Peptide inhibitors that inhibit HIV capsid

Abstract

Provided herein are cyclic or linear peptides that bind to HIV-1 capsid protein and inhibit HIV-1 capsid assembly and / or disassembly. Also provided are methods for preparing the peptides and pharmaceutical compositions and methods for treating HIV infection.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 858,666, filed on June 7, 2019, the entire contents of which are incorporated herein by reference.

[0003] Reference to electronically submitted sequence listings

[0004] The contents of the sequence listing submitted electronically in an ASCII text file (name UMC_196127_Seq_List_ST25.txt; size: 5026 bytes; creation date: June 8, 2020) are incorporated herein by reference along with the entire contents of this application. Background Art

[0005] The remarkable development of combined antiretroviral therapy (cART) has transformed HIV / AIDS from a deadly epidemic to a chronic and manageable disease (Antiviral Therapy Cohort, 2017). If administered effectively, cART can significantly reduce morbidity and mortality in HIV-infected individuals in resource-rich countries as well as low- and middle-income countries (Quinn, 2008; Sabin, 2013; May et al. 2014, Harries et al. 2016; and Teeraanachai et al., 2017). However, emerging drug-resistant mutations (DRMs) continue to threaten the expected effects of cART.

[0006] HIV-capsid (CA) has emerged as an important potential antiviral target over the past 15 years due to the increased understanding of its involvement in the viral life cycle. The CA protein has two distinct domains, the N-terminal domain (CA-NTD) and the C-terminal domain (CA-CTD), which are connected by a flexible linker of approximately 5 amino acid residues ( Figure 1A Both the CA-NTD and CA-CTD domains contain mainly α-helical secondary structures. CA-NTD consists of seven α-helices (α1-α7), whereas CA-CTD consists of four α-helices (α8-α11) and a short 3 10 -helices. The structures of CA revealed interactions between CA promoters in the form of hexameric and pentamer building blocks (Pornillos et al., 2009; and Pornillos et al., 2011). These structures also revealed that the fullerene-like structure of the capsid core is composed of approximately 1,500 CA copies organized into a lattice of approximately 250 hexamers and 12 pentamers, which promotes the closure of the capsid core.

[0007] The capsid core is involved in multiple steps of HIV replication. After infection and fusion of the viral and cellular membranes, the capsid core enters the cytosol and undergoes controlled disassembly in the cytosol as it passes through the cytosol and docks at the nuclear pore, a process known as uncoating. The timing, process, and extent of capsid core uncoating in the cytosol are not fully understood, but it has been shown that uncoating is associated with the initiation of reverse transcription (Campbell and Hope, 2015). Once the capsid core reaches the nuclear pore, CA promotes nuclear entry and enters the nucleus with the pre-integration complex. CA may also be involved in the integration of viral DNA into host chromosomes, but the specific role of CA in this process has not been clarified (Schaller et al., 2011; Chen et al., 2016; and Francis and Melikyan, 2018). During viral assembly (late stage of viral replication), Gag polyprotein, a precursor of capsid protein, assembles on the plasma membrane and buds out as spherical, immature, non-infectious viruses. After activation of viral proteases during maturation, Gag is processed into several structural proteins and small peptides, including the multifunctional CA ( Figure 1A ), forming a conical capsid core (also called capsid or core). Therefore, CA plays an important role in HIV biology.

[0008] Many reports have shown that the stability of the capsid core and its interactions with host factors affect reverse transcription. Mutations that alter the stability of the lattice or the interactions between the CA building blocks alter replication events and may severely affect viral infectivity. For example, the stabilizing mutations E45A and E128A / R132A or the destabilizing mutations R18A / N21A, P38A, Q63A, L136D, K170A, and Q129A lead to either slow or fast uncoating, respectively, ultimately reducing viral infectivity (Forshey et al, 2002). Although destabilization must occur for successful replication, a balance must be maintained between stability and dissociation, demonstrating the complex relationship between capsid and viral infectivity. Similarly, disruption of CA interactions with various host factors, including CPSF6 (cleavage and polyadenylation specificity factor 6, TNPO3 (transporter 3), NUP153 (nucleoporin 153), NUP 358 (nucleoporin 358), and cyclophilin A, has been shown to affect infectivity during replication (Krishnan et al., 2010; Lee et al., 2010; Schaller et al., 2011; Ambrose et al., 2012; Bichel et al., 2013; and Matreyek et al., 2013).

[0009] Given the role of CA in multiple steps of viral replication, it is a notable antiviral target, providing a new strategy for therapeutic intervention (Prevelige, 2011; Bocanegra et al., 2012; and Li et al., 2013). Several drug-like compounds that bind to either CA-NTD or CA-CTD have been identified (Tang et al., 2003; Sticth et al., 2005; Ternois et al., 2005; Kelly et al., 2007; Blair et al., 2010; Curreli et al., 2011; and Bocanegra et al., 2012). Among them are benzodiazepine (BD) and benzimidazole (BM) compounds (Fader et al., 2011; Lemke et al., 2012; Tremblay et al., 2012), pyrrolopyrazolones (BI-1 and BI-2) (Lamorte et al., 2013) and CAP-1 (Kelly et al., 2007 and Curreli et al., 2011). One of the best-studied CA inhibitors is PF-3450074 (PF74) ( Figure 1A). Crystal structures of PF74 bound to CA (Blair et al., 2010; Bhattacharya et al., 2014; and Price et al., 2014) have shown that PF74 occupies a pocket located between the CA-NTD and CA-CTD. This binding pocket is also shared by the small molecule BI-2 and the host factors CPSF6 and NUP153 (Price et al., 2014). Therefore, both PF74 and BI-2 are predicted to prevent the interaction between CA and CPSF6 or NUP153 (Price et al., 2012 and Price et al., 2014). PF74 appears to modulate subunit interactions in vitro, interfere with capsid assembly, and increase the rate of CA multimerization. PF74 interferes with both early and late events in the HIV replication cycle (Blair et al., 2010 and Thenin-Houssier and Valente, 2016). Accumulation of late reverse transcription products, 2LTRs, and integrated proviruses suggests that PF74 has antiviral activity at early stages of viral replication (Blair et al., 2010; Shi et al., 2011; and Fricke et al., 2014), whereas disruption of native-like particle formation suggests additional roles for PF74 at later stages of HIV replication (Blair et al., 2010; Thenin-Houssier and Valente, 2016). In addition to the chemical inhibitors described above, peptide inhibitors (e.g., NYAD-1) have also been reported. In cell-based assembly assays, NYAD-1 disrupts the formation of immature and mature-like virions (Zhang et al., 2013). Despite promising leads, none of these compounds or their derivatives have progressed to clinical trials.

[0010] The recently reported CA inhibitors GS-CA1 and GS6207 (an analog of GS-CA1, also known as GS-CA2) ( Figure 1B ) is more potent than currently approved anti-HIV drugs (Blair et al., 2010; Tse et al., 2017; and Zheng et al., 2018). 50 GS-6270 showed anti-HIV activity in MT-4 cells, EC 50 The average EC of 23 HIV-1 clinical isolates from different subtypes was 100 pM in PBMCs. 5050 pM (20-160 pM) (Zheng et al., 2018). In contrast, the most studied capsid inhibitor PF74 exhibited an isoform-dependent EC50 range of 80 nM to 640 nM in PBMCs. In MT-2 cells, PF74 had an EC50 of 570 nM (Blair et al., 2010). In addition, studies in rats and dogs have shown that a single subcutaneous injection can maintain plasma concentrations of GS-CA1 and GS-6207 above the plasma binding-regulated effective concentration required for 95% inhibition of HIV-1 replication for >12 weeks, indicating their potential as long-acting drugs (Jarvis, 2017; Tse et al., 2017; Carnes et al., 2018; and Sager et al., 2019). Similar to PF74, GS-CA1 inhibits both early and late stages of viral replication.

[0011] Nevertheless, there is still a need to discover new antiviral drugs that act through new mechanisms and / or target new targets. Summary of the invention

[0012] The present disclosure relates to a 1 –F–X 2 –P–V–X 3 -F (SEQ ID NO: 2) of the amino acid sequence of a cyclic peptide or a linear peptide, wherein X 1 , X 2 and X 3 Each independently is glycine (G), lysine (K) or a polar uncharged amino acid. 2 is glycine, X 1 and X 3 Each is a polar uncharged amino acid. In certain aspects, X 1 is threonine (T) and / or X 3 is asparagine (R). Thus, in certain aspects, X 1 is threonine (T) and / or X 3 is asparagine (R), X 2 It's glycine.

[0013] The present disclosure also relates to the above-mentioned cyclic peptide or linear peptide, wherein the peptide comprises X 4 -X 5 -X 6 -FX 1 -FX 2 -PVX 3 -FX 7 -X 8 (SEQ ID NO: 3), and wherein X 4, X 5 , X 6 , X 7 and X 8 Each is independently selected from the group consisting of glycine (G), proline (P), lysine (K), polar uncharged amino acids and hydrophobic amino acids. In certain aspects; X 4 is a polar uncharged amino acid, such as serine (S); X 6 is a hydrophobic amino acid, such as valine (V); X 7 is proline (P); and / or X 5 and / or X 8 It is glycine (G).

[0014] In certain aspects of the above peptides, X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 or X 8 At least one of them is lysine (K).

[0015] The present disclosure also relates to cyclic or linear peptides, wherein the peptide comprises or consists of the amino acid sequence SGVFTFGPVNFPG (SEQ ID NO: 4) or SGVFYFWPVNFPG (SEQ ID NO: 5).

[0016] In certain aspects of any of the above peptides, the peptide further comprises one or more additional amino acids, wherein at least one of the additional amino acids is lysine (K).

[0017] The present disclosure also relates to a cyclic peptide or a linear peptide comprising or consisting of an amino acid sequence of SGVFTFGPVNFPG (SEQ ID NO: 4) or SGVFYFWPVNFPG (SEQ ID NO: 5), except that the sequence has one or two changes selected from amino acid substitutions, single amino acid additions; single amino acid deletions, or a combination of both. In some aspects, the sequence has one or two amino acid substitutions, but no amino acid additions or amino acid deletions. In some aspects, the sequence has only one amino acid substitution. Moreover, in some aspects, the amino acid substitutions are conservative amino acid substitutions.

[0018] In certain aspects of any of the above peptides, the peptide further comprises a dye, a chelator, a radionuclide, or any combination of a dye, a chelator, and a radionuclide.

[0019] In some aspects of any of the above peptides, the peptide binds to HIV-1 capsid protein. In some aspects, the peptide binds to the binding pocket of HIV-1 capsid protein between the C-terminal domain (CTD) and the N-terminal domain (NTD) of the capsid protein. In some aspects, the HIV-1 capsid protein has an amino acid sequence comprising:

[0020]

[0021] And the peptide interacts with residues of a capsid protein comprising L56 of SEQ ID NO:1, N57 of SEQ ID NO:1, M66 of SEQ ID NO:1, or any combination thereof. In some aspects, the binding affinity of the peptide to HIV-1 capsid protein is at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times higher than that of the capsid inhibitor PF74. In some aspects, the peptide inhibits assembly and / or disassembly of HIV-1 capsids. In some aspects, the IC50 of the peptide is about 2 μM to about 4 μM. In some aspects, the IC50 of the peptide is about 3.2 μM.

[0022] In certain aspects of any of the above peptides, the peptide is 50 amino acids or less, 40 amino acids or less, 30 amino acids or less, 25 amino acids or less, 20 amino acids or less, 19 amino acids or less, 18 amino acids or less, 17 amino acids or less, 16 amino acids or less, 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, or has a length of 8 amino acids.

[0023] In certain aspects, the peptide is a linear peptide.

[0024] In some aspects, the peptide is a cyclic peptide. A method for producing a cyclic peptide of the present disclosure is also provided, the method comprising producing a linear peptide of the desired sequence of the cyclic peptide, and cyclizing the linear peptide to produce the cyclic peptide. In some aspects, the linear peptide is chemically synthesized. In some aspects, the linear peptide is translated in a host cell. Therefore, a polynucleotide isolated from a nucleic acid encoding any peptide of the present disclosure is also provided. In some aspects, the isolated polynucleotide also comprises a heterologous nucleic acid. In some aspects, the heterologous nucleic acid comprises a promoter operably connected to the nucleic acid encoding the peptide. A vector comprising any of the above-mentioned polynucleotides is provided in some aspects. In some aspects, the vector is a plasmid. A host cell comprising such a vector is also provided in some aspects, such as a bacterium, an insect cell, a mammalian cell or a plant host cell.

[0025] Also provided herein are pharmaceutical compositions comprising any of the peptides disclosed herein and methods of treating HIV infection in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of the composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A 、B. Figure 1A and 1B The structures of HIV-1 CA protein and representative CA inhibitors are shown. (A) This figure was generated from the X-ray crystal structure of the native HIV-1 capsid protein bound to PF74 (PDB entry 4XZF) (Gres et al. 2015). NTD: N-terminal domain, CA-CTD: C-terminal domain. (B) Chemical structures of selective CA inhibitors. The structures of the CA inhibitors shown here have either been solved in complex with CA or we have used them for docking protocols.

[0027] Figure 2A-D. Figure 2 shows the molecular model of the CA / GS-CA1 complex. (A) Docked pose of GS-CA1 in the CA-hexamer (dimer only shown). (B) Close-up view of the predicted GS-CA1 binding site in the CA-hexamer. The side chains of CA and GS-CA1 are presented as spheres and sticks. The main chain of CA is presented as a ribbon. Residues with orange carbons depict the location of GS-CA1 resistance mutations (Perrier et al., 2017). (C) Detailed view of these residues and their proximity to GS-CA1. (D) Other residues of CA interact with GS-CA1. GS-CA1 carbons in this and subsequent figures are shown in green. Nitrogen, oxygen, sulfur, and fluorine atoms are shown in blue, red, yellow, and turquoise, respectively.

[0028] Figure 3A-E. (A) Superposition of GS-CA1 and GS-6207. The attachment position of the cyclopentapyrazole ring is shown with a dotted circle. (B) Differences in the side chain conformation of K70 and R173 between the CA / GS-CA1 and CA / GS-6207 complexes. The solid arrows show the displacement of the Nζ atom of K73 in the two complexes, while the dotted lines show the H-bond formed by GS-6207 with K70. This interaction is missing in the CA / GS-CA1 complex. (C) Superposition of the CA / PF74 crystal structure (PDB entry 4XZF) on the CA / GS-CA1 molecular model. Resistance mutations associated with PF74 near the binding pocket are shown in cyan carbons. Resistance-associated residues of GS-CA1 are shown in Figure 2. (D) Rotated view of the A face about 45°. The dotted circles show the superposition of the three structural components of GS-CA1 and PF74. (E) Superposition of GS-CA1, PF74, and BI-2.

[0029] Figure 4A,B .(A) Superposition of the CA / CPS6 crystal structure on the CA / GS-CA1 molecular model. For clarity, residues P313-P316 and the backbone atoms of CPSF6 are omitted. The figure shows the superposition of the difluorobenzyl ring of GS-CA1 on F321 of CPSF6. (B) Superposition of the CA / NUP153 crystal structure complex on the CA / GS-CA1 molecular model. For clarity, only residues F1415-G1418 are shown. The figure shows the superposition of the difluorobenzyl ring of GS-CA1 on F1417 of NUP153. In addition, F1415 of NUP153 and the mesyl group of GS-CA1 have a common interaction with P38 of the CA-hexamer. For clarity, the atoms of P38 are not shown.

[0030] Figure 5.Superposition of the CA / CAP-1 crystal structure complex on the CA / GS-CA1 molecular model. For clarity, only M66 is shown in both structures (orange – CA / GS-CA1; cyan – CA / CAP-1).

[0031] Figure 6A,B Figure 6 shows that Pep-2-loop binds HIV-1 capsid better than the well-studied inhibitor PF74. Binding affinities of CA for pep-2-loop (A) and PF74 (B) determined by microthermophoresis. The figure shows the fluorescence changes due to thermophoresis at increasing concentrations of Pep-2-loop and PF74 (1 nM to 2000 nM) in the presence of 200 nM CA-hexamer.

[0032] Figure 7 . Figure 7 The inhibitory activity of Pep-2-loop in cell culture was shown.

[0033] Figure 8 . Figure 8 The inhibitory activity of Pep-2-loop in a humanized mouse model was shown to be comparable to that of the approved drug Rilpivirine (NNRTI) and the capsid inhibitor PF74. Pep-2-loop inhibits HIV-1, EC 50 Comparable to rilpivirine but much better than the most studied capsid inhibitor PF74 (lines represent median EC50) (results from 15 mice) - 2 mice each in the rilpivirine and PF74 arms were euthanized, and 1 mouse died in the Pep-2-ring group.

[0034] Fig. 9 . Fig. 9 The changes in plasma Pep-2-ring and PF74 concentrations over time in the efficacy study are shown. Symbols are the mean ± SD times of the mouse serum protein-binding-modulated EC95 (paEC95) value for each compound (results from 3 mice).

[0035] Fig.10 . Fig.10 is a list of representative peptide sequences of the present disclosure. DETAILED DESCRIPTION

[0036] definition

[0037] It should be noted that the term "a" or "an" entity refers to one or more of the entity; for example, "a cyclic peptide" is understood to represent one or more cyclic peptides. Similarly, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

[0038] In addition, "and / or" as used herein should be considered as a specific disclosure of each specific feature or component with or without the others. Therefore, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B and / or C" is intended to include each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0039] It will be understood that wherever the language “comprising” or “comprises” is used herein to describe any aspect, additional provision is also made for describing similar aspects using the terms “consisting of,” “consists of,” “consisting essentially of,” and / or “consists essentially of.”

[0040] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0041] Numerical ranges include the numbers defining the range. Even when not explicitly identified with "and any range therebetween" etc., where a series of values, such as 1, 2, 3 or 4, is recited, the disclosure specifically includes any range between the values, such as 1 to 3, 1 to 4, 2 to 4, etc., unless otherwise indicated.

[0042] The headings provided herein are for convenience of reference only and are not limitations of the various aspects or aspects of the disclosure, which can be obtained by reference to the specification as a whole.

[0043] As used herein, the term "polypeptide" is intended to include a single "polypeptide" as well as a plurality of "polypeptides", and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also referred to as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids, rather than a product of a specific length. Therefore, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms used to refer to one or more chains of two or more amino acids are included in the definition of "polypeptide", and the term "polypeptide" can be used to replace any of these terms or to be used interchangeably therewith. The term "polypeptide" also means a product modified after the expression of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage or modification by non-standard amino acids. The polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.

[0044] As used herein, "cyclic peptide" refers to a peptide that is not composed of a linear amino acid sequence but is cyclized. For example, in certain aspects, a cyclic peptide can be formed by a lactam bond between a carboxyl C-terminus and an amine N-terminus.

[0045] As used herein, "polar uncharged amino acids" refer to amino acids whose side chains carry polar chemical functions that are different from charged amino acids. Thus, polar uncharged amino acids include: serine (S), threonine (T), cysteine ​​(C), asparagine (N), glutamine (Q) and tyrosine (Y).

[0046] As used herein, "hydrophobic amino acid" refers to an amino acid with a hydrophobic side chain. Thus, hydrophobic amino acids include: glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M) and tryptophan (W).

[0047] As used herein, "protein" may refer to a single polypeptide, ie, a single amino acid chain as defined above, but may also refer to two or more polypeptides associated by, for example, disulfide bonds, hydrogen bonds or hydrophobic interactions to produce a multimeric protein.

[0048] An "isolated" polypeptide or fragment, variant or derivative thereof is one that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. As disclosed herein, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated, as are recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0049] The term "polynucleotide" is intended to include a single nucleic acid as well as multiple nucleic acids, and refers to an isolated nucleic acid molecule or structure, such as messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide may contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, as found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid fragments present in a polynucleotide, such as a DNA or RNA fragment. An "isolated" nucleic acid or polynucleotide refers to a nucleic acid molecule, DNA or RNA removed from its natural environment. For example, as disclosed herein, a recombinant polynucleotide encoding a polypeptide subunit contained in a vector is considered to be isolated. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or polynucleotides purified (partially or in large quantities) in a solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides. Isolated polynucleotides or nucleic acids also include such molecules produced synthetically. In addition, a polynucleotide or nucleic acid may be or may include a regulatory element, such as a promoter, a ribosome binding site or a transcription terminator.

[0050] In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide may be RNA.

[0051] The terms used herein, "sequence identity" or "% identity" to a reference sequence, etc., refer to the relationship between two or more polynucleotide sequences or between two or more polypeptide sequences. When a position in a sequence is occupied by the same nucleic acid base or amino acid at the corresponding position of the comparison sequence, the sequences are considered to be "identical" at that position. The "sequence identity" percentage is calculated by measuring the number of positions where the same nucleic acid base or amino acid appears in the two sequences to obtain the number of "identical" positions. The number of "identical" positions is then divided by the total number of positions in the comparison window and multiplied by 100 to obtain the percentage of "sequence identity". The percentage of "sequence identity" is determined by comparing two optimally aligned sequences and a reference polynucleotide or polypeptide within a comparison window. In order to optimally align the sequences for comparison, the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions called gaps, while the reference sequence remains constant. Optimal alignment refers to an alignment that produces as many "identical" positions as possible between the reference sequence and the comparison sequence, even if there are gaps. The percentage of "sequence identity" between two sequences can be determined using the program "BLAST 2 Sequences" version available from the National Center for Biotechnology Information as of September 1, 2004, which incorporates the programs BLASTN (for nucleotide sequence comparisons) and BLASTP (for polypeptide sequence comparisons), both of which are based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993). When using "BLAST 2 Sequences", parameters that are the default parameters as of September 1, 2004 may be used for word length (3), open gap penalty (11), extension gap penalty (1), gap reduction (50), expectation value (10), and any other desired parameters, including but not limited to the matrix option.

[0052] A "vector" is a nucleic acid molecule that is introduced into a host cell to produce a transformed host cell. A vector may include a nucleic acid sequence that allows it to replicate in the host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic factors known in the art.

[0053] A "transformed" cell or "host" cell is a cell into which a nucleic acid molecule is introduced by molecular biology techniques. As used herein, the term transformation encompasses those techniques by which nucleic acid molecules can be introduced into such cells, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. The transformed cell or host cell can be a bacterial cell or a eukaryotic cell.

[0054] The term "expression" as used herein refers to the process by which a gene produces a biochemical substance (e.g., a polypeptide). The process includes any manifestation of the functional presence of a gene in a cell, including but not limited to gene knockout, transient expression, and stable expression. It includes but is not limited to transcribing a gene into a messenger RNA (mRNA), and translating this mRNA into a polypeptide. If the final desired product is a biochemical substance, expression includes the production of the biochemical product and any precursors. The expression of a gene produces a "gene product". As used herein, a gene product can be a nucleic acid, such as a messenger RNA produced by gene transcription, or a polypeptide translated from a transcript. Gene products described herein also include nucleic acids with post-transcriptional modifications (e.g., polyadenylation), or polypeptides with post-translational modifications (e.g., methylation, glycosylation, lipid addition, association with other protein subunits, proteolytic cleavage, etc.).

[0055] As used herein, the terms "treat," "treatment," or "treatment of" (e.g., in the phrase "treating a subject") refer to reducing the likelihood of disease pathology, reducing the occurrence of disease symptoms, such as to a certain extent resulting in a longer survival rate or reduced discomfort in the subject. For example, treatment can refer to the ability of a treatment to reduce disease symptoms, signs, or causes when administered to a subject. Treatment also refers to alleviating or reducing at least one clinical symptom and / or inhibiting or delaying the progression of a condition and / or preventing or delaying the onset of a disease or disorder.

[0056] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis or treatment is desired. Mammalian subjects include humans, livestock, farm animals, sports animals, and zoo animals, including, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears, etc.

[0057] The term "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective and which contains no other components which are unacceptably toxic to the subject to which the composition is administered. Such compositions may be sterile.

[0058] An "effective amount" of an antibody disclosed herein is an amount sufficient to achieve a specific stated purpose (eg, a "therapeutically effective amount"). An "effective amount" can be determined empirically and in a routine manner, and is relevant to the stated purpose.

[0059] As used herein, "a cyclic peptide or a linear peptide of the present disclosure" and the like refers to any and all aspects or embodiments of the cyclic peptide or the linear peptide described herein, unless otherwise specified.

[0060] As used herein, HIV-1 capsid "assembly" refers to the formation of the capsid core from multiple subunits, and "disassembly" refers to the dissolution of the capsid core to release the HIV genome into the cytoplasm.

[0061] Overview

[0062] The crystal structure of a CA hexamer bound to GS-CA1 has been reported but not yet published. GS-CA1 is reported to bind CA in the same site as PF74, CPSF6, and NUP153 (Tse et al., 2017). The crystal structure of CA in complex with GS-6207 has not been reported. Although not fully understood, it is possible that GS-CA1 and GS-6207 interact more extensively with CA than PF74, providing a stronger binding affinity and thus being more potent than PF74. Here, we present details of the interaction between GS-CA compounds and CA using computational methods and reported inhibitor-bound CA structures. We found that GS-CA compounds contain similar features also present in PF74, BI-2, NUP153, and CPSF6. Using the same structural features in our computational model, we designed a cyclic peptide (Pep-2-loop) that docked in the GS-CA binding site with comparable docking scores. We validated the binding of Pep-2-loop to CA by determining its CA binding affinity using microscale thermophoresis (MST) experiments, which showed that CA bound to Pep-2-loop with approximately 7-fold higher affinity than the well-known CA inhibitor PF74.

[0063] Interaction of GS-CA1 and GS-6207 with CA in simulated CA / GS-CA complexes.

[0064] To gain insight into the interaction between CA hexamer and GS-CA1, Induced-Fit Docking (IFD) (Examples) in conjunction with the Maestro of Schrodinger Suite (Schrodinger LLC, New York) was used. FIG2 shows the docking of GS-CA1 (with the best Glide score) in the crystal structure of the native form of CA-hexamer (PDB entry 4XFZ) (Gres et al., 2015). The figure shows that GS-CA1 binds tightly to residues L56, M66, Q67, N74, and A105 (FIG2B and FIG2C). In vitro screening studies have identified the GS-CA1 resistance mutations L56I, M66I, Q67H, N74D, and A105E (Perrier et al., 2017), suggesting that these mutations may affect the binding of GS-CA1 to CA hexamer. Notably, IFD docking was performed without any bias toward L56, M66, Q67, L74, or A105. In addition, in our model of the CA / GS-CA1 complex, CA-NTD residues I37, P38, S41, N53, T54, N57, Q63, L69, K70, I73, T106, T107, Y130, Y169, L172, R173, and Q179 also interact directly with GS-CA1 (Figure 2D). Many of these residues are essential for the binding of small molecules or peptides derived from the host factors CPSF6 and NUP153 (Price et al., 2014).

[0065] The antiviral activity of GS-CA1 was reported to be comparable among clinical isolates of different subtypes in a limited cohort (n = 15) (Tse et al., 2017), indicating that the amino acid residues in the GS-CA1 binding pocket are highly conserved. To assess whether the GS-CA1 binding pocket is conserved among subtypes, the Los Alamos HIV sequence database (World Wide Web URL: https: / / www.hiv.lanl.gov) was used to generate the consensus sequence of CA from HIV-1 subtype C (HIV-1C), which accounts for more than 50% of all HIV-1 infections. The results showed that the GS-CA1 binding site in HIV-1C is highly conserved. Only one substitution was found in HIV-1C (F169) compared to HIV-1B (Y169). The nearest (Cδ) atom of Y169 (or F169 in HIV-1C) is located at an interaction distance of 1.3 Å to GS-CA1. within 3.0 ng / mL, indicating a weak interaction with GS-CA1.

[0066] GS-6207 differs from GS-CA1 in three modifications: (i) the cyclopropane moiety on the sulfonamide group is replaced by a methyl group, (ii) the difluoroethyl group on the indazole ring is replaced by a trifluoroethyl group, and (iii) the difluoromethyl group on the cyclopentadienylpyrazole ring is replaced by a trifluoