reverse mode with maximum distance between C⍺ atoms to be aligned of 10.0 A. Each gray point represents the structural alignment of CTC- 640 with a different structure in the database. Each black point represents the structural alignment of CTC-640 with different structures of ACE2. For both plots, structural alignments of <50 total residues were discarded. A) Structural alignments are performed using TMalign, which aligns structures based on the order of the secondary structure elements in the polypeptide chain. Sequence identity is computed based on the structurally aligned residues. Although CTC-640 mimics the ACE2 binding surface, it does not align well in lineal structure or sequence to any protein in the database, including ACE2. B) Structural alignment is performed using MICAN in rewiring/reverse mode, which allows inverse direction of secondary structures, alternative alignments, and non-sequential alignments. The highest sequence identity observed for CTC-640 is with ACE2 where H1+H2+H3 correctly align to their counterparts in ACE2 with sequence identity of 33.7% for aligned residues. [0024] Figure 19A-C – Figure 19A-C shows the kinetics of binding of ACE2 protein decoy CTC-708 (CTC-445.2t) via Octet binding assays to SARS-CoV-2 (19A), SARS- CoV-1 (19B), and results of a competition assay [0025] Figure 20A-C – Figure 20A-C provides results from deep mutational scanning of ACE2 protein decoy CTC445.2 and plotted as sequence logo using logomaker [ref: https://www.biorxiv.org/content/10.1101/635029v1]. Letters are scaled according to their
probability and ordered from highest probability (top) to lowest (bottom). The native sequence of CTC445.2 is shaded in black. Residues 1-54 are shown in 20A, residues 55-107 are shown in 20B and residues 108-160 are shown in panel 20C. [0026] Figure 21A-C - Figure 21A shows neutralization of SARS-CoV-2 infection with ACE2 protein decoys CTC445.2d and CTC445.3d in engineered HEK293T cells overexpressing hACE2 determined using a non-replicative VSV pseudovirus carrying a luciferase reporter gene and expressing the spike protein of SARS-CoV-2 (GenBank: QHD43416.1) on its surface. Figure 21B shows a neutralization assay using control pseudovirus expressing VSVg instead of spike protein. Figure 21C shows cell viability of engineered HEK293T cells incubated with ACE2 protein decoys CTC-445.2d and CTC- 445.3d. SUMMARY [0027] The present inventors have built de novo proteins that can accurately recapitulate the natural binding surface targeted by some coronaviruses. In particular, the present inventors have built de novo proteins that present a binding surface recognized by coronaviruses, in particular, coronaviruses that use ACE2 to mediate entry into host cells. By binding to the coronavirus, these de novo proteins can act as decoys for host ACE2 protein and, in certain aspects, prevent the virus from binding to its receptor, ACE2. Accordingly, provided herein are, inter alia, de novo proteins, ACE2 protein decoys, that bind to the coronavirus spike protein of SARS-CoV and SARS-CoV-2. The proteins of the present invention are useful, inter alia, for inhibiting or neutralizing the activity of the virus. In some embodiments, the proteins are useful for blocking binding of the virus to its host cell receptor and for preventing the entry of the coronavirus into host cells. In some embodiments, the proteins function by inhibiting the cell-to-cell transmission of the virus. In certain embodiments, the proteins are useful in preventing, treating or ameliorating at least one symptom of coronavirus infection in a subject. In certain embodiments, the proteins may be administered prophylactically or therapeutically to a subject having or at risk of having coronavirus infection. [0028] In a first aspect, the present invention provides de novo proteins, ACE2 protein decoys, that bind specifically to coronavirus spike protein, in particular, spike protein from those coronaviruses that use ACE2 as their receptor to facilitate viral entry into target cells, for example, SARS-CoV and SARS-CoV-2. Also provided are de novo proteins, ACE2 protein decoys, that block (e.g., partially or fully) coronavirus spike protein binding to its
native receptor and, in particular, block coronavirus spike protein binding to ACE2. In some embodiments, the present invention provides de novo proteins, ACE2 protein decoys, that block the binding of coronavirus to its native human, camel or bat ACE2 receptor. [0029] The de novo proteins of the present invention are non-naturally occurring and are comprised of peptide domains, including at least two alpha helical domains, H1 and H2, and an optional beta hairpin domain, H3. These three domains interface with the coronavirus spike protein. Exemplary de novo proteins of the present invention further comprise at least one structural domain that facilitates protein folding and binding- competent presentation of the H1 and H2 alpha helices and H3 beta hairpin domains to the coronavirus spike protein. In some aspects, exemplary de novo proteins of the present invention comprise at least two structural domains that facilitate protein folding and binding-competent presentation of the H1 and H2 alpha helices and H3 beta hairpin domains to the coronavirus spike protein. The H1 and H2 alpha helical domains and optional beta hairpin comprise amino acid residues that interact with/act as binding sites to the coronavirus spike protein. [0030] The de novo proteins of the present invention interact with amino acid residues in the receptor binding domain of coronavirus spike protein. Without wishing to be bound by theory, for SARS-CoV, the expected binding residues on the RBD (receptor binding domain) are: 442, 443, 461, 462, 463, 470, 471, 472, 473, 475, 476, 479, 481, 482, 483, 486, 487, 488, 489 and 491 and for SARS-CoV-2, the expected binding residues are: 455, 456, 475, 476, 477, 486, 487, 489, 490, 493, 496, 497, 498, 500, 501, 502, 504, and 505. [0031] In a related aspect, the present invention provides not only the proteins comprising the peptide domains, H1, H2 and H3, but the peptide domains themselves. [0032] In a second aspect, the present invention provides nucleic acid molecules encoding the de novo proteins and peptide domains of the present invention. For example, the present invention provides nucleic acid molecules encoding any of the proteins and peptide domains described herein. [0033] In a related aspect, the present invention provides recombinant expression vectors capable of expressing the proteins of the present invention. For example, the present invention includes recombinant expression vectors comprising any of the nucleic acid molecules mentioned above. Also included within the scope of the present invention are host cells into which such vectors have been introduced, as well as methods of producing
the proteins by culturing the host cells under conditions permitting production of the proteins, and recovering the proteins so produced. [0034] In a third aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a de novo ACE2 protein decoy of the present invention and a pharmaceutically acceptable carrier. In a related aspect, the invention features a composition which is a combination of a de novo protein of the present invention and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with a de novo protein of the present invention. Exemplary agents include, without limitation, other agents that inhibit viral activity including infectivity of host cells. [0035] In a fourth aspect, the invention provides therapeutic methods for treating a disease or disorder associated with a coronavirus that uses ACE2 as its receptor to facilitate viral entry into target cells. The methods include, for example, treating a viral infection in a subject using de novo ACE2 protein decoy of the invention, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising the ACE2 protein decoy to the subject in need thereof. The disorder treated is any disease or condition which is improved, ameliorated, inhibited or prevented by inhibition of SARS-CoV or SARS-CoV-2 coronavirus activity or activity of any other coronavirus that gains access to its target cells using the ACE2 receptor. In certain embodiments, the present invention provides methods to prevent, treat or ameliorate at least one symptom of coronavirus infection, the method comprising administering a therapeutically effective amount of a protein of the present invention to a subject in need thereof. In some embodiments, the present invention provides methods to ameliorate or reduce the severity of at least one symptom or indication of coronavirus infection in a subject by administering a protein of the invention, wherein at least one symptom or indication is selected from the group consisting of: inflammation in the lung, alveolar damage, fever, cough, shortness of breath, diarrhea, heart failure, arrhythmias, multiple organ dysfunction, pneumonia, septic shock and/or death. In certain embodiments, the invention provides methods to decrease viral load in a subject, the methods comprising administering to the subject an effective amount of a ACE2 protein decoy of the invention that binds the coronavirus spike protein from SARS-CoV or SARS-CoV-2 or another coronavirus spike protein from a coronavirus that gains entry into its target cells by use of the ACE2 receptor and blocks binding of the spike protein to its host cell receptor. In some embodiments, the de novo protein may be administered prophylactically or therapeutically
to a subject having or at risk of having a coronavirus infection. The subjects at risk include, but are not limited to, an immunocompromised person, an elderly adult (more than 65 years of age), healthcare workers, adults or children in close contact with a person(s) with confirmed or suspected coronavirus infection, and people with underlying medical conditions such as pulmonary disease or infection, heart disease or diabetes. In certain embodiments, the de novo proteins of the present invention are administered in combination with a second therapeutic agent to a subject in need thereof. The second therapeutic agent may be, for example, selected from the group consisting of an anti-inflammatory drug (such as corticosteroids, and non-steroidal anti-inflammatory drugs), an anti-infective drug, or an anti-viral drug. In certain embodiments, the second therapeutic agent may be an agent that helps to counteract or reduce any possible side effect(s) associated with a de novo protein of the invention, if such side effect(s) should occur. For example, in some aspects, the second therapeutic is one to treat cytokine release syndrome (e.g., a cytokine storm). The de novo protein thereof may be administered, for example, subcutaneously, intravenously, intradermally, intraperitoneally, orally, or intramuscularly. In some aspects, the de novo proteins are inhaled. [0036] Also included in the present invention are methods for treating a viral infection in a subject using the nucleic acids of the invention. The methods comprise administering a therapeutically effective amount of a nucleic acid encoding an ACE2 protein decoy of the present invention to a subject in need thereof. [0037] The present invention also includes use of protein or nucleic acid of the invention in the manufacture of a medicament for the treatment of a disease or disorder that would benefit from the blockade of coronavirus binding and/or activity. [0038] In a fifth aspect, the invention provides methods for detecting coronavirus spike protein in a biological sample. The methods comprise the steps of contacting the biological sample with an ACE2 protein decoy of the present invention and detecting coronavirus spike protein in the biological sample. [0039] Other embodiments will become apparent from a review of the ensuing detailed description. DEFINITIONS [0040] The term “SARS-CoV” refers to the Severe Acute Respiratory Syndrome coronavirus that emerged in China in 2002. It binds via the viral spike protein to human host cell receptor ACE2. The term “SARS-CoV-S”, also called “S protein”, refers to the
spike protein of the SARS coronavirus (S1 and S2). The SARS-CoV spike protein mediates receptor recognition and membrane fusion. During viral infection, the S protein is cleaved into two subunits, S1 and S2. The receptor binding domain is found in the S1 subunit and it directly binds to the peptidase domain (PD) of ACE2. S2 is responsible for membrane fusion. When S1 binds to ACE2, S2 is cleaved by host proteases. This cleavage of S2 is believed to be critical for viral infection. An exemplary SARS-CoV-S protein is provided herein as SEQ ID NO:2. The signal peptide is amino acids 1-13, S1 spike protein is amino acids 14-667, and S2 spike protein is amino acids 668-1255. The term SARS-CoV-S and SARS-CoV spike protein includes protein variants of SARS-CoV spike protein isolated from different SARS-CoV isolates. SARS-CoV isolates include, for example, Isolate BJ01, Isolate BJ02, Isolate BJ03, Isolate BJ04, Isolate GZ50, Isolate CUHK-W1, Isolate HKU- 36871, Isolate GD01, Isolate GD03, Isolate Shanghai LY, Isolate Frankfurt 1, Isolate FRA, Isolate SZ23, Isolate SZ3, and Isoalte Tor2. [0041] The term “COVID-19” or “2019-nCov” or “SARS-CoV-2” refers to a new SARS- like coronavirus that emerged from Wuhan, China in 2019 and was labeled by the WHO as a pandemic on March 11, 2020. As with SARS-CoV, it binds via the viral spike protein to human host cell receptor ACE2. The term “SARS-CoV-2-S”, also called “S protein” refers to the spike protein of the SARS-CoV-2 coronavirus. An exemplary SARS-CoV-2 S protein is provided herein as SEQ ID NO:3. The term SARS-CoV-2-S and SARS-CoV-2 spike protein includes protein variants of SARS-CoV-2 spike protein isolated from different SARS-CoV-2 isolates. The term “coronavirus ACE2-binding spike protein” or “ACE2- binding spike protein” as used herein refers to a coronavirus spike protein that uses ACE2 to mediate its entry into host cells. [0042] “ACE2” refers to the angiotensin-converting enzyme 2 that acts as a receptor for select coronaviruses. The full length of the human ACE2 protein is provided herein as SEQ ID NO:1. The signal peptide is amino acid residues 1-17; the extracellular PD domain is amino acid residues 18-740; the transmembrane segment is residues 741-761; and the intracellular domain is residues 762-805. [0043] The primary physiological role of ACE2 is in the maturation of angiotensin, however, it has also been hijacked as a cellular receptor for some coronaviruses. The ACE2 protein decoys of the present invention were designed to act as decoys for human ACE2, nevertheless, in some embodiments, they can act as protein decoys for ACE2 from other mammalian species.
[0044] The term “coronavirus infection” or “SARS-CoV infection” or “SARS-CoV-2 infection”, as used herein, refers to infection by a coronavirus that use the ACE2 receptor to gain entry into host cells, and in particular, SARS or COVID-19 coronavirus. Severe acute respiratory illness is associated with both SARS and COVID-19 coronavirus infection. A wide range of additional complications are associated with COVID-19 coronavirus, including thrombotic events and neurological disease. Symptoms of infection include fever, cough, shortness of breath pneumonia, gastro-intestinal symptoms such as diarrhea, organ failure (kidney failure, heart failure, and renal dysfunction), septic shock and death in severe cases. [0045] The term “recombinant”, as used herein, refers to proteins of the invention created, expressed, isolated or obtained by technologies or methods known in the art as recombinant DNA technology which include, e.g., DNA splicing and transgenic expression. [0046] The term “specifically binds,” or “binds specifically to”, or the like, means that a first moiety has a greater affinity for a second moiety than it does for other moieties. Specific binding does not, however, require exclusive binding. In some embodiments, a first moiety specifically binds a second moiety and the resulting complex is relatively stable under physiologic conditions. Specific binding can be characterized, in some embodiments, by an equilibrium dissociation constant of about 650 nM or less, or in some embodiments, 100 nM or less (e.g., a smaller KD denotes a tighter binding). Methods for determining whether two moieties specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, proteins have been identified which bind specifically to coronavirus spike protein, in particular, proteins that bind specifically to coronavirus ACE2-binding spike protein. [0047] The phrase “therapeutically effective amount” refers to an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). [0048] As used herein, the term “subject” refers to an animal, preferably a mammal, more preferably a human, in need of amelioration, prevention and/or treatment of a disease or disorder such as viral infection. The term includes human subjects who have or are at risk of having a coronavirus infection.
[0049] As used herein, the terms “treat”, “treating”, or “treatment” refer to the reduction or amelioration of the severity of at least one symptom or indication of a coronavirus infection due to the administration of a therapeutic agent such as a protein of the present invention to a subject in need thereof. The terms include inhibition of progression of disease or of worsening of infection. The therapeutic agent may be administered at a therapeutic dose to the subject. [0050] The term “prevent”, “preventing” or “prevention” refers to inhibition of the onset of symptoms of a coronavirus infection. In some embodiments, prevention encompasses inhibition of a coronavirus infection and/or inhibition of the spread of coronavirus infection from a subject to another individual. [0051] As used herein, the term “anti-viral drug” refers to any anti-infective drug or therapy used to treat, prevent, or ameliorate a viral infection in a subject. The term “anti- viral drug” includes, but is not limited to ribavirin, remdesivir, oseltamivir, zanamivir, interferon-alpha2b, analgesics and corticosteroids. In the context of the present invention, the viral infections include infection caused by human coronaviruses, including SARS-CoV and SARS-CoV-2. [0052] The term “identity”, as used herein in reference to polypeptide sequences, refers to the amino acid sequence identity between two molecules. When an amino acid position in both molecules is occupied by the same amino acid, then the molecules are identical at that position. The identity between two polypeptides is a direct function of the number of identical positions. In general, the sequences are aligned so that the highest order match is obtained (including gaps if necessary). Identity can be calculated using published techniques and widely available computer programs, such as the GCG program package (Devereux et al., Nucleic Acids Res.12:387, 1984), BLASTP, FASTA (Atschul et al., J. Molecular Biol.215:403, 1990), etc. Sequence identity can be measured, for example, using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, WI 53705), with the default parameters thereof. When determining identity for the present invention, it is also important to consider positioning of the binding interface residues with the coronavirus spike protein. If amino acids are added or deleted, it should be done in such a way that doesn’t substantially interfere with presentation of the protein to its binding partner or with secondary structure. Unless indicated otherwise, percent identity is determined across the length of the reference sequence.
[0053] In some aspects, amino acid substitutions relative to the reference peptide domains can be, for example, conservative amino acid substitutions. As used herein, “conservative amino acid substitution” means a given amino acid can be replaced by an amino acid having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as Ile, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity is retained. Amino acids can be grouped according to similarities in the properties of their side chains. Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties. Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class. Particular conservative substitutions include, for example; Ala to Gly or Ser; Arg to Lys; Asn to Gln or H; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and/or Phe to Val, Ile or Leu. A common hydrophobic grouping is glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), and phenylalanine (Phe). [0054] As used herein, the natural amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). As used herein “any amino acid” typically refers to the 20 natural amino acids. The skilled practitioner will appreciate, however, that one or more, (e.g., from 1 to 10, 1 to 5, 1 to 3, or 1 or 2) unnatural amino acids can be used in place of a natural amino acid. As used herein, the term “unnatural amino acid” refers to an amino acid other than the 20 amino acids that occur naturally in protein. Unnatural amino acids are known in the art. [0055] As used herein, the terms "polypeptide”, “protein” or “peptide” refer to any chain of amino acid residues, regardless of its length or post-translational modification (e.g., glycosylation or phosphorylation).
[0056] “Operably linked” is intended to mean that the nucleotide sequence of is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). “Regulatory sequences” include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). The expression constructs of the invention can be introduced into host cells to thereby produce the proteins disclosed herein. [0057] The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell but are still included within the scope of the term as used herein. [0058] As used herein, the terms “transformation” and “transfection” refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, particle gun, or electroporation. [0059] As used herein, the term “pharmaceutically acceptable carrier” includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics) can also be incorporated into the compositions. GENERAL [0060] The inventors have described herein de novo ACE2 protein decoys that specifically bind to the spike protein on coronaviruses and modulate the interaction of the spike proteins with their innate receptor. In particular, the de novo ACE2 protein decoys bind to the spike protein on those coronaviruses that use ACE2 as their receptor to facilitate viral entry into target cells. Notably, although the de novo ACE2 protein decoys of the present invention contain the domains necessary for interacting with and binding to coronavirus spike protein, they typically do not contain domains associated with other enzymatic activities of native ACE2, including for example the catalytic domain (e.g., metalloprotease catalytic domain) of ACE2. Exemplary de novo ACE2 protein decoys of the present invention do not catalyze the cleavage of angiotensin (i.e., any forms of angiotensin, including angiotensin I and II).
In some embodiments, the ACE2 protein decoys provided herein have no more than 60%, 55%, 50%, 45%, 40%, or 35% sequence identity to human ACE2 (SEQ ID NO: 1). With regard to identity to ACE2, percent identity is calculated with the ACE2 protein decoy as query and ACE2 as reference, over the length of the query. [0061] In some embodiments, protein decoys of the present invention bind to the spike protein (e.g., spike protein from SARS-CoV-2) with a Kd of 700 nM or less, Kd of 600 nM or less, Kd of 500 nM or less, Kd of 400 nM or less, Kd of 300 nM or less, Kd of 200 nM or less, K
d of 100 nM or less, K
d of 50 nM or less, preferably about 20 nM or less, or even 15 nM, 10 nM, or 5 nM or less. Methods of determining K
d are known in the art and described in the examples. In certain embodiments, the de novo protein decoys of the present invention are blocking proteins in that they may bind to the coronavirus spike protein (e.g., from SARS-CoV-2) and block the interaction of the spike protein with their native receptor (i.e., ACE2). As used herein, blocking proteins may completely block the interaction of the spike protein with their native receptor or may partially block the interaction of the spike protein with their native receptor. In certain embodiments, the de novo protein decoys inhibit the interaction of the spike protein with their native receptor (i.e., ACE2) with an IC
50 of 100 nM or less. In some embodiments, the de novo proteins inhibit the interaction of the spike protein with their native receptor (i.e., ACE2) with an IC
50 of 50, 40, 30, 20, 10, or 5 nM or less. In some embodiments, the blocking proteins of the invention block the binding of the coronavirus spike protein to its receptor and/or inhibit or neutralize or reduce viral infectivity of host cells. In some embodiments, the blocking proteins may be useful for treating a subject suffering from a coronavirus infection (e.g., COVID-19 coronavirus infection). In some embodiments, The de novo protein decoys of the present invention, when administered to a subject in need thereof, reduce the infection by a coronavirus such as SARS-CoV-2 in the subject. They may be used to decrease viral loads in a subject. Protein decoys of the present invention may be used alone or as adjunct therapy with other therapeutic moieties or modalities known in the art for treating viral infection. The ability of the de novo proteins of the invention to bind to and inhibit/neutralize the activity of SARS-CoV or SARS-CoV-2 may be measured using any standard method known to those skilled in the art, including binding assays, or activity assays, as described herein. For example, in vitro assays for measuring binding and inhibition and/or blocking activity are illustrated in examples. [0062] The de novo protein decoys of the present invention may contain no additional labels or moieties, or they may contain labels (e.g., an N-terminal or C-terminal label or
moiety). In one embodiment, the label or moiety is biotin. In a binding assay, the location of a label (if any) may determine the orientation of the peptide relative to the surface upon which the peptide is bound. For example, if a surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide will be distal to the surface. The label may be, for example, an enzyme, a radionuclide, a fluorescent dye or a MRI-detectable label. Such labeled proteins may be used in diagnostic assays including imaging assays. In some aspects, for ACE-2 protein decoys that do not contain tryptophan, the label may be a C-terminal peptide that allows for detection of protein by absorbance at 280 nm (e.g., GSGWGSG, SEQ ID NO:248). STRUCTURAL AND SEQUENCE CHARACTERISTICS OF THE ACE2 PROTEIN DECOYS [0063] Provided herein are de novo proteins of the present invention, referred to herein as ACE2 protein decoys. These protein decoys are, by nature, non-naturally occurring proteins, and comprise at least two alpha helical domains that interface with the coronavirus spike protein and preferably at least one structural domain that facilitates protein folding and binding-competent presentation of the alpha helices. In some embodiments, the de novo proteins further comprise an optional beta hairpin domain. The alpha helical domains that interface with the coronavirus spike protein and optional beta-hairpin domain interact with/act as binding sites to the coronavirus spike protein. These domains, referred to herein as H1, H2, and H3, comprise both amino acid residues that engage in binding interactions with the coronavirus spike protein and amino acid residues that do not engage in binding interactions with the coronavirus spike protein. Generally, with respect to the H1 and H2 domains, those amino acid residues that do not engage in binding interactions with the coronavirus ACE2- binding spike protein are at positions that can be very promiscuous with respect to the identity of the amino acid that sits at that position. A number of these residues are also at solvent exposed positions. In some embodiments, when replacing amino acids at solvent exposed positions, the use of hydrophilic amino acids are particularly desirable, although non-hydrophilic amino acids are acceptable as well. [0064] The skilled artisan will appreciate that the de novo proteins of the present invention, the ACE2 protein decoys, were designed such that the binding domains align structurally to the corresponding binding sites in the native ACE2 protein whereas the supporting structural domains do not structurally or sequentially align to any other secondary structures in ACE2. In some aspect, the de novo proteins of the present invention
structurally align to the native ACE2 binding motifs within, for example, 2.75 A RMSD (root mean square deviation) and contain one or more secondary structure elements that do not structurally or sequentially align to any other secondary structure elements in ACE2. Methods of determining RMSD are known in the art, for example using the MICAN protein structure alignment algorithm. MICAN identifies the best structural alignment between protein pairs by disregarding the connectivity between secondary structure elements. [0065] In some aspects, the de novo proteins of the present invention comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein H1 comprises the amino acid sequence SX
1X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L (SEQ ID NO:4) or X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L (SEQ ID NO:176); H2 comprises the amino acid sequence NX
14X
15NX
16X
17X
18KX
19X
20X
21FX
22X
23EQX
24X
25LX
26X
27MY (SEQ ID NO:5); and H3, if present, comprises the amino acid sequence X
28X
29X
30X
31X
32X
33KGDX
34RX
35X
36 (SEQ ID NO:6), wherein X
1, X
2, X
3, X
4, X
5, X
6, X
7, X
8, X
9, X
10, X
11, X
12, X
13, X
14, X
15, X
16, X
17, X
18, X
19, X
20, X
21, X
22, X
23, X
24, X
25, X
26, X
27, X
28, X
29, X
30, X
31, X
32, X
33, X
34, X
35, and X
36, are each independently selected from any amino acid. [0066] Such de novo proteins further comprise one or more structural domain that facilitates protein folding and binding-competent presentation of H1, H2, and H3. In some embodiments of the present invention, the coronavirus-binding amino acid residues of the ACE2 protein decoys are the same as the coronavirus binding amino acid residues of the ACE2 protein. For example, in some such embodiments, the binding residues of H1, H2 and H3 are identical to the amino acid at the same structural position in the native ACE2 protein. In addition, in some exemplary embodiments, all, or all but one to six, or one to five, or one to four, or one to three of the solvent exposed amino acids of H1 and all, or all but one to six, or one to five, or one to four, or one to three of the solvent exposed amino acids of H2, whether or not they are involved in binding, are the same as the amino acids at the same structural position in the native ACE2 protein. By presenting a virtually identical surface as the one that the virus targets in the protein ACE2, the ability of the virus to mutate to avoid binding to the ACE2 protein decoys of the present invention is minimized. In SEQ ID NO:4, the amino acid residues at positions 1, 5, 6, 9, 10, 12, 13, 16, 17, 19, 20, 23, 24 and 27 are, for the most part, solvent exposed and/or involved in binding to
coronavirus ACE2-binding spike protein; in SEQ ID NO: 176, the amino acid residues at positions 3, 4, 7, 8, 10, 11, 14, 15, 17, 18, 21, 22 and 25 are, for the most part, solvent exposed and/or involved in binding to coronavirus ACE2-binding spike protein; in SEQ ID NO:5, the amino acid residues at positions 1, 4, 8, 12, 15, 16, 19, 22 and 23 are, for the most part, solvent exposed and/or involved in binding to coronavirus ACE2-binding spike protein; and in SEQ ID NO:6 the amino acid residues at positions 7, 8, and 9 are, for the most part, solvent exposed and/or involved in binding to coronavirus ACE2-binding spike protein. In some aspects, when making amino acid substitutions, these residues are not substituted. In other aspects, it may be desirable to modify these residues. For example, modifications to these residues can be made in order to create a protein that binds to ACE2 with a higher affinity than native coronavirus spike protein. Such a protein can be used, for example, for diagnostic purposes. In some aspects, no more than 4, no more than 3, no more than 2 or no more than 1 of the residues at positions 1, 5, 6, 9, 10, 12, 13, 16, 17, 19, 20, 23, 24 and 27 of SEQ ID NO:4 are substituted or no more than 4, no more than 3, no more than 2 or no more than 1 of the residues at positions 3, 4, 7, 8, 10, 11, 14, 15, 17, 18, 21, 22 and 25 of SEQ ID NO:176 are substituted; no more than 3, no more than 2 or no more than 1 of the residues at positions 1, 4, 8, 12, 15, 16, 19, 22 and 23 of SEQ ID NO:5, are substituted; and no more than 1 of the residues of 7, 8, 9 or 11 in SEQ ID NO:6 is substituted or no more than 1 of the residues of 7, 8, or 9 in SEQ ID NO:6 is substituted. In some such aspects, such substitutions are with amino acids selected from D, E, G, K, N, P, Q, R, S, or T. In some such aspects, substitutions are with conservative amino acids. In other aspects, one or more of the following substitutions are made in SEQ ID NO: 4: S1I; E5D; E5Q; E5V; D12V, D12E; Q24K; and Q24L. In other aspects, one or more of the following substitutions are made in SEQ ID NO: 176: E3D; E3Q; E3V; D10V, D10E; Q22K; and Q22L. With some exceptions, the residues identified in SEQ ID NOs: 4, 176, 5, and 6 as X (with a subscript numeral) are not solvent exposed and/or are not directly involved in binding to coronavirus ACE2-binding spike protein. These residues typically may be different from the corresponding amino acids in ACE2. Included in the present invention are those proteins wherein not more than half of, or no more than 8 of, the amino acids represented as X (with a subscript numeral) are the same amino acid as the corresponding position in native ACE2 represented by SEQ ID NO:1. In some aspects, not more than 4, 3 or 2, of the amino acids represented as X (with a subscript numeral) in H1; not more than 4, 3 or 2, of the amino acids represented as X (with a subscript numeral) in H2; and/or not more than 5, 4, 3 or 2, of the amino acids represented as X (with a subscript numeral) in
H3, are the same as the corresponding position in native ACE2. An exemplary corresponding sequence of H1 in ACE2 is ST IEEQAKTFLD KFNHEAEDLF YQSSL (SEQ ID NO:7); an exemplary corresponding sequence of H2 in ACE2 is NMNNAGDKWS AFLKEQSTLA QMY (SEQ ID NO:8); an exemplary corresponding sequence of H3 in ACE2 is TAWD LGKGDFRIL (SEQ ID NO:9). ALPHA HELICAL DOMAIN H1 [0067] The de novo proteins of the present invention (i.e., ACE2 protein decoys) can comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein: H1 comprises the amino acid sequence: SX
1X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L (SEQ ID NO:4) or X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L (SEQ ID NO:176); wherein X
1, X
2, X
3, X
4, X
5, X
6, X
7, X
8, X
9, X
10, X
11, X
12, X
13 are each independently selected from any amino acid. [0068] Included in the present invention are de novo proteins of the present invention wherein H1 comprises the amino acid sequence: SX
1X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L (SEQ ID NO:4) or X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L (SEQ ID NO:176); wherein: X
1 is an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, C, E, F, G, I, L, M, N, Q, R, S, T, V, or Y); X
2 is an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably V, A, D, H, I, N, P, T, or W); X
3, is an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y (preferably R, L,C, H, I, K, M, S, T, or Y); X
4 is an amino acid selected from C, I, L, M, T, or V ( preferably L, I, T, or V); X5, is an amino acid selected from A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, K, C, E, I, L, N, R, V, or Y); X
6 is an amino acid selected from A, C, G, L, M, S, T, or V (preferably A, C, S, or T); X
7 is an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, Q, S, T, or V (preferably A, C, E, F, I, L, N, Q, S, T, or V);
X
8 is an amino acid selected from A, C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, or Y (preferably F, D, E, H, L, M, N, Q, or W); X
9 is an amino acid selected from A, C, D, F, G, H, I, L, M, N, Q, S, T, V, W, or Y (preferably M, A, C, F, G, L, S, T, or V); X
10 is an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably R, A, C, E, F, G, H, I, M, P, Q, T, V, W, or Y); X
11, is an amino acid selected from N, C, F, G, H, L, M, W, or Y (preferably F, N, W, or Y); X
12 is an amino acid selected from A, C, F, G, H, I, L, M, N, Q, S, T, V, W, or Y (preferably A, C, S, or T); and X
13 is an amino acid selected from A, C, D, E, F, G, H, I, L, M, N, Q, S, T, or V (preferably A, C, F, G, L, M, N, S, T, or V). [0069] Included in the present invention are de novo proteins of the present invention wherein H1 comprises the amino acid sequence: SX
1X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L (SEQ ID NO:4) or X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L (SEQ ID NO:176); wherein: X
1 is an amino acid selected from D, E, G, K, N, P, Q, R, S, or T; X
2,X
6,X
9,X
11,X
12,and X
13, are each independently an amino acid selected from A, F, I, L, M, P or V; X
5,X
7,X
8, and X
10 are each independently an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X
3 is an amino acid selected from A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y or is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X
4 is an amino acid selected from A, F, I, L, M, P, V, D, E, G, K, N, P, Q, R, S, or T. De novo ACE2 protein decoys of the present invention include those above wherein phenylalanine is included in the list of amino acids for X
1, asparagine is included in the list of amino acids for X
2, histidine is included in the list of amino acids for X
8, and asparagineis included in the list of amino acids for X
11. De novo ACE2 protein decoys of the present invention include those above wherein phenylalanine is included in the list of
amino acids for X
1, asparagine is included in the list of amino acids for X
2, asparagine is removed from the list of amino acid substitutions for X
3, X
4 is an amino acid selected from I, L, M, P, V, or T; aspartic acid is removed from the list of amino acid substitutions for X
5, X
6 is an amino acid selected from A, L, M, or V; proline is removed from the list of amino acid substitutions for X
7, histidine is included in the list of amino acids for X
8 and lysine and proline are removed, proline is removed from the list of amino acids for X
9, asparagine is included in the list of amino acids for X
11 and alanine, isoleucine, proline and valine are removed, and proline is removed from the list of amino acid substitutions for X
12 and X
13. [0070] Included in the present invention are de novo ACE2 protein decoys of the present invention wherein (i) X
4 is an amino acid selected from A, F, I, L, M, P or V (preferably I, L, M or V); and X
1, X
2, X
3, X
5, X
6, X
7, X
8, X
9, X
10, X
11, X
12, and X
13 are as described in any of the embodiments provided herein; (ii) X
4 is an amino acid selected from A, F, I, L, M, P or V (preferably I, L, M or V); and X
5 is an amino acid selected from D, E, G, K, N, P, Q, R, S, or T (preferably E, G, K, N, P, Q, R, S, or T); and X
1, X
2 ,X
3, X
6, X
7, X
8,X
9, X
10,X
11,X
12, and X
13 are as described in any of the embodiments provided herein; (iii) X
1 is an amino acid selected from R or S or F; and X
2,X
3, X
4, X5, X
6, X
7, X
8,X
9, X
10,X
11, X
12, and X
13 are as described in any of the embodiments provided herein; (iv) X
3 is an amino acid selected from R, L, A, F, I, M, P, or V; and X
1, X
2, X
4, X
5, X
6, X
7, X
8, X
9, X
10, X
11, X
12, and X
13 are as described in any of the embodiments provided herein; (v) X
3 is an amino acid selected from R, L, C, or S,; and X
1, X
2, X
4, X
5, X
6, X
7, X
8, X
9, X
10, X
11, X
12, and X
13 are as described in any of the embodiments provided herein; (vi) X
7 is an amino acid selected from A or T; and X
1,X
2,X
3, X
4, X5, X
6, X
8,X
9, X
10, X
11, X
12, and X
13 are as described in any of the embodiments provided herein; (vii) X
10 is an amino acid selected from R, S or L or an amino acid selected from R or S; and X
1,X
2,X
3, X
4, X5, X
6, X
7, X
8,X
9,X
11, X
12, and X
13 are as described in any of the embodiments provided herein; (viii) X
1 is an amino acid selected from R or S or F; X
3 is an amino acid selected from R or L; X
7 is an amino acid selected from A or T; and X
10 is an amino acid
selected from R, S or L; and X
2, X
4, X
5, X
6, X
8, X
9, X
11, X
12, and X
13 are as described in any of the embodiments provided herein; (ix) X5 is an amino acid selected from D, E, G, K, N, P, Q, R, S, or T (preferably E, G, K, N, P, Q, R, S, or T); and X
1,X
2,X
3, X
4, X
6, X
7, X
8,X
9, X
10,X
11, X
12, and X
13 are as described in any of the embodiments provided herein; (x) X5 is lysine; and X
1,X
2,X
3, X
4, X
6, X
7, X
8,X
9, X
10,X
11, X
12, and X
13 are as described in any of the embodiments provided herein; (xi) X
9 is an amino acid selected from M or L; and X
1, X
2, X
3, X
4, X
5, X
6, X
7, X
8, X
10, X
11, X
12, and X
13 are as described in any of the embodiments provided herein; (xii) X
1 is serine; X
3 is L; and X
2, X
4, X
5, X
6, X
7, X
8, X
9, X
10, X
11, X
12, and X
13 are as described herein in any of the embodiments provided herein; or (xiii) X
8 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, W, or Y (preferably A, D, E, F, G, I, L, M, N, Q, R, S, T, W, or Y) or X
8 is an amino acid selected from F, D, E, G, K, N, P, Q, R, S, or T (preferably F, D, E, G, N, Q, R, S, or T) or X
8 is phenylalanine or X
8 is phenylalanine or histidine ; and X
1,X
2,X
3, X
4, X
5, X
6, X
7, X
9, X
10, X
11, X
12, and X
13 are as described in any of the embodiments provided herein. [0071] The de novo proteins of the present invention can comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein: H1 comprises the amino acid sequence: SX
1X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L (SEQ ID NO:4) or X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L (SEQ ID NO:176); wherein: X
1 and X
5 are each independently an amino acid selected from D, E, G, K, N, P, Q, R, S, or T; X
2,X
4, X
6,X
9,X
11,X
12,and X
13, are each independently an amino acid selected from A, F, I, L, M, P or V; X
3,X
7, and X
10,are each independently an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X
8 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, W, or Y (more preferably from F, D, E, G, K, N, P, Q, R, S, or T). De novo proteins of the present invention include those above wherein phenylalanine is included in the list of amino
acids for X
1, asparagine is included in the list of amino acids for X
2, histidine is included in the list of amino acids for X
8, and asparagine is included in the list of amino acids for X
11. De novo proteins of the present invention include those above wherein phenylalanine is included in the list of amino acids for X
1, asparagine is included in the list of amino acids for X
2, asparagine is removed from the list of amino acid substititions for X
3, X
4 is an amino acid selected from I, L, M, P, or V; asparatic acid is removed from the list of amino acid substitions for X
5, X
6 is an amino acid selected from A, L, M, or V; proline is removed from the list of amino acid substitions for X
7, histidine is included in the list of amino acids for X
8 and lysine and proline are removed; proline is removed from the list of amino acids for X
9, asparagine is included in the list of amino acids for X
11 and alanine, isoleucine, proline and valine are removed, and proline is removed from the list of amino acid substitions for X
12 and X
13. [0072] The de novo proteins of the present invention can comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein H1 comprises the amino acid sequence SX
1X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L (SEQ ID NO:4) or X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L (SEQ ID NO:176); wherein: X
1 is an amino acid selected from R or S; X
3 is an amino acid selected from R or L; X
7 is an amino acid selected from A or T; X
10 is an amino acid selected from R or S or L (preferably R or S); X
2, X
4, X
6, X
9, X
11, X
12, and X
13, are each independently an amino acid selected from A, F, I, L, M, P or V; X
5 is an amino acid selected from D, E, G, K, N, P, Q, R, S, or T; and X
8 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, W, or Y ( preferably from F, D, E, G, K, N, P, Q, R, S, or T). De novo proteins of the present invention include those above wherein phenylalanine is included in the list of amino acids for X
1, asparagine is included in the list of amino acids for X
2, histidine is included in the list of amino acids for X
8, and asparagine is included in the list of amino acids for X
11. De novo proteins of the present invention include those above wherein phenylalanine is included in the list of amino acids for X
1, asparagine is included in the list of amino acids
for X
2, asparagine is removed from the list of amino acid substititions for X
3, X
4 is an amino acid selected from I, L, M, P, or V; asparatic acid is removed from the list of amino acid substitions for X
5, X
6 is an amino acid selected from A, L, M, or V; proline is removed from the list of amino acid substitions for X
7, histidine is included in the list of amino acids for X
8 and lysine and proline are removed; proline is removed from the list of amino acids for X
9, asparagine is included in the list of amino acids for X
11 and alanine,isoleucine, proline and valine are removed, and proline is removed from the list of amino acid substitions for X
12 and X
13. [0073] The de novo proteins of the present invention can comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein H1 comprises the amino acid sequence SX
1X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L (SEQ ID NO:4) or X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L (SEQ ID NO:176); wherein: X
1 is an amino acid selected from R or S; X
2 is V; X
3 is an amino acid selected from R or L; X
4 is leucine; X
5 is lysine; X
6 is alanine; X
7 is an amino acid selected from A or T; X
8 is phenylalanine; X
9 is methionine; X
10 is an amino acid selected from R or S or L (preferably R or S); X
11 is phenylalanine; X
12 is A; and X
13 is A. [0074] The de novo proteins of the present invention can comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein H1 comprises the amino acid sequence SX
1X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L (SEQ ID NO:4) or X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L (SEQ ID NO:176); wherein: X
1 is an amino acid selected from R or S; X
2 is valine; X
3 is an amino acid selected from R or L or C or S; X
4 is leucine; X
5 is lysine; X
6 is alanine; X
7 is an amino acid selected from A or T; X
8 is phenylalanine; X
9 is M or L; X
10 is an amino acid selected from R or S or L (preferably R or S); X
11 is phenylalanine; X
12 is alanine; and X
13 is alanine. [0075] The de novo proteins of the present invention can comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein H1 comprises the amino acid sequence SX
1X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L (SEQ ID NO:4) or X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L (SEQ ID NO:176); wherein:
X
1 is an amino acid selected from R or S or F; X
2 is an amino acid selected from N or V; X
3 is an amino acid selected from R or L or C or S; X
4 is leucine; X
5 is lysine; X
6 is alanine; X
7 is an amino acid selected from A or T; X
8 is an amino acid selected from F or H; X
9 is an amino acid selected from M or L; X
10 is an amino acid selected from R or S or L (preferably R or S); X
11 is an amino acid selected from F or N; X
12 is alanine; and X
13 is alanine. [0076] The de novo proteins of the present invention can comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein: H1 comprises the amino acid sequence: SX
1X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L (SEQ ID NO:4) or X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L (SEQ ID NO:176); wherein (i) X
8 is not valine or (ii) X
8 is not an an amino acid selected from V, A, I, L, M, or P or (iii) X
8 is phenylalanine; and X
1, X
2, X
3, X
4, X
5, X
6, X
7, X
9, X
10, X
11, X
12, and X
13 are as described herein in any of the embodiments provided herein. [0077] The de novo proteins of the present invention can comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein H1 comprises the amino acid sequence: SX
1X
2X
3EQX
4X
5TFX
6DKX
7X
8HEX
9EDX
10X
11YQX
12X
13L X
37X
38X
39X
40X
41X
42 X
43X
44X
45X
46 (SEQ ID NO:10); wherein X
1- X
13 are as provided in the any of the embodiments herein for H1; and X
37,X
38, X
41, X
42, X
45, and X
46 are each independently selected from any amino acid. In some aspects, X
1- X
13 are as provided in the any of the embodiments herein for H1; and X
37, X
38, X
41, X
42, X
45, and X
46 are each independently an amino acid selected from A, F, I, L, M, P or V; X
40 and X
44 are each independently an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X
39 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, F, I, L, M, P or V); X
43 is an amino acid selected from D, E, G, K, N, P, Q, R, S, or T. De novo proteins of the present invention include those above wherein isoleucine is removed from the list of amino acids for X
37, proline is removed from the list of amino acids for X
38; X
39 is an amino acid selected from F, I, L, M, V, W, or Y; histidine is included in the list of amino
acids for X
40 and proline is removed, proline is removed from the list of amino acids for X
41; and the amino acids, T and Y are included in the list of amino acids for X
42. In some such aspects X
42 is an amino acid selected from A or V. [0078] Also included in the present invention are those ACE2 protein decoys wherein X
1, X
2, X
3, X
4, X5, X
6, X
7, X
8, X
9, X
10, X
11, X
12, X
13 are as described in any of the embodiments herein and one or more (preferably not more than 1 to 3) of the amino acids at positions 1, 5, 6, 9, 10, 12, 13, 16, 17, 19, 20, 23, 24 and 27 of SEQ ID NO:4 are substituted (or the equivalent positions in SEQ ID NO:176). For example, in some aspects, one or more of the following substitutions are made: S1I; E5D; E5Q; E5V; D12V, D12E; Q24L; and Q24K. In some aspects, one of the following substitutions is made in SEQ ID NO:4: S1I; E5D; E5Q; E5V; D12V, D12E; Q24L; and Q24K (or the equivalent positions in SEQ ID NO:176). In some aspects, one or more of the following substitutions are made in SEQ ID NO:4: T9F, D12I, D12N, E17I, Y23H or the equivalent positions in SEQ ID NO:176). [0079] In some embodiments, de novo proteins of the present invention include those described herein provided that X
4 is not an amino acid selected from D, E, G, H, K, N, P, Q, R, S, W, or Y; X
6 is not an amino acid selected from D, E, F, H, K, P, Q, R, W, or Y; X
7 is not P; X
8 is not proline; X
9 is not an amino acid selected from K, P, or R; X
11 is not an amino acid selected from D E, K, P, R, T or V; X
12 is not an amino acid selected from K, P, or R; X
13 is not an amino acid selected from P or W; X
38 is not an amino acid selected from H, K, P, or R;X
39 is not an amino acid selected from D, E, G, K, or P; and/or X
41 is not proline. [0080] ACE2 protein decoys of the present invention include those wherein H1 comprises an amino acid sequence having at least 70%, 80%, 90%, 95% or 100% identity to an amino acid sequence set forth in SEQ ID NO:11-17, 177-183, 198, or 199: SRVLEQLKTFADKAFHEMEDRFYQAAL (SEQ ID NO:11) SSVREQLKTFADKAFHEMEDRFYQAAL (SEQ ID NO:12)ƒ44 SRVREQLKTFADKTFHEMEDRFYQAAL (SEQ ID NO:13) SRVREQLKTFADKAFHEMEDSFYQAAL (SEQ ID NO:14) SSVLEQLKTFADKAFHEMEDRFYQAAL (SEQ ID NO:15) SSVLEQLKTFADKTFHEMEDSFYQAAL (SEQ ID NO:16) SRVREQLKTFADKAFHEMEDRFYQAAL (SEQ ID NO:17) VLEQLKTFADKAFHEMEDRFYQAAL (SEQ ID NO:177) VREQLKTFADKAFHEMEDRFYQAAL (SEQ ID NO:178)
VREQLKTFADKTFHEMEDRFYQAAL (SEQ ID NO:179) VREQLKTFADKAFHEMEDSFYQAAL (SEQ ID NO:180) VLEQLKTFADKAFHEMEDRFYQAAL (SEQ ID NO:181) VLEQLKTFADKTFHEMEDSFYQAAL (SEQ ID NO:182) VREQLKTFADKAFHEMEDRFYQAAL (SEQ ID NO:183) SSVLEQLKTFADKAFHEMEDLFYQAAL (SEQ ID NO:198) VLEQLKTFADKAFHEMEDLFYQAAL (SEQ ID NO:199). [0081] ACE2 protein decoys of the present invention include those wherein H1 comprises an amino acid sequence having at least 70%, 80%, 90%, 95% or 100% identity to an amino acid sequence set forth in SEQ ID NO:15 (SSVLEQLKTFADKAFHEMEDRFYQAAL) (SEQ ID NO:15) wherein the amino acid at position 1 is S or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein the amino acid at position 2 is S or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein the amino acid at position 3 is V or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein the amino acid at position 4 is L or if substituted is A, C, D, E, F, G, H, I, K, M, P, Q, R, S, T, V, W, or Y wherein the amino acid at position 5 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y wherein the amino acid at position 6 is Q or if substituted is A, C, D, E, F, G, H, I, L, M, N, P, S, T, V, or W; wherein the amino acid at position 7 is L or if substituted is C, I, M, T, or V; wherein the amino acid at position 8 is K or if substituted is A, C, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein the amino acid at position 9 is T or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein the amino acid at position 10 is F or if substituted is A, C, H, V, W, or Y; wherein the amino acid at position 11 is A or if substituted is C, G, L, M, S, T, or V; wherein the amino acid at position 12 is D or if substituted is A, C, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;
wherein the amino acid at position 13 is K or if substituted is A, C, F, H, I, L, M, N, Q, R, S, V, W, or Y; wherein the amino acid at position 14 is A or if substituted is C, D, E, F, G, H, I, K, L, M, N, Q, S, T, or V; wherein the amino acid at position 15 is F or if substituted is A, C, D, E, G, H, I, L, M, N, Q, R, S, T, V, W, or Y; wherein the amino acid at position 16 is H or if substituted is A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein the amino acid at position 17 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein the amino acid at position 18 is M or if substituted is A, C, D, F, G, H, I, L, N, Q, S, T, V, W, or Y; wherein the amino acid at position 19 is E or if substituted is D, M, N, P, Q, T, or V; wherein the amino acid at position 20 is D or if substituted is E, F, G, H, L, N, or Q; wherein the amino acid at position 21 is R or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein the amino acid at position 22 is F or if substituted is C, G, H, L, M, N, W, or Y; wherein the amino acid at position 23 is Y or if substituted is H, D, or F; wherein the amino acid at position 24 is Q or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein the amino acid at position 25 is A or if substituted is C, F, G, H, I, L, M, N, Q, S, T, V, W, or Y; wherein the amino acid at position 26 is A or if substituted is C, D, E, F, G, H, I, L, M, N, Q, S, T, or V; and wherein the amino acid at position 27 is L or if substituted is A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, or Y. Also included are those ACE-2 protein decoys wherein the amino acids at positions 1 and 2 are absent. In some particulary preferred embodiments, no more 3, 2, or 1 of the amino acids at positions 1, 5, 6, 9, 10, 12, 13, 16, 17, 19, 20, 23, 24 and 27 are substituted. [0082] In some aspects, H1 comprises additional amino acids at the C terminuts (e.g., an additional 11 amino acids at the C terminus). In any of the embodiments for H1 herein (including H1 having an amino acid sequence having at least 70%, 80%, 90%, 95% or
100% identity to an amino acid sequence set forth in SEQ ID NO:11-17, 177-183, 198, or 199), the amino acid sequence AVFEAAEAAAG (SEQ ID NO:18), AVWEAAEAAAG (SEQ ID NO:19), AVFEAVEAAAG (SEQ ID NO:249) or AVWEAVEAAAG (SEQ ID NO:250) can be optionally present at the C terminus. [0083] Accordingly, in some aspects, H1 comprises a sequence having at least 70%, 80%, 90%, 95% or 100% identity to the sequence set forth in SEQ ID NOS.240-243 or 251-254: SSVLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG (SEQ ID NO:240), VLEQLKTFADKAFHEMEDRFYQAALAVFEAAEAAAG (SEQ ID NO:241); SSVLEQLKTFADKAFHEMEDLFYQAALAVFEAAEAAAG (SEQ ID NO:242); VLEQLKTFADKAFHEMEDLFYQAALAVFEAAEAAAG (SEQ ID NO:243); SSVLEQLKTFADKAFHEMEDRFYQAALAVFEAVEAAAG (SEQ ID NO:251), VLEQLKTFADKAFHEMEDRFYQAALAVFEAVEAAAG (SEQ ID NO:252); SSVLEQLKTFADKAFHEMEDLFYQAALAVFEAVEAAAG (SEQ ID NO:253); or VLEQLKTFADKAFHEMEDLFYQAALAVFEAVEAAAG (SEQ ID NO:254). ALPHA HELICAL DOMAIN H2 [0084] The de novo proteins of the present invention can comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein H2 comprises the amino acid sequence: NX
14X
15NX
16X
17X
18KX
19X
20X
21FX
22X
23EQX
24X
25LX
26X
27MY (SEQ ID NO:5) wherein X
14, X
15, X
16, X
17, X
18, X
19, X
20, X
21, X
22, X
23, X
24, X
25, X
26, X
27 are each independently selected from any amino acid. [0085] Included in the present invention are de novo proteins of the present invention wherein H2 comprises the amino acid sequence: NX
14X
15NX
16X
17X
18KX
19X
20X
21FX
22X
23EQX
24X
25LX
26X
27MY (SEQ ID NO:5) wherein X
14 is an amino acid selected from A, C, D, G, H, I, L, M, N, P, R, S, T, V, or W (preferably A, C, G, P, T, or V); wherein X
15 is an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, C, D, E, F, I, K, L, M, N, Q, R, S, T, or V); wherein X
16 is an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, C, E, H, I, L, M, N, Q, R, S, V, W, or Y); wherein X
17 is an amino acid selected from A, C, D, E, G, I, L, M, N, P, Q, S, T, V, W, or Y (preferably A, C, G, I, or S);
wherein X
18 is an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y, or W (preferably A, C, F, G, H, I, K, L, M, N, Q, R, S, T, V, or Y); wherein X
19 is an amino acid selected from from A, C, D, G, I, L, Q, S, T, V, or W (preferably A, L, T, or V); wherein X
20 is an amino acid selected from from A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y (preferably A, C, D, E, F, G, H, I, K, Q, R, S, T, V, W, or Y); wherein X
21 is an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, D, E, F, G, H, I, K, L, M, N, R, S, T, V, or W); wherein X
22 is an amino acid selected from A, C, D, F, G, I, L, M, N, S, T, V, W, or Y (preferably A, C, F, G, I, L, S, or T); wherein X
23 is an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y (preferably A, C, D, E, G, H, I, L, M, N, R, S, T, V, or Y); wherein X
24 is an amino acid selected from A, C, E, F, G, I, L, M, Q, S, T, V, or W (preferably A, I, or S) wherein X
25 is an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y (preferably K, D, G, H, I, Q, or R); wherein X
26 is an amino acid selected from A, C, F, G, I, L, S, T, V, or Y (preferably A, or I); wherein X
27 is an amino acid selected from A, C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, or Y (preferably D, C, F, I, S, or T). [0086] Included in the present invention are de novo proteins of the present invention wherein H2 comprises the amino acid sequence: NX
14X
15NX
16X
17X
18KX
19X
20X
21FX
22X
23EQX
24X
25LX
26X
27MY (SEQ ID NO:5) wherein X
15, X
18, X
21, X
23, X
25, and X
27 are each independently an amino acid selected from D, E, G, K, N, P, Q, R, S, or T; X
14, X
16, X
17, X
19, X
22, X
24, and X
26 are each independently an amino acid selected from A, F, I, L, M, P or V; and X
20 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. De novo proteins include those above wherein phenylalanine is removed from the list of amino acids for X
14 and X
17, tyrosine is included in the list of amino acids for X
18, glutamic acid and proline are removed from the list of amino acids for X
19, proline is removed from the list of amino acids for X
20, X
22, X
23, X
24 and X
25, methionine is
included in the list of amino acids for X
21, histidine is included in the list of amino acids for X
23,methionine and proline are removed from the list of amino acids for X
26, and isoleucine is included in the list of amino acids for X
27 and lysine and proline are removed. [0087] Included in the present invention are de novo proteins of the present invention wherein H2 comprises the amino acid sequence: NX
14X
15NX
16X
17X
18KX
19X
20X
21FX
22X
23EQX
24X
25LX
26X
27MY (SEQ ID NO:5) wherein X
15, X
18, X
21, X
23, X
25, and X
27 are each independently an amino acid selected from D, E, G, K, N, P, Q, R, S, or T; X
14, X
17, X
22, X
24, and X
26 are each independently an amino acid selected from A, F, I, L, M, P or V; X
16 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X
19 is an amino acid selected from A, F, I, L, M, P, E, T, or V; and X
20 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. De novo proteins include those above wherein phenylalanine is removed from the list of amino acids for X
14 and X
17, tyrosine is included in the list of amino acids for X
18, glutamic acid and proline are removed from the list of amino acids for X
19, proline is removed from the list of amino acids for X
20, X
22, X
23, X
24 and X
25, methionine is included in the list of amino acids for X
21, histidine is included in the list of amino acids for X
23,methionine and proline are removed from the list of amino acids for X
26, and isoleucine is included in the list of amino acids for X
27 and lysine and proline are removed. [0088] Included in the present invention are de novo proteins of the present invention wherein H2 comprises the amino acid sequence: NX
14X
15NX
16X
17X
18KX
19X
20X
21FX
22X
23EQX
24X
25LX
26X
27MY (SEQ ID NO:5) wherein X
15, X
18, X
21, X
23, X
25, and X
27 are each independently an amino acid selected from D, E, G, K, N, P, Q, R, S, or T; X
14, X
17, X
19, X
22, X
24, and X
26 are each independently an amino acid selected from A, F, I, L, M, P or V; X
16 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably from A, F, I, L, M, P, E, or V), and X
20 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. De novo proteins include those above wherein phenylalanine is removed from the list of
amino acids for X
14 and X
17, tyrosine is included in the list of amino acids for X
18, glutamic acid and proline are removed from the list of amino acids for X
19, proline is removed from the list of amino acids for X
20, X
22, X
23, X
24 and X
25, methionine is included in the list of amino acids for X
21, histidine is included in the list of amino acids for X
23,methionine and proline are removed from the list of amino acids for X
26, and isoleucine is included in the list of amino acids for X
27 and lysine and proline are removed.. [0089] The de novo proteins of the present invention can comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein H2 comprises the amino acid sequence: NX
14X
15NX
16X
17X
18KX
19X
20X
21FX
22X
23EQX
24X
25LX
26X
27MY (SEQ ID NO:5) wherein X
14 is A or V X
16 is A or E (or X
16 is A or E or N) X
20 is K or Q X
15, X
18, X
21, X
23, X
25, and X
27 are each independently an amino acid selected from D, E, G, K, N, P, Q, R, S, or T; and X
17, X
19, X
22, X
24, and X
26 are each independently an amino acid selected from A, F, I, L, M, P or V. De novo proteins of the present invention include those above wherein for X
17, tyrosine is included in the list of amino acids for X
18, proline is removed from the list of amino acids for X
19, proline is removed from the list of amino acids for X
22, X
23, X
24 and X
25, methionine is included in the list of amino acids for X
21, histidine is included in the list of amino acids for X
23,methionine and proline are removed from the list of amino acids for X
26, andisoleucine is included in the list of amino acids for X
27 and lysine and proline are removed. [0090] The de novo proteins of the present invention can comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein H2 comprises the amino acid sequence: NX
14X
15NX
16X
17X
18KX
19X
20X
21FX
22X
23EQX
24X
25LX
26X
27MY (SEQ ID NO:5) wherein: X
14 is A or V; X
15 is E; X
16 is A or E; X
17 is A; X
18 is R; X
19 is A; X
20 is K or Q;
X
21 is E; X
22 is A; X
23 is E X
24 is A; X
25 is K; X
26 is A; and X
27 is D. [0091] The de novo proteins of the present invention can comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein H2 comprises the amino acid sequence: NX
14X
15NX
16X
17X
18KX
19X
20X
21FX
22X
23EQX
24X
25LX
26X
27MY (SEQ ID NO:5) wherein X
14, X
17, X
19, X
22, X
24, and X
26 are each independently an amino acid selected from A, F, I, L, M, P or V; X
15 E; X
16 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably from A, F, I, L, M, P, E, or V); X
18 is R X
20 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X
21 is E; X
23 is E; X
25 is K; and X
27 is D. De novo proteins of the present invention include those above wherein phenylalanine is removed from the list of amino acids for X
14 and X
17, proline is removed from the list of amino acids for X
20, X
22, and X
24, and methionine and proline are removed from the list of amino acids for X
26. [0092] In some such aspects, X
14 is an amino acid selected from A or V; X
16 is an amino acid selected from A or E; and X
20 is an amino acid selected from K or Q. [0093] The de novo proteins of the present invention can comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein H2 comprises the amino acid sequence: NX
14X
15NX
16X
17X
18KX
19X
20X
21FX
22X
23EQX
24X
25LX
26X
27MY (SEQ ID NO:5) wherein X
14 is an amino acid selected from A or V X
16 is an amino acid selected from A or E X
20 is an amino acid selected from K or Q
X
15, X
17, X
18, X
19, X
21, X
22, X
23, X
24, X
26 X
25, X
26 and X
27 can be as provided in any of the embodiments herein. [0094] The de novo proteins of the present invention can comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein H2 comprises the amino acid sequence: NX
14X
15NX
16X
17X
18KX
19X
20X
21FX
22X
23EQX
24X
25LX
26X
27MY (SEQ ID NO:5) wherein (i) X
15 is E and X
14, X
16, X
17, X
18, X
19, X
20, X
21, X
22, X
23, X
24, X
25, X
26, and X
27 are as provided in the any of the embodiments herein for H2; (ii) X
18 is R and X
14, X
15, X
16, X
17, X
19, X
20, X
21, X
22, X
23, X
24, X
25, X
26, and X
27 are as provided in the any of the embodiments herein for H2; (iii) X
21 is E and X
14, X
15, X
16, X
17, X
18, X
19, X
20, X
22, X
23, X
24, X
25, X
26, and X
27 are as provided in the any of the embodiments herein for H2; (iv) X
23 is E and X
14, X
15, X
16, X
17, X
18, X
19, X
20, X
21, X
22, X
24, X
25, X
26, and X
27 are as provided in the any of the embodiments herein for H2; (v) X
25 is K and X
14, X
15, X
16, X
17, X
18, X
19, X
20, X
21, X
22, X
23, X
24, X
26, and X
27 are as provided in the any of the embodiments herein for H2; (vi) X
26 is A and X
14, X
15, X
16, X
17, X
18, X
19, X
20, X
21, X
22, X
23, X
24, X
25, and X
27, are as provided in the any of the embodiments herein for H2; (vii) X
27 is D and X
14, X
15, X
16, X
17, X
18, X
19, X
20, X
21, X
22, X
23, X
24, X
25, and X
26, are as provided in the any of the embodiments herein for H2; (viii) X
16 is A and X
14, X
15, X
17, X
18, X
19, X
20, X
21, X
22, X
23, X
24, X
25, X
26, and X
27 are as provided in the any of the embodiments herein for H2; or (ix) at least one, two, three, four, five or six of the following are true: X
15 is E; X
18 is R; X
21 is E; X
23 is E; X
25 is K; and X
27 is D. [0095] The de novo proteins of the present invention can comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein H2 comprises the amino acid sequence: X
47X
48X
49X50X51NX
14X
15NX
16X
17X
18KX
19X
20X
21FX
22X
23EQX
24X
25LX
26X
27MYX
52 X53X54X
55X56 (SEQ ID NO:20) wherein X
14- X
27 are as provided in the any of the embodiments herein for H2; and X
47, X
48, X
49, X
50, X
51, X
52, X
53, X
54, X
55, and X
56 are are each independently selected from any amino
acid. In some aspects, X
14- X
27 are as provided in the any of the embodiments herein for H2; and X
49, X
52, and X
55 are each independently an amino acid selected from A, F, I, L, M, P or V; X50 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X
54 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, F, I, L, M, P or V); and X
47, X
48, X
51, X
53, and X
56 are each independently an amino acid selected from D, E, G, K, N, P, Q, R, S, or T. De novo proteins of the present invention include those above wherein cysteine and glutamine are included in the list of amino acids for X
47 and lysine is removed, phenylalanine and tyrosine are included in the list of amino acids for X
48, histidine is included in the list of amino acids for X
52 and X
54, and aspartic acid is removed from the list of amino acids for X
56. [0096] Included in the present invention are ACE2 protein decoys wherein X
14- X
27 are as provided in the any of the embodiments herein for H2; X
49, X
52, and X
55 are each independently an amino acid selected from A, F, I, L, M, P or V; X
50 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X
54 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, F, I, L, M, P or V); and X
48, X
51, X
53, and X
56 are each independently an amino acid selected from D, E, G, K, N, P, Q, R, S, or T; and X
47 is G. De novo proteins of the present invention include those above wherein cysteine and glutamine are included in the list of amino acids for X
47 and lysine is removed, phenylalanine and tyrosine are included in the list of amino acids for X
48, histidine is included in the list of amino acids for X
52 and X
54, and aspartic acid is removed from the list of amino acids for X
56. [0097] Included in the present invention are ACE2 protein decoys wherein wherein X
14- X
27 are as provided in the any of the embodiments herein for H2; X
49, X
52, and X
55 are each independently an amino acid selected from A, F, I, L, M, P or V; X50 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
X
54 is L; and X
48, X
51, X
53, and X
56 are each independently an amino acid selected from D, E, G, K, N, P, Q, R, S, or T; and X
47 is G. De novo proteins of the present invention include those above wherein phenylalanine and tyrosine are included in the list of amino acids for X
48, histidine is included in the list of amino acids for X
52, and aspartic acid is removed from the list of amino acids for X
56. [0098] In some aspects, X
47 is G or E; X
48 is D; X
49 is A; X
50 is A; and X
51 is R. In some such aspects, X
47 is G. In other such aspects, X
47 is E. [0099] In some aspects, X
52 is A; X53 is E; X54 is L or F or N; X
55 is A; and X56 is K. In some such aspects, X54 is L. In other such aspects, X54 is F. In yet other aspects, X54 is N. [00100] Also included in the present invention are those ACE2 protein decoys wherein one or more (preferably not more than 1 to 3) of the amino acids at positions 1, 4, 8, 12, 15, 16, 19, 22 and 23 of SEQ ID NO:5 are substituted and X
14, X
15, X
16, X
17, X
18, X
19, X
20, X
21, X
22, X
23, X
24, X
25, X
26, X
27 are as described in any of the embodiments provided herein. For example, in some aspects, the following substitution is made E15G. In some aspects, one or more of the following substitions are made: Q16N, Q16Y, L19Y, or M22H. [00101] De novo proteins of the present invention include those wherein X
4 is not an amino acid selected from K or R; X
22 is not an amino acid selected from E, K, Q, or R; X
23 is not an amino acid selected from P; X
24 is not an amino acid selected from D or P; X
26 is not an amino acid selected from D, E, H, K, P, Q, or R; X
9 is not K, P, or R; and X
27 is not P. [00102] De novo proteins of the present invention include those wherein H2 comprises an amino acid sequence having at least 70%, 80%, 90%, 95% or 100% identity to an amino acid sequence set forth in: NAENAARKAKEFAEEQAKLADMY (SEQ ID NO:21) NVENEARKAQEFAEEQAKLADMY (SEQ ID NO:22) [00103] ACE2 protein decoys of the present invention include those wherein H2 comprises an amino acid sequence having at least 70%, 80%, 90%, 95% or 100% identity to an amino acid sequence set forth in SEQ ID NO:21 wherein the amino acid at position 1 is N or if substituted is A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein the amino acid at position 2 is A or if substituted is C, D, G, H, I, L, M, N, P, R, S, T, V, or W; wherein the amino acid at position 3 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
wherein the amino acid at position 4 is N or if substituted is A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y wherein the amino acid at position 5 is A or if substituted is C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y wherein the amino acid at position 6 is A or if substituted is C, D, E, G, I, L, M, N, P, Q, S, T, V, W, or Y; wherein the amino acid at position 7 is R or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, Y, or W; wherein the amino acid at position 8 is K or if substituted is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein the amino acid at position 9 is A or if substituted is C, D, G, I, L, Q, S, T, V, or W; wherein the amino acid at position 10 is K or if substituted is A, C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, or W; wherein the amino acid at position 11 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein the amino acid at position 12 is F or if substituted is A, C, D, E, G, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein the amino acid at position 13 is A or if substituted is C, D, F, G, I, L, M, N, S, T, V, W, or Y; wherein the amino acid at position 14 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein the amino acid at position 15 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein the amino acid at position 16 is Q or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, R, S, T, Y, or V; wherein the amino acid at position 17 is A or if substituted is C, E, F, G, I, L, M, Q, S, T, V, or W; wherein the amino acid at position 18 is K or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein the amino acid at position 19 is L or if substituted is A, C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, Y, or W; wherein the amino acid at position 20 is A or if substituted is C, F, G, I, L, S, T, V, or Y;
wherein the amino acid at position 21 is D or if substituted is A, C, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, or Y; wherein the amino acid at position 22 is M or if substituted is A, C, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W, or Y; and wherein the amino acid at position 23 is Y or if substituted is D, F, H, I, L, M, or V. [00104] Also included are those ACE-2 protein decoys wherein no more 3, 2, or 1 of the amino acids at positions 1, 4, 8, 12, 15, 16, 19, 22 and 23 are substituted. [00105] In some aspects H2 comprises additional amino acids at the N terminus (e.g., at least 5 additional amino acids at the N terminus). In some aspects, the amino acid sequence EDAAR (SEQ ID NO:23) or GDAAR (SEQ ID NO:24) is present at the N terminus. Accordingly, in some aspects H1 comprises a sequence having at least 70%, 80%, 90%, 95% or 100% identity to the sequence GDAAR NAENAARKAKEFAEEQAKLADMY AELAK (SEQ ID NO:244). [00106] In some aspects H2 comprises additional amino acids at the C terminus (e.g., at least 5 additional amino acids at the C terminus). In some aspects, the amino acid sequence AELAK (SEQ ID NO:25) or AEFAK (SEQ ID NO:26) or AENAK (SEQ ID NO:27) is present at the C terminus. [00107] In some aspects, the amino acid sequence AELAK (SEQ ID NO:25) is present at the C terminus and the amino acid sequence GDAAR (SEQ ID NO:24) is present at the N terminus. BETA DOMAIN H3 [00108] The de novo proteins of the present invention can comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein H3, if present, comprises the amino acid sequence: X
28X
29X
30X
31X
32X
33KGDX
34RX
35X
36 (SEQ ID NO:6); wherein X
28X
29X
30X
31X
32X
33 , X
34, X
35, and X
36, are each independently selected from any amino acid. [00109] Included in the present invention are de novo proteins of the present invention wherein H3 comprises the amino acid sequence: X
28X
29X
30X
31X
32X
33KGDX
34RX
35X
36 (SEQ ID NO:6); wherein wherein:
X
28 is an amino acid selected from A, C, D, E, G, I, L, M, P, Q, R, S, T, V, or W (preferably A, G, I, L, M, Q, T, V, or W); X
29 is an amino acid selected from A, C, D, E, G, L, M, P, R, S, T, V, or W (preferably E, L, M, P, S, T, V, or W); X
30 is an amino acid selected from C, F, I, L, M, T, V, or W (preferably I, F, or V); X
31 is an amino acid selected from A, C, D, E, G, I, K, L, M, N, S, T, or V (preferably D, M, or N); X
32 is an amino acid selected from F, I, L, M, or V (preferably L, M, or F); X
33 is an amino acid selected from D, G, or L; X
34 is an amino acid selected from A, C, E, F, G, I, K, L, Q, R, S, T, V, W, or Y (preferably F, A, E, I, K, L, Q, R, S, or V); X
35 is an amino acid selected from A, C, D, E, G, H, K, L, M, P, R, S, T, V, W, or Y (preferably E, D, G, L, M, P, S, V, or W); and X
36 is an amino acid selected from A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, or W (preferably I, C, F, M, P, Q, S, T, V, or W). [00110] Included in the present invention are de novo proteins of the present invention wherein H3, if present, comprises the amino acid sequence: X
28X
29X
30X
31X
32X
33KGDX
34RX
35X
36 (SEQ ID NO:6); wherein X
31 is an amino acid selected from D, E, G, K, N, P, Q, R, S, or T; X
35 is an amino acid selected from D, E, G, K, N, P, Q, R, S, V, or T X
29 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably D, E, G, K, N, P, Q, R, S, T, or V) X
33 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably G, A, F, I, L, M, P or V ); X
30, X
32, and X
36 are each independently an amino acid selected from A, F, I, L, M, P or V; X
28 is an amino acid selected from A, F, I, L, M, P, T, or V; and X
34 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y and optionally C (preferably F, D, E, G, K, N, P, Q, R, S, Y, or T). De novo proteins of the present invention include those above wherein glutamine is included in the list of amino
acids for X
28, alanine and proline are removed from the list of amino acids for X
30, proline, glutamine, and arginine are removed from the list of amino acids for X
31, alanine and proline are removed from the list of amino acids for X
32, X
33 is an amino acid selected from D, G, or L; and aspartic acid and proline are removed from the list of amino acids for X
34. [00111] Included in the present invention are de novo proteins of the present invention wherein H3, if present, comprises the amino acid sequence: X
28X
29X
30X
31X
32X
33KGDX
34RX
35X
36 (SEQ ID NO:6); wherein X
31 and X
35 are each independently an amino acid selected from D, E, G, K, N, P, Q, R, S, or T; X
29 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably D, E, G, K, N, P, Q, R, S, T, or V) X
33 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably G, A, F, I, L, M, P or V ); X
28, X
30, X
32, and X
36 are each independently an amino acid selected from A, F, I, L, M, P or V; and X
34 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y and optionally C (preferably F, D, E, G, K, N, P, Q, R, S, Y, or T). De novo proteins of the present invention include those above wherein glutamine is included in the list of amino acids for X
28, alanine and proline are removed from the list of amino acids for X
30, proline, glutamine, and arginine are removed from the list of amino acids for X
31, alanine and proline are removed from the list of amino acids for X
32, X
33 is an amino acid selected from D, G, or L; and aspartic acid and proline are removed from the list of amino acids for X
34. [00112] The de novo proteins of the present invention can comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein H3, if present, comprises the amino acid sequence: X
28X
29X
30X
31X
32X
33KGDX
34RX
35X
36 (SEQ ID NO:6); wherein X
35 is an amino acid selected from D, E, G, K, N, P, Q, R, S, or T; X
28 is A or V; X
29 is E or V;
X
31 is D X
32 is M or L; X
33 is G; X
30, and X
36 are each independently an amino acid selected from A, F, I, L, M, P or V; and X
34 is an amino acid selected from F, Y, K or C. De novo proteins of the present invention include those above wherein alanine and proline are removed from the list of amino acids for X
30. [00113] The de novo proteins of the present invention can comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein H3, if present, comprises the amino acid sequence: X
28X
29X
30X
31X
32X
33KGDX
34RX
35X
36 (SEQ ID NO:6); wherein X
28 is A or V; X
29 is E or V; X
30 is I; X
31 is D X
32 is M or L; X
33 is G; X
34 is an amino acid selected from F, Y, K or C; X
35 is E; and X
36 is I. [00114] The de novo proteins of the present invention can comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein H3, if present, comprises the amino acid sequence: X
28X
29X
30X
31X
32X
33KGDX
34RX
35X
36 (SEQ ID NO:6); wherein X
28 is A or V or T; X
29 is E or V; X
30 is I; X
31 is D X
32 is M or L or I;
X
33 is G; X
34 is an amino acid selected from F, Y, K, I, or C; X
35 is E or V; and X
36 is I. [00115] The de novo proteins of the present invention can comprise two alpha helical domains, H1 and H2, and an optional beta hairpin domain H3, wherein H3, if present, comprises the amino acid sequence: X
57X
28X
29X
30X
31X
32X
33KGDX
34RX
35X
36X
58 ; (SEQ ID NO:28) wherein X
28-X
36 are as provided in the any of the embodiments herein for H3; X
57 is an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X
58 is an amino acid selected from D, E, G, K, N, P, Q, R, S, C or T. De novo proteins of the present invention include those above wherein cysteine is included in the list of amino acids for X
28 and the amino aicds D, K, M, N, P, Q, and Y are removed from the list of amino acids for X
28, and the amino acids A, C, F, H, I, L, M, V, or W are included in the list of amino acids for X
58.Also included in the present invention are those ACE2 protein decoys wherein X
28, X
29, X
30, X
31, X
32, X
33, X
34, X
35, and X
36, are as described in any of the embodiments herein and the amino acids at position 7 and/or position 8 of SEQ ID NO:6 are substituted. For example, in some aspects, one or more of the following substitutions are made: K7M or G8R. [00116] Proteins of the present invention include those wherein H3 comprises an amino acid sequence having at least 70%, 80%, 90%, 95% or 100% identity to an amino acid sequence set forth in SEQ ID NOS: 29-34 or 200. AEIDLGKGDFREI (SEQ ID NO:29) AEIDLGKGDCREI (SEQ ID NO:30) VVIDLGKGDFREI (SEQ ID NO:31) VVIDLGKGDCREI (SEQ ID NO:32) AEIDMGKGDCREI (SEQ ID NO:33) AEIDMGKGDFREI (SEQ ID NO:34) VEIDLGKGDFREI (SEQ ID NO: 200).
[00117] ACE2 protein decoys of the present invention include those wherein H3 comprises an amino acid sequence having at least 70%, 80%, 90%, 95% or 100% identity to an amino acid sequence set forth in SEQ ID NO:29 wherein the amino acid at position 1 is A or if susbstituted is C, D, E, G, I, L, M, P, Q, R, S, T, V, or W; wherein the amino acid at position 2 is E or if susbstituted is A, C, D, G, L, M, P, R, S, T, V, or W; wherein the amino acid at position 3 is I or if substituted is C, F, L, M, T, V, or W; wherein the amino acid at position 4 is D or if substituted is A, C, E, G, I, K, L, M, N, S, T, or V; wherein the amino acid at position 5 is L or if substituted is F, I, M, or V; wherein the amino acid at position 6 is G or if substituted is D, or L; wherein the amino acid at position 7 is K or if substituted is I, M, N, Q, R, or T; wherein the amino acid at position 8 is G or if substituted is D, E, M, R, or S; wherein the amino acid at position 9 is D or if substituted is E, K, or T; wherein the amino acid at position 10 is F or if substituted is A, C, E, G, I, K, L, Q, R, S, T, V, W, or Y; wherein the amino acid at position 11 is R or if substituted is K, M, Q, or S; wherein the amino acid at position 12 is E or if substituted is A, C, D, G, H, K, L, M, P, R, S, T, V, W, or Y; and wherein the amino acid at position 13 is I or if substituted is A, C, D, F, G, H, L, M, N, P, Q, R, S, T, V, or W. Also included are those ACE-2 protein decoys wherein no more 3, 2, or 1 of the amino acids at positions 7, 8, 9, and 11 are substituted, wherein numbering is in accordance with SEQ ID NO: 29. [00118] Also included in the present invention are ACE2 protein decoys comprising a H3 domain wherein the amino acid at position X
34 is not cysteine. Also included in the present invention are ACE2 protein decoys comprising a H3 domain wherein if X
34 is cysteine, X
32 is leucine. Also included in the present invention are ACE2 protein decoys including a H3 domain wherein the amino acid at position X
34 is selected from D, E, G, K, N, P, Q, R, S, or T. Also included in the present invention ar ACE2 protein decoys including a H3 domain wherein the amino acid at position X
34 is an amino acid selected from F, Y, K or C.
[00119] In some aspects, H3 comprises at least one additional amino acid at the N terminus, preferably an amino acid that doesn’t negatively impact binding to the coronavirus spike protein. In some aspects, the amino acid is selected from D, E, G, K, N, P, Q, R, S, Y, or T. In some aspects, the amino acid is selected from A, C, E, F, G, I, L, R, S, T, V, or W. In other aspects, the amino acid is selected from S, P, T, or Y or from L, S, P, T, or Y. In some aspects, the amino acid is S or P (preferably S). Accordingly, in some aspects H3 comprises a sequence having at least 70%, 80%, 90%, 95% or 100% identity to the sequence SAEIDLGKGDFREIR (SEQ ID NO: 245) or SVEIDLGKGDFREIR (SEQ ID NO:246) [00120] In some aspects H3 comprises at least one additional amino acid at the C terminus, preferably an amino acid that doesn’t negatively impact binding to the coronavirus spike protein. In some aspects, the amino acid is selected from L, D, E, G, K, N, P, Q, R, S, or T or A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V or W . In some aspects, the amino acid is R. [00121] It will be understood by the skilled practitioner than any of the H1, H2, and optional H3 domains described herein can be combined for use in the present invention. [00122] De novo proteins of the present invention comprise at least one structural domain that facilitates protein folding and binding-competent presentation of the alpha helices and beta hairpin domains to the coronavirus spike protein. Preferred structural domains provide the de novo proteins with a hydrophobic core and serve to stabilize the relative position and orientation of the binding motifs in a manner that is competent for binding. The supporting structures can be computationally generated and placed by an available method (e.g. Rosetta fragment assembly, parametric generation, and the like) or extracted from existing structures (see, e.g., examples herein). Unlike the H1, H2, and H3 regions, these structural domains do not substantially map to the structural domains of the ACE2 protein (i.e., do not structurally or sequentially align to other secondary structure elements in ACE2). Using the teachings of the present invention, in addition to the skill in the art, a skilled practitioner could use protein design principles to create structural domains for use in the present invention. [00123] Structural domains that facilitate protein folding and binding-competent presentation of H1, H2, and H3, if present, can comprise an amino acid sequence set forth below for D1 and D2:
D1 – X
AX
AX
BX
BX
CX
BX
BX
BX
AX
BX
BX
AX
CX
BX
CX
AX
BX
CX
AX
CX
CX
AX
AX
BX
CX
AX
A (SEQ ID NO:35). D2 – XAXCXCXAXBXBXAXCXBXBXAXBXBXAXCXBXBXAXBXBXDXAXBXBXAXCXBXBXA X
C (SEQ ID NO:36) wherein each X
A is independently an amino acid selected from D, E, G, K, N, P, Q, R, S, C, and T; each X
B is independently an amino acid selected from A, F, I, L, M, C, and P (preferably A, F, I, L, M, and P), each X
C is independently an amino acid selected from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, Y or C; and each X
D is independently an amino acid from A, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, Y or C. In some embodiments X
D is an amino acid selected from A, F, I, L, M, P, or V. In other embodiments X
D is an amino acid selected from D, E, G, K, N, P, Q, R, S, T, or C. [00124] In some embodiments, D1 comprises the amino acid sequence set forth below: X
AX
AAAX
AALAX
AA AX
AAMKX
AALX
AI IX
AX
AIAX
AX
A (SEQ ID NO:37)
; wherein each X
A is independently an amino acid selected from D, E, G, K, N, P, Q, R, S, C, or T. [00125] In some embodiments, D1 comprises an amino acid sequence at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence set forth below: REAAEALAEAARAMKEALEIIREIAEK (SEQ ID NO:38) REAAEALAEAARAMKEALEILREIAEK (SEQ ID NO:222). [00126] ACE2 protein decoys of the present invention include those wherein at least one structural domain (e.g., D1) comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% identity to an amino acid sequence set forth in SEQ ID NO:38 wherein the amino acid at position 1 is R or if substituted is A, C, E, F, G, I, K, L, M, P, S, T, V, or W (preferably C, E, F, G, K, L, M, P, S, T, or W ); wherein the amino acid at position 2 is E or if substituted is A, C, D, F, G, I, K, L, M, P, Q, R, S, T, V, W, or Y (preferably A, G, K, M, V, W, or Y); wherein the amino acid at position 3 is A or if substituted is C, E, G, K, L, M, P, Q, R, S, T, V, W, or Y (preferably C, K, P, Q, or V); wherein the amino acid at position 4 is A or if substituted is D, E, G, I, K, L, M, N, P, R, S, T, V, or W (preferably E, N, T, V, or W) ;
wherein the amino acid at position 5 is E or if substituted is C, D, G, K, L, Q, R, S, T, V, W, or Y (preferably C, D, Q, S, V, W, or Y); wherein the amino acid at position 6 is A or if substituted is C, D, E, G, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably D, K, P, R, V, or Y); wherein the amino acid at position 7 is L or if substituted is A, C, F, I, M, Q, S, T, or V (preferably T); wherein the amino acid at position 8 is A or if substituted is C, D, E, F, G, H, I, K, Q, L, M, R, S, T, V, or W (preferably D, E, G, H, I, L, Q, R, S, V, or W); wherein the amino acid at position 9 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, P, R, S, V, W, or Y (preferably A, D, H, L, M, N, R, S, or V); wherein the amino acid at position 10 is A or if substituted is C, G, L, M, Q, S, T, V, or W (preferably C, G, M, or S); wherein the amino acid at position 11 is A or if substituted is C, D, G, L, M, N, Q, R, S, T, or V (preferably C, G, M, S, T, or V); wherein the amino acid at position 12 is R or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y (preferably A, C, D, E, F, G, H, I, K, M, N, P, Q, S, T, V, W, or Y); wherein the amino acid at position 13 is A or if substituted is C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y (preferably D, E, K, M, R, S, or V); wherein the amino acid at position 14 is M or if substituted is A, C, D, E, G, H, I, K, L, Q, R, S, T, V, W, or Y (preferably A, C, D, E, G, K, R, S, T, V, or Y ); wherein the amino acid at position 15 is K or if substituted is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y (preferably E, G, H, M, R, S, or Y); wherein the amino acid at position 16 is E or if substituted is A, C, D, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y (preferably A, C, D, G, K, L, M, Q, T, V, or W); wherein the amino acid at position 17 is A or if substituted is C, G, P, S, T, or V (preferably C, G, or T); wherein the amino acid at position 18 is L or if substituted is C, F, H, I, K, M, N, Q, R, T, V, W, or Y (preferably C, F, H, or V); wherein the amino acid at position 19 is E or if substituted is A, C, D, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y (preferably A, C, F, G, L, Q, R, S, T, V, or Y);
wherein the amino acid at position 20 is I or if substituted is A, C, D, E, F, G, H, K, L, M, N, Q, R, S, T, V, W, or Y (preferably C, E, G, L, Q, R, S, T, V, or Y); wherein the amino acid at position 21 is I or if substituted is A, C, D, E, F, G, K, L, M, N, Q, S, T, V, W, or Y (preferably A, C, D, F, L, M, N, S, T, V, or Y); wherein the amino acid at position 22 is R or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, Q, S, T, V, Y, or W (preferably A, C, D, E, F, G, I, L, M, Q, S, T, V, or Y); wherein the amino acid at position 23 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, C, D, F, H, I, M, N, P, T, or W); wherein the amino acid at position 24 is I or if substituted is A, C, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y (preferably C, S, T, or V); wherein the amino acid at position 25 is A or if substituted is C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably D, G, H, I, M, N, S, V, or Y); wherein the amino acid at position 26 is E or if substituted is A, C, D, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y (preferably C, F, I, L, S, T, or Y); and wherein the amino acid at position 27 is K or if substituted is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y (preferably C, D, E, F, G, H, M, N, S, or Y). [00127] In some embodiments D2 comprises an amino acid sequence set forth in SEQ ID NO: 39 or 40: XAAXAXAAAXAXA IAXAAIXAXAAAXA AIAXAAAXAIAA XAA (SEQ ID NO:39) XAAXAXAAAXAXA VAXAAIXAXAAAXA AIVXAAAXAIAA XAA (SEQ ID NO:40); wherein each X
A is independently an amino acid selected from D, E, G, K, N, P, Q, R, S, C, or T. [00128] In some embodiments D2 comprises the amino acid sequence set forth below: RASEAAKRX
59AX
60AIRKAAD AIX
61X
62AAKIAA RA (SEQ ID NO:41), wherein X
59 is I or V, X
60 is K or R or C, X
61 is A or V or C, X
62 is E or C. [00129] In some embodiments D2 comprises an amino acid sequence at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% identical to an amino acid sequence set forth in SEQ ID NO:42-46: RASEAAKR IAKAIRKAAD AIAEAAKIAA RA (SEQ ID NO:42); RASEAAKR IACAIRKAAD AIAEAAKIAA RA (SEQ ID NO:43); RASEAAKR IAKAIRKAAD AIACAAKIAA RA (SEQ ID NO:44); RASEAAKR VARAIRKAAD AIVEAAKIAA RA (SEQ ID NO:45);
RASEAAKR VACAIRKAAD AIVEAAKIAA RA (SEQ ID NO:46). [00130] ACE2 protein decoys of the present invention include those wherein at least one structural domain (e.g., D2) comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% identity to an amino acid sequence set forth in SEQ ID NO:42 wherein the amino acid at position 1 is R or if susbstituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y (preferably A, C, D, E, H, K, L, N, Q, S, or Y); wherein the amino acid at position 2 is A or if susbstituted is C, G, I, L, M, N, P, Q, S, T, V, or Y (preferably C, M, Q, T, or V); wherein the amino acid at position 3 is S or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y (preferably C, E, F, G, I, L, M, Q, or R); wherein the amino acid at position 4 is E or if substituted is A, C, D, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably C, D, I, N, S, or W); wherein the amino acid at position 5 is A or if substituted is C, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W, or Y (preferably D, E, I, M, or Y); wherein the amino acid at position 6 is A or if substituted is C, F, G, S, or T (preferably C or S); wherein the amino acid at position 7 is K or if substituted is A, C, D, E, G, H, L, M, P, Q, R, S, T, V, W, or Y (preferably A, C, D, E, G, H, L, M, R, S, T, V, W, or Y); wherein the amino acid at position 8 is R or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, Q, S, T, V, W, or Y (preferably A, C, D, E, G, H, L, M, Q, S, T, V, or Y); wherein the amino acid at position 9 is I or if substituted is A, C, F, G, K, L, M, Q, S, T, V, W, or Y (preferably A, C, F, G, L, M, S, T, W, or Y); wherein the amino acid at position 10 is A or if substituted is D, G, T, or V (preferably D, G, or V); wherein the amino acid at position 11 is K or if substituted is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, C, D, E, F, G, H, L, M, Q, R, S, V, W, or Y); wherein the amino acid at position 12 is A or if substituted is C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y (preferably E, G, I, S, or T); wherein the amino acid at position 13 is I or if substituted is A, C, D, E, F, G, H, L, M, N, Q, S, T, V, or Y (preferably A, C, D, F, G, L, M, N, Q, S, T, or V);
wherein the amino acid at position 14 is R or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, Q, S, T, V, W, or Y (preferably F, H, K, L, N, V, or W); wherein the amino acid at position 15 is K or if substituted is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, or Y); wherein the amino acid at position 16 is A or if substituted is C, F, G, M, P, S, T, V, or Y (preferably G, T, or Y); wherein the amino acid at position 17 is A or if substituted is C, D, E, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y (preferably G, T, or V); wherein the amino acid at position 18 is D or if substituted is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, F, G, I, L, N, R, S, T, W, or Y) ; wherein the amino acid at position 19 is A or if substituted is C, D, E, F, G, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably G, K, M, Q, S, or T); wherein the amino acid at position 20 is I or if substituted is A, C, F, G, H, L, M, Q, T, V, W, or Y (preferably G, L, M, T, V, or Y); wherein the amino acid at position 21 is A or if substituted is C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y, or W (preferably D, E, G, I, K, M, N, Q, S, T, V, W, or Y); wherein the amino acid at position 22 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, D, G, K, M, P, S, T, V, W, or Y); wherein the amino acid at position 23 is A or if substituted is C, G, N, S, T, or V (preferably G, S, or T); wherein the amino acid at position 24 is A or if substituted is C, D, E, G, K, M, N, S, T, or V (preferably C, G, or T); wherein the amino acid at position 25 is K or if substituted is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, C, D, E, G, I, L, P, Q, R, S, V, W, or Y); wherein the amino acid at position 26 is I or if substituted is A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably C, D, E, K, L, M, P, Q, R, S, or V); wherein the amino acid at position 27 is A or if substituted is C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y (preferably F, G, M, N, S, T, or V); wherein the amino acid at position 28 is A or if substituted is C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y, or W (preferably C, D, E, G, H, Q, S, T, V, or Y);
wherein the amino acid at position 29 is R or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y (preferably A, C, D, G, H, I, K, L, M, N, P, S, T, V, W, or Y); wherein the amino acid at position 30 is A or if substituted is C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably C, D, G, H, K, N, P, Q, R, S, T, or V). [00131] It will be understood by the skilled practitioner that the D1 and D2 domains described herein can be combined with any of the H1, H2, and H3 domains described herein. [00132] The de novo proteins of the present invention optionally comprise amino acid linkers between the domains. The amino acid linkers may be of any length as deemed appropriate for an intended use. The linkers can be, for example, from 1 to 100 amino acids in length, such as 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, or 1-5 amino acids in length. As with the variability permitted in the amino acid residues and flexibility in domain order, the flexibility in linker stems from the use of de novo protein design to construct the proteins of the present invention. In these proteins, the majority of the contributions to protein folding come from interactions among the secondary structure elements rather than from the linkers. At least for that reason, the linkers can generally be modified in de novo designed proteins without compromising protein folding. In some embodiments, the linkers result in variable loop regions between the domains. [00133] An exemplary ACE2 protein decoy of the present invention is CTC-445 and is as set forth in SEQ ID NO:47. SAEIDMGKGDFREIRASEDAREAAEALAEAARAMKEALEIIREIAEKLRDSSR ASEAAKRIAKAIRKAADAIAEAAKIAARAAKDEDAARNAENAARKAKEFAEE QAKLADMYAEFAKNGDKSRVREQLKTFADKAFHEMEDRFYQAALAVFEAA EAAAG (SEQ ID NO:47) ACE2 protein decoy CTC-445 comprises a H1, H2, and H3 domain as well as two structural domains that facilitate protein folding and binding-competent presentation of H1, H2, and H3. In CTC-445, the order of the domains is H3-D1-D2-H2-H1. H3 is from amino acid 1-15; D1 is from amino acid 21-47; D2 is from amino acid 53-82; H2 is from amino acid 86-118; and H1 is from amino acid 123-160. The ACE2 protein decoy also includes four linkers linking together the various domains. The first linker is from amino acid 16-20, the second linker is from amino acid 48-52, the third linker is from amino acid 83-85 and the fourth linker is from amino acid 119-122. Numbering is according to SEQ ID NO:47. The teachings provided herein with respect to the H1, H2, H3, D1 and D2 domains, in
addition to the examples provided herein and the skill in the art, can be used to make ACE2 protein decoys that are variants of CTC-445. In some embodiments, exemplary variants are those that have introduced amino acid substitutions that play a role in optimizing the stability and/or folding of the protein. Typically, these substitutions are not at the binding interface but at other locations in the protein. Although these substitutions are not at the binding interface, they can lead to improved binding affinity, in addition to improved activity. Methods of testing proteins for improved binding, stability, and or protein folding are known in the art and are described herein. CTC-445 has been demonstrated to specifically bind to the SARS-COV-2 spike protein but only weakly bind to the SARS- CoV-1 spike protein. In some embodiments, ACE2 protein decoys that are variants of CTC- 445 are capable of binding to both the SARS-COV-2 spike protein and the SARS-COV-1 spike protein with higher affinity as compared to CTC-445. Exemplary ACE2 protein decoys having identity to CTC-445 include those set forth in SEQ ID NOS:48-68, 184-188, 104-172, and 224-239. Also included in the present invention are those ACE2 protein decoys comprising a sequence at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 99% or 100% identical to an amino acid sequence set forth in SEQ ID NOS:47-68, 184-188, 104-172, 224-239, 255-257 or 265. The teachings provided herein with respect to the H1, H2, H3, D1 and D2 domains, in addition to the examples provided herein and the skill in the art, can be used to make variants of such ACE2 protein decoys. Table 1
[00139] In some embodiments, the amino acid residue at position 88 of any of SEQ ID NOS: 69-90, 258-259, or 189-192, if not A, is selected from F, I, L, M, P or V; wherein position 88 is in reference to SEQ ID NO:47 with fixed linker lengths. In some embodiments, the amino acid residue at position 137 of any of SEQ ID NOS: 69-90 or 189- 192, if not F, is D, E, G, K, N, P, Q, R, S, or T. In some embodiments, if the amino acid residue at position 11 of any of SEQ ID NOS: 69-90, 258-259, or 189-192 is cysteine, the amino acid residue at position 6 is L and/or the amino acid residue at position 126 is L and/or the amino acid residue at position 124 is S. Positions 88, 137, 11, 124, and 126 referred to above mean the positions in SEQ ID NOs: 69-90, 258-259, or 189-192 that correspond to positions 88, 137, 11, 124, and 126, respectively, in SEQ ID NO:47, and not the actual positions in SEQ ID NOS: 69-90 or 189-193, which may vary due to the non- fixed length of the linkers X
L. Thus, reference to “position 88 of any one of SEQ ID NOs: 69-90 or 189-192” means the position in any one of SEQ ID NOs: 69-90, 258-259, or 189- 193 corresponding to position 88 in SEQ ID NO: 47. [00140] Exemplary de novo proteins of the present invention include those comprising a sequence at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 99%
or 100% identical to the amino acid sequence set forth in SEQ ID NO: 69-90, 258-259, or 189-192 wherein a. the amino acid residue at position 2 is V; or b. the amino acid residue at position 3 is V; or c. the amino acid residue at position 6 is L; or d. the amino acid residue at position 11 is F or C; or e. the amino acid residue at position 41 is L; or f. the amino acid residue at position 61 is V; or g. the amino acid residue at position 63 is R or C; or h. the amino acid residue at position 73 is V; or i. the amino acid residue at position 74 is E or C; or j. the amino acid residue at position 86 is G; or k. the amino acid residue at position 92 is V; or l. the amino acid residue at position 95 is E; or m. the amino acid residue at position 100 is Q; or n. the amino acid residue at position 116 is L ; or o. the amino acid residue at position 124 is S; or p. the amino acid residue at position 126 is L; or q. the amino acid residue at position 136 is T; or r. the amino acid residue at position 143 is S or L; or any combination of (a) – (r) – above, wherein the noted positions are according to the numbering of SEQ ID NO:47 not of SEQ ID NOS: 69-90 or 189-192 due to the non-fixed length of the linkers X
L, as discussed above. [00141] Exemplary de novo proteins of the present invention include those comprising a sequence at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 69-90, 258-259, or 189-192 wherein no more than 1 of the amino acids at positions 8, 9, or 10 is substituted; no more than 3, no more than 2 or no more than 1 of the amino acids at positions 91, 94, 98, 102, 105, 106, 109, 112, or 113 is substituted; no more than 3, no more than 2 or no more than 1 of the amino acids at positions 123, 127, 128, 131, 132, 134, 138, 139, 141, 142, 145, 146, or 149 is substituted. The noted positions are according to the numbering of SEQ
ID NO:47 not of SEQ ID NOS: 69-90, 258-259, or 189-192 due to the non-fixed length of the linkers X
L, as discussed above. [00142] Exemplary de novo proteins of the present invention include those comprising a sequence at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 69-90, 258-259, or 189-192 wherein no more than 4, 3, 2, or 1 of the amino acids at positions 8, 9, 10, 91, 94, 98, 102, 105, 106, 109, 112, 113123, 127, 128, 131, 132, 134, 138, 139, 141, 142, 145, 146, or 149 is substituted. The noted positions are according to the numbering of SEQ ID NO:47 not of SEQ ID NOS: 69-90, 258-259, or 189-192 due to the non-fixed length of the linkers X
L, as discussed above. [00143] Exemplary de novo proteins of the present invention include those comprising a sequence at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 69-90, 258-259, or 189-192 wherein: a. the amino acid residue at position 8 is K; b. the amino acid residue at position 9 is G; c. the amino acid residue at position 10 is D d. the amino acid residue at position 91 is N e. the amino acid residue at position 94 is N f. the amino acid residue at position 98 is K g. the amino acid residue at position 102 is F h. the amino acid residue at position 105 is E i. the amino acid residue at position 106 is Q j. the amino acid residue at position 109 is L k. the amino acid residue at position 112 is M l. the amino acid residue at position 113 is Y m. the amino acid residue at position 123 is S n. the amino acid residue at position 127 is E o. the amino acid residue at position 128 is Q p. the amino acid residue at position 131 is T q. the amino acid residue at position 132 is F
r. the amino acid residue at position 134 is D s. the amino acid residue at position 138 is H t. the amino acid residue at position 139 is E u. the amino acid residue at position 141 is E v. the amino acid residue at position 142 is D w. the amino acid residue at position 145 is Y x. the amino acid residue at position 146 is Q; and y. the amino acid residue at position 149 is L; wherein the noted positions are according to the numbering of SEQ ID NO:47 not of SEQ ID NOS: 69-90 or 189-192 due to the non-fixed length of the linkers X
L, as discussed above. [00144] Including in the present invention are ACE2 protein decoys comprising a sequence at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:47 provided that the following substitutions are present (i) M6L_R126L; (ii) F116L_R124S; (iii) M6L_F116L_R124S_R126L; (iv) M6L_E86G_F116L_R124S_R126L; (v) A2V_E3V_M6L_I61V_K63R_A73V_K84T_E86G_A92V_A95E_ K100Q_F116L_R124S_R126L _A136T_R143S; (vi) M6L_F11C_R126L; (vii) M6L_K63C_R126L; (viii) M6L_E74C_R126L; (ix) F11C_F116L_R124S; (x) K63C_F116L_R124S; (xi) E74C_F116L_R124S; (xii) M6L_F11C_F116L_R124S_R126L; (xiii) M6L_K63C_F116L_R124S_R126L; (xiv) M6L_E74C_F116L_R124S_R126L; (xv) A2V_E3V_M6L_F11C_I61V_K63R_A73V_K84T_E86G_A92V_ A95E_K100Q_F116L_R124S_ R126L_A136T_R143S; (xvi) A2V_E3V_M6L_I61V_K63C_A73V_K84T_E86G_A92V_A95E_ K100Q_F116L_ R124S_R126L_A136T_R143S; (xvii) A2V_E3V_M6L_I61V_K63R_A73V_E74C_K84T_E86G_A92V_ A95E_K100Q_F116L_ R124S_R126L_A136T_R143S; (xviii) M6L_F11C_E86G_F116L_R124S_R126L; (xix) M6L_K63C_E86G_F116L_R124S_R126L; (xx) M6L_E74C_E86G_F116L_R124S_R126L; (xxi) A2V_M6L_E86G_F116L_R124S_R126L; (xxii) A2V_M6L_E86G_F116L_R124S_R126L_R143L; (xxiii) M6L_I41L_E86G_F116L_R124S_R126L_R143L; or (xxiv) A2V_M6L_I41L_E86G_F116L_R124S_R126L_R143L.
[00145] In some preferred embodiments of the present invention, when the ACE2 protein decoy comprises a sequence at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 99% or 100% identical to the amino acid sequence for (i) CTC 637: the following amino acids are present: 6L and 126L; (ii) CTC-638; the following amino acids are present: 116L and 124S; (iii) CTC-639; the following amino acids are present: 6L, 116L, 124S, and 126L (iv) CTC-640; the following amino acids are present: 6L, 86G, 116L, 124S, and 126L (v) CTC-641; the following amino acids are present: 2V, 3V, 6L, 61V, 63R, 73V, 84T, 86G, 92V, 95E, 100Q, 116L, 124S, 126L , 136T, and 143S (vi) CTC-642 ; the following amino acids are present: 6L, 11C, and 126L (vii) CTC-643; the following amino acids are present: 6L, 63C, and 126L (viii) CTC-644; the following amino acids are present: 6L, 74C, and 126L (ix) CTC-645; the following amino acids are present: 11C, 116L, and 124S (x) CTC-646; the following amino acids are present: 63C, 116L, and 124S (xi) CTC-647; the following amino acids are present:74C, 116L, and 124S (xii) CTC-648; the following amino acids are present: 6L, 11C, 116L, 124S, and 126L (xiii) CTC-649; the following amino acids are present: 6L, 63C, 116L, 124S, and 126L (xiv) CTC-650; the following amino acids are present: 6L, 74C, 116L, 124S, and 126L (xv) CTC-651; the following amino acids are present: 2V, 3V, 6L, 11C, 61V, 63R, 73V, 84T, 86G, 92V, 95E, 100Q, 116L, 124S, 126L, 136T, and 143S (xvi) CTC-652; the following amino acids are present: 2V, 3V, 6L, 61V, 63C, 73V, 84T, 86G, 92V, 95E, 100Q, 116L, 124S, 126L, 136T, and 143S (xvii) CTC-653; the following amino acids are present: 2V, 3V, 6L, 61V, 63R, 73V, 74C, 84T, 86G, 92V, 95E, 100Q, 116L, 124S, 126L, 136T, and 143S (xviii) CTC-656: the following amino acids are present:1P, 11K, 42S, 46S, 81H, 88N, 116N, 124S, 137V, and 143L; (xix) CTC-693: the following amino acids are present: 6L, 11C, 86G, 116L, 124S, and 126L; (xx) CTC-694: the following amino acids are present: 6L, 63C, 86G, 116L, 124S, and 126L; (xxi) CTC-695: the following amino acids are present: 6L, 74C, 86G, 116L, 124S, and 126L; (xxii) CTC-699; the following amino acids are present 2V, 6L, 86G, 116L, 124S, and 126L; (xxiii) CTC-700; the following amino acids are present: 2V, 6L, 86G, 116L, 124S, 126L, 143L; (xxiv) CTC-701; the following amino acids are present 6L, 41L, 86G, 116L, 124S, 126L, and 143L; and (xxv) CTC-702; the following amino acids are present 2V, 6L, 41L, 86G, 116L, 124S, 126L, and R143. [00146] Included in the present invention are de novo proteins of the present invention comprising a decoy unit comprises an amino acid sequence at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in SEQ ID NO: 50 (CTC-640) wherein wherein the amino acid at position 1 is S or if substituted is A, C, E, F, G, I, L, R, S, T, V, or W (preferably C, E, G, I, or L); wherein the amino acid at position 2 is A or if substituted is C, D, E, G, I, L, M, P, Q, R, S, T, V, or W (preferably G, I, L, M, Q, T, V, or W); wherein the amino acid at position 3 is E or if substituted is A, C, D, G, L, M, P, R, S, T, V, or W (preferably L, M, P, S, T, V, or W); wherein the amino acid at position 4 is I or if substituted is C, F, L, M, T, V, or W (preferably F, or V); wherein the amino acid at position 5 is D or if substituted is A, C, E, G, I, K, L, M, N, S, T, or V (preferably M or N); wherein the amino acid at position 6 is L or if substituted is F, I, M, or V (preferably M or F); wherein the amino acid at position 7 is G or if substituted is D or L; wherein the amino acid at position 8 is K or if substituted is I, M, N, Q, R, or T (preferably M); wherein the amino acid at position 9 is G or if substituted is D, E, M, R, or S (preferably R); wherein the amino acid at position 10 is D or if substituted is E, K, or T (preferably unsubstituted); wherein the amino acid at position 11 is F or if substituted is A, C, E, G, I, K, L, Q, R, S, T, V, W, or Y (preferably A, E, I, K, L, Q, R, S, or V); wherein the amino acid at position 12 is R or if substituted is K, M, Q, or S (preferably unsubstituted); wherein the amino acid at position 13 is E or if substituted is A, C, D, G, H, K, L, M, P, R, S, T, V, W, or Y (preferably D, G, L, M, P, S, V, or W); wherein the amino acid at position 14 is I or if substituted is A, C, D, F, G, H, L, M, N, P, Q, R, S, T, V, W (preferably I, C, F, M, P, Q, S, T, V, or W); wherein the amino acid at position 15 is R or if substituted is A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W (preferably C, D, E, F, H, K, L, Q, S, V, W); wherein the amino acid at position 16 is A or if substituted is A, D, E, F, G, L, M, R, S, T,
V, W (preferably E, F, G, L, M, S, T, V, W); wherein the amino acid at position 17 is S or if substituted is A, C, D, E, G, K, L, M, P, Q, R, S, T, V, W (preferably A, C, D, E, G, L, P, T, V, W); wherein the amino acid at position 18 is E or if substituted is A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y (preferably C, G, I, L, M, N, P, R, S, T, V); wherein the amino acid at position 19 is D or if substituted is A, C, D, E, G, I, K, L, M, N, P, R, S, T, V, W, Y (preferably C, E, G, M, P, R, V, W); wherein the amino acid at position 20 is A or if substituted is A, C, E, G, I, L, P, Q, R, S, T, V, W (preferably L, R, S, T, V, W); wherein the amino acid at position 21 is R or if substituted is A, C, E, F, G, I, K, L, M, P, S, T, V, or W (preferably C, E, F, G, K, L, M, P, S, T, or W ); wherein the amino acid at position 22 is E or if substituted is A, C, D, F, G, I, K, L, M, P, Q, R, S, T, V, W, or Y (preferably A, G, K, M, V, W, or Y); wherein the amino acid at position 23 is A or if substituted is C, E, G, K, L, M, P, Q, R, S, T, V, W, or Y (preferably C, K, P, Q, or V); wherein the amino acid at position 24 is A or if substituted is D, E, G, I, K, L, M, N, P, R, S, T, V, or W (preferably E, N, T, V, or W); wherein the amino acid at position 25 is E or if substituted is C, D, G, K, L, Q, R, S, T, V, W, or Y (preferably C, D, Q, S, V, W, or Y); wherein the amino acid at position 26 is A or if substituted is C, D, E, G, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably D, K, P, R, V, or Y); wherein the amino acid at position 27 is L or if substituted is A, C, F, I, M, Q, S, T, or V (preferably T); wherein the amino acid at position 28 is A or if substituted is C, D, E, F, G, H, I, K, Q, L, M, R, S, T, V, or W (preferably D, E, G, H, I, L, Q, R, S, V, or W); wherein the amino acid at position 29 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, P, R, S, V, W, or Y (preferably A, D, H, L, M, N, R, S, or V); wherein the amino acid at position 30 is A or if substituted is C, G, L, M, Q, S, T, V, or W (preferably C, G, M, or S); wherein the amino acid at position 31 is A or if substituted is C, D, G, L, M, N, Q, R, S, T, or V (preferably C, G, M, S, T, or V);
wherein the amino acid at position 32 is R or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y (preferably A, C, D, E, F, G, H, I, K, M, N, P, Q, S, T, V, W, or Y); wherein the amino acid at position 33 is A or if substituted is C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y (preferably D, E, K, M, R, S, or V); wherein the amino acid at position 34 is M or if substituted is A, C, D, E, G, H, I, K, L, Q, R, S, T, V, W, or Y (preferably A, C, D, E, G, K, R, S, T, V, or Y ); wherein the amino acid at position 35 is K or if substituted is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y (preferably E, G, H, M, R, S, or Y); wherein the amino acid at position 36 is E or if substituted is A, C, D, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y (preferably A, C, D, G, K, L, M, Q, T, V, or W); wherein the amino acid at position 37 is A or if substituted is C, G, P, S, T, or V (preferably C, G, or T); wherein the amino acid at position 38 is L or if substituted is C, F, H, I, K, M, N, Q, R, T, V, W, or Y (preferably C, F, H, or V); wherein the amino acid at position 39 is E or if substituted is A, C, D, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y (preferably A, C, F, G, L, Q, R, S, T, V, or Y); wherein the amino acid at position 40 is I or if substituted is A, C, D, E, F, G, H, K, L, M, N, Q, R, S, T, V, W, or Y (preferably C, E, G, L, Q, R, S, T, V, or Y); wherein the amino acid at position 41 is I or if substituted is A, C, D, E, F, G, K, L, M, N, Q, S, T, V, W, or Y (preferably A, C, D, F, L, M, N, S, T, V, or Y); wherein the amino acid at position 42 is R or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, Q, S, T, V, Y, or W (preferably A, C, D, E, F, G, I, L, M, Q, S, T, V, or Y); wherein the amino acid at position 43 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, C, D, F, H, I, M, N, P, T, or W); wherein the amino acid at position 44 is I or if substituted is A, C, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y (preferably C, S, T, or V); wherein the amino acid at position 45 is A or if substituted is C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably D, G, H, I, M, N, S, V, or Y); wherein the amino acid at position 46 is E or if substituted is A, C, D, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y (preferably C, F, I, L, S, T, or Y);
wherein the amino acid at position 47 is K or if substituted is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y (preferably C, D, E, F, G, H, M, N, S, or Y); wherein the amino acid at position 48 is L or if substituted is A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y (preferably C, D, F, G, H, I, P, Q, S, T, W, Y); wherein the amino acid at position 49 is R or if substituted is A,C, D, E, F, G, I, K, L, M, P, Q, R, S, T, V, W, Y (preferably A, D, K, M, Q, S, T, V, Y); wherein the amino acid at position 50 is D or if substituted is A, C, D, E, F, G, K, L, M, N, Q, R, S, T, V, W (preferably C, E, M, T, V); wherein the amino acid at position 51 is S or if substituted is A, C, D, E, F, G, I, K, L, M, P, Q, R, S, T, V, W, Y (preferably A, D, E, F, K, M, P, T, V, Y); wherein the amino acid at position 52 is S or if substituted is A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y (preferably A, E, M, Q); wherein the amino acid at position 53 is R or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y (preferably A, C, D, E, H, K, L, N, Q, S, or Y); wherein the amino acid at position 54 is A or if substituted is C, G, I, L, M, N, P, Q, S, T, V, or Y (preferably C, M, Q, T, or V); wherein the amino acid at position 55is S or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y (preferably C, E, F, G, I, L, M, Q, or R); wherein the amino acid at position 56 is E or if substituted is A, C, D, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably C, D, I, N, S, or W); wherein the amino acid at position 57 is A or if substituted is C, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W, or Y (preferably D, E, I, M, or Y); wherein the amino acid at position 58 is A or if substituted is C, F, G, S, or T (preferably C or S); wherein the amino acid at position 59 is K or if substituted is A, C, D, E, G, H, L, M, P, Q, R, S, T, V, W, or Y (preferably A, C, D, E, G, H, L, M, R, S, T, V, W, or Y); wherein the amino acid at position 60 is R or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, Q, S, T, V, W, or Y (preferably A, C, D, E, G, H, L, M, Q, S, T, V, or Y); wherein the amino acid at position 61 is I or if substituted is A, C, F, G, K, L, M, Q, S, T, V, W, or Y (preferably A, C, F, G, L, M, S, T, W, or Y); wherein the amino acid at position 62 is A or if substituted is D, G, T, or V (preferably D, G, or V);
wherein the amino acid at position 63 is K or if substituted is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, C, D, E, F, G, H, L, M, Q, R, S, V, W, or Y); wherein the amino acid at position 64 is A or if substituted is C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y (preferably E, G, I, S, or T); wherein the amino acid at position 65 is I or if substituted is A, C, D, E, F, G, H, L, M, N, Q, S, T, V, or Y (preferably A, C, D, F, G, L, M, N, Q, S, T, or V); wherein the amino acid at position 66 is R or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, Q, S, T, V, W, or Y (preferably F, H, K, L, N, V, or W); wherein the amino acid at position 67 is K or if substituted is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, or Y); wherein the amino acid at position 68 is A or if substituted is C, F, G, M, P, S, T, V, or Y (preferably G, T, or Y); wherein the amino acid at position 69 is A or if substituted is C, D, E, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y (preferably G, T, or V); wherein the amino acid at position 70 is D or if substituted is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, F, G, I, L, N, R, S, T, W, or Y) ; wherein the amino acid at position 71 is A or if substituted is C, D, E, F, G, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably G, K, M, Q, S, or T); wherein the amino acid at position 72 is I or if substituted is A, C, F, G, H, L, M, Q, T, V, W, or Y (preferably G, L, M, T, V, or Y); wherein the amino acid at position 73 is A or if substituted is C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y, or W (preferably D, E, G, I, K, M, N, Q, S, T, V, W, or Y); wherein the amino acid at position 74 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, D, G, K, M, P, S, T, V, W, or Y); wherein the amino acid at position 75 is A or if substituted is C, G, N, S, T, or V (preferably G, S, or T); wherein the amino acid at position 76 is A or if substituted is C, D, E, G, K, M, N, S, T, or V (preferably C, G, or T); wherein the amino acid at position 77 is K or if substituted is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, C, D, E, G, I, L, P, Q, R, S, V, W, or Y);
wherein the amino acid at position 78 is I or if substituted is A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably C, D, E, K, L, M, P, Q, R, S, or V); wherein the amino acid at position 79 is A or if substituted is C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y (preferably F, G, M, N, S, T, or V); wherein the amino acid at position 80 is A or if substituted is C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y, or W (preferably C, D, E, G, H, Q, S, T, V, or Y); wherein the amino acid at position 81 is R or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y (preferably A, C, D, G, H, I, K, L, M, N, P, S, T, V, W, or Y); wherein the amino acid at position 82 is A or if substituted is C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably C, D, G, H, K, N, P, Q, R, S, T, or V); wherein the amino acid at position 83 is A or if substituted is C, D, E, G, K, L, Q, R, S, T, V, W, or Y (preferably C, E, L, S, T, or Y); wherein the amino acid at position 84 is K or if substituted is A, C, D, E, F, G, L, M, N, P, Q, R, S, T, V, or W (preferably E, F, G, M, N, S, or W); wherein the amino acid at position 85 is D or if substituted is A, C, E, G, H, L, M, N, P, Q, R, S, T, V, W, or Y (preferably C, L, M, N, Q, S, T, V, or W); wherein the amino acid at position 86 is G or if substituted is A, C,D, E, I, L, M, N, P, Q, R, S, T, V, or W (preferably C, D, E, I, M, N, P, Q, R, S, or T); wherein the amino acid at position 87 is D or if substituted is A, C, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably E, F, G, H, I, M, P, Q, R, S, V, W, or Y); wherein the amino acid at position 88 is A or if substituted is C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably D, E, H, L, M, N, R, T, V, W, or Y); wherein the amino acid at position 89 is A or if substituted is C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably E, F, G, L, N, P, Q, S, T, V, or W); wherein the amino acid at position 90 is R or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y (preferably A, C, D, E, H, K, N, P, Q, T, V, W, or Y); wherein the amino acid at position 91 is N or if substituted is A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y (preferably A, D, E, G, K, L, M, P, Q, R, S, T, V, or W); wherein the amino acid at position 92 is A or if substituted is C, D, G, H, I, L, M, N, P, R, S, T, V, or W (preferably C, G, P, T, or V); wherein the amino acid at position 93 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, C, D, F, I, K, L, M, N, Q, R, S, T, or V);
wherein the amino acid at position 94 is N or if substituted is A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y (preferably D, F, G, I, L, M, Q, S, T, V, or W) wherein the amino acid at position 95 is A or if substituted is C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably C, E, H, I, L, M, N, Q, R, S, V, W, or Y) wherein the amino acid at position 96 is A or if substituted is C, D, E, G, I, L, M, N, P, Q, S, T, V, W, or Y(preferably C, G, I, or S); wherein the amino acid at position 97 is R or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, Y, or W (preferably A, C, F, G, H, I, K, L, M, N, Q, S, T, V, or Y); wherein the amino acid at position 98 is K or if substituted is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, F, G, I, L, P, Q, T, V); wherein the amino acid at position 99 is A or if substituted is C, D, G, I, L, Q, S, T, V, or W (preferably L, T, or V); wherein the amino acid at position 100 is K or if substituted is A, C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, or W (preferably A, C, D, E, F, G, H, I, Q, R, S, T, V, W, or Y); wherein the amino acid at position 101 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, D, F, G, H, I, K, L, M, N, R, S, T, V, or W); wherein the amino acid at position 102 is F or if substituted is A, C, D, E, G, I, K, L, M, P, Q, R, S, T, V, W, or Y (C, G, I, K, P, V, W, Y); wherein the amino acid at position 103 is A or if substituted is C, D, F, G, I, L, M, N, S, T, V, W, or Y (preferably C, F, G, I, L, S, or T); wherein the amino acid at position 104 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y (preferably A, C, D, G, H, I, L, M, N, R, S, T, V, or Y); wherein the amino acid at position 105 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, C, F, G, H, K, L, M, Q, R, S); wherein the amino acid at position 106 is Q or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, R, S, T, Y, or V (preferably C, D, I, K, N, R, T, Y); wherein the amino acid at position 107 is A or if substituted is C, E, F, G, I, L, M, Q, S, T, V, or W (preferably I or S); wherein the amino acid at position 108 is K or if substituted is A, C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, or Y (preferably D, G, H, I, Q, or R); wherein the amino acid at position 109 is L or if substituted is A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, Y, or W (preferably A, C, D, F, G, H, I, K, M, N, Q, R, S, T, V, W, Y);
wherein the amino acid at position 110 is A or if substituted is C, F, G, I, L, S, T, V, or Y (preferably I); wherein the amino acid at position 111 is D or if substituted is A, C, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, or Y (preferably C, F, I, S, or T); wherein the amino acid at position 112 is M or if substituted is A, C, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W, or Y (preferably A, D, E, F, H, I, K, L, Q, R, S, T, V, Y); wherein the amino acid at position 113 is Y or if substituted is D, F, H, I, L, M, or V (preferably F); ; wherein the amino acid at position 114 is A or if substituted is C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or W (preferably C, D, G, H, N, R, S, or V); wherein the amino acid at position 115 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, G, L, M, Q, R, S, T, or V); wherein the amino acid at position 116 is L or if substituted is A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y (preferably A, C, D, E, F, G, H, I, M, N, Q, S, T, V, or Y); wherein the amino acid at position 117 is A or if substituted is C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y (preferably C, D, F, G, I, N, P, or T); wherein the amino acid at position 118 is K or if substituted is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, C, D, E, F, G, H, L, M, N, R, S, T, V, or Y); wherein the amino acid at position 119 is N or if substituted is A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y (preferably A, D, F, G, H, I, K, M, Q, R, S, T, or Y); wherein the amino acid at position 120 is G or if substituted is A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, D, E, N, or Q); wherein the amino acid at position 121 is D or if substituted is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably E, F, or N); wherein the amino acid at position 122 is K or if substituted is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, or W); wherein the amino acid at position 123 is S or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y (preferably A, C, D, E, G, H, I, K, L, M, N, P, R, T, V, or W); wherein the amino acid at position 124 is S or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y (preferably A, C, E, F, G, I, L, M, N, Q, R, T, V, or Y); wherein the amino acid at position 125 is V or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y (preferably A, D, H, I, N, P, T, or W);
wherein the amino acid at position 126 is L or if substituted is A, C, D, E, F, G, H, I, K, M, P, Q, R, S, T, V, W, or Y (preferably R, C, H, I, K, M, S, T, or Y) wherein the amino acid at position 127 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, C, D, F, G, H, M, P, Q, R, T, V, W, or Y) wherein the amino acid at position 128 is Q or if substituted is A, C, D, E, F, G, H, I, L, M, N, P, S, T, V, or W (preferably N or P); wherein the amino acid at position 129 is L or if substituted is C, I, M, T, or V (preferably I, T, or V); wherein the amino acid at position 130 is K or if substituted is A, C, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y(preferably A, C, E, I, L, N, R, V, or Y); wherein the amino acid at position 131 is T or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y (preferably A, C, F, H, I, K, L, M, Q, R, S, V, W, or Y); wherein the amino acid at position 132 is F or if substituted is A, C, H, V, W, or Y (preferably W); wherein the amino acid at position 133 is A or if substituted is C, G, L, M, S, T, or V preferably C, S, or T); wherein the amino acid at position 134 is D or if substituted is A, C, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, E, H, I, L, N, Q, R, S, T, V, Y); wherein the amino acid at position 135 is K or if substituted is A, C, F, H, I, L, M, N, Q, R, S, V, W, or Y (preferably A, H, M, N, R, S, V, W, or Y); wherein the amino acid at position 136 is A or if substituted is C, D, E, F, G, H, I, K, L, M, N, Q, S, T, or V (preferably C, E, F, I, L, N, Q, S, T, or V); wherein the amino acid at position 137 is F or if substituted is A, C, D, E, G, H, I, L, M, N, Q, R, S, T, V, W, or Y (preferably D, E, H, L, M, N, Q, or W); wherein the amino acid at position 138 is H or if substituted is A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, D, G, I, K, M, P, Q, R, S, or Y); wherein the amino acid at position 139 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably D, G, I, L, N, Q, S, T, V); wherein the amino acid at position 140 is M or if substituted is A, C, D, F, G, H, I, L, N, Q, S, T, V, W, or Y (preferably A, C, F, G, L, S, T, or V); wherein the amino acid at position 141 is E or if substituted is D, M, N, P, Q, T, or V (preferably D, N, or T);
wherein the amino acid at position 142 is D or if substituted is E, F, G, H, L, N, or Q (preferably E); wherein the amino acid at position 143 is R or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y (preferably A, C, E, F, G, H, I, M, P, Q, T, V, W, or Y);; wherein the amino acid at position 144 is F or if substituted is C, G, H, L, M, N, W, or Y (preferably N, W, or Y); wherein the amino acid at position 145 is Y or if substituted is H, D, or F (preferably H); wherein the amino acid at position 146 is Q or if substituted is A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y (preferably A, C, D, F, G, H, I, K, M, N, S, or V); wherein the amino acid at position 147 is A or if substituted is C, F, G, H, I, L, M, N, Q, S, T, V, W, or Y (preferably C, S, or T); wherein the amino acid at position 148 is A or if substituted is C, D, E, F, G, H, I, L, M, N, Q, S, T, or V(preferably C, F, G, L, M, N, S, T, or V); wherein the amino acid at position 149 is L or if substituted is A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, or Y (preferably C, F, I, K, R, T, or V) wherein the amino acid at position 150 is A or if substituted is A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably K, M, N, S, or T); wherein the amino acid at position 151 is V or if substituted is A, C, D, F, G, I, L, M, N, Q, S, or T (preferably A, I, L, or T); wherein the amino acid at position 152 is F or if substituted is C, H, I, L, M, V, W, or Y (preferably H, or W); wherein the amino acid at position 153 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y (preferably D, G, H, I, K, M, N, Q, S, or T); wherein the amino acid at position 154 is A or if substituted is C, E, F, G, I, L, M, N, Q, R, S, T, V, W, or Y (preferably C, L, Q, or V); wherein the amino acid at position 155 is A or if substituted is C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y, or W (preferably C, D, E, G, H, I, L, M, P, Q, S, T, V, W, or Y); wherein the amino acid at position 156 is E or if substituted is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably D, H, I, M, N, P, Q, R, S, or T); wherein the amino acid at position 157 is A or if substituted is E, S, or V; wherein the amino acid at position 158 is A or if substituted is E, G, S, T, or V (preferably V);
wherein the amino acid at position 159 is A or if substituted is E, G, S, T, or V (preferably S or T); and wherein the amino acid at position 160 is G or if substituted is E, R, or V (preferably R). In some preferred embodiments, not more than 6, 5, 4, 3, 2 or 1 of positions 8, 9, 10, 12, 91, 94, 98, 102, 105, 106, 109, 112, 113, 123, 127, 128, 131, 132, 134, 135, 138, 139, 141, 142, 145, 146, and 140 are substituted. [00147] In some exemplary embodiments, amino acid residues are added at the N terminus of the protein decoys to add stability. For example, in exemplary embodiments, a PG sequence (i.e., proline-glycine) is added to the N terminus. Any of the amino acid sequence set forth in SEQ ID NOs.47-90, 104-172, 184-193, 224-239, 255-260 and 265-266 and SEQ ID NOs.91-95, 194-197, 223, and 261-263 (as provided below) can be preceded, for example, by a PG sequence, or a MPG sequence. [00148] The ACE2 protein decoys may comprise one, two, three, four, or more decoy units. In some embodiments, the ACE2 protein decoys may comprise one, two, three, or four decoy units. In some embodiments, a decoy unit comprises (i) at least two alpha helical domains, H1 and H2, (ii) an optional beta hairpin domain, H3, and (iii) at least one structural domain. In some embodiments, a decoy unit comprises (i) two alpha helical domains, H1 and H2, (ii) one beta hairpin domain, H3, and (iii) two structural domains. In exemplarly embodiments, the H1, H2, H3 and structural domains are as described herein. Nonlimiting exemplary decoy units are provided in SEQ ID NOS: 47-90, 104-172, 184-193, 224-239, and 255-260. In some aspects, an ACE2 protein decoy is multivalent (e.g. bivalent, trivalent, tetravelent), which means it comprises at least two decoy units. In some embodiments, the ACE2 protein decoy comprises two, three, four, or more amino acid sequences independently selected from SEQ ID NOS: 47-90, 104-172, 184-193, 224-239, 255-260, or 265-266. In some embodiments, the ACE2 protein decoy comprises multiple copies of the same decoy unit. In some embodiments, the ACE2 protein decoy comprises 2 to 4 copies of the same or a different decoy unit. [00149] In some aspects, a the C terminus of a first decoy unit is linked to the N terminus of a second decoy unit. Linkage can be via a chemical or enzymatic crosslinking (e.g. bismaleimide). The two or more decoy units may directly abut each other in the translational fusion or may be linked by a polypeptide linker suitable for the intended purpose. Exemplary such linkers include, but are not limited, to those disclosed in WO2016178905, WO2018153865, and WO 2018170179. In other embodiments, suitable linkers include, but are not limited to peptide linkers, such as, for example, GGGGG (SEQ
ID NO: 96), GSGGG (SEQ ID NO: 97), GGGGGG (SEQ ID NO: 98), GGSGGG (SEQ ID NO: 99), GGSGGSGGGSGGSGSG (SEQ ID NO: 100), GSGGSGGGSGGSGSG (SEQ ID NO: 101), GGSGGSGGGSGGSGGGGSGGSGGGSGGGGS (SEQ ID NO: 102), GGGGSGGSGSGGSGGGS (SEQ ID NO: 175), [GGGGX]n (SEQ ID NO: 103), where X is Q, E or S and n is 2-5, and GGGSGGSGSGGSGGGS (SEQ ID NO: 264). In some embodiments, an amino acid linker between decoy units is from 1-100, from 1-80, from 1- 60, from 1-50, from 1-40, from 1-30, or from 1-20 amino acids in length. [00150] In some aspects, two or more ACE2 protein decoys are different. Any of the ACE2 protein decoys provided herein can be linked together for use in the present invention. [00151] Exemplary ACE2 protein decoys include those comprising one or more of CTC- 640, CTC-693, CTC-694, CTC-695, CTC-702, CTC-705, or CTC-726. In some aspects, an ACE2 protein decoy comprises (i) CTC-640 and/or CTC-693; (ii) CTC-640 and/or CTC- 694; (iii) CTC-640 and/or CTC-694. In some aspects, an ACE2 protein decoy of the present comprises a multivalent, serially duplicated version of CTC-640; a multivalent ACE2 protein decoy wherein each individual decoy unit comprises a sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, or 99% identity to the amino acid sequence set forth for CTC-640; a multivalent, serially duplicated version of CTC-702; a multivalent ACE2 protein decoy wherein each individual decoy unit comprises a sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, or 99% identity to the amino acid sequence set forth for CTC-702; a multivalent, serially duplicated version of CTC-705; a multivalent ACE2 protein decoy wherein each individual decoy unit comprises a sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, or 99% identity to the amino acid sequence set forth for CTC-705; a multivalent, serially duplicated version of CTC-726; or a multivalent ACE2 protein decoy wherein each individual decoy unit comprises a sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, or 99% identity to the amino acid sequence set forth for CTC-726. [00152] It will be appreciated by the skilled artisan that, in addition to the linkers, additional amino acid residues may be added to the N or C terminus of each individual decoy unit prior to being fused together to create multivalent ACE2 protein decoy, e.g, bivalent, trivalent, tetravalent, etc. In addition, the multivalent ACE2 protein decoys can be cyclized. Methods for cyclizing proteins are known in the art, see, for example, Wood et al., Journal of Biological Chemistry, 289:21; 14512-14519, 2014.
[00153] Exemplary multivalent ACE2 protein decoys include those comprising a sequence at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in SEQ ID NOS: 91-95, 194-197, 223, or 261-263: Table 3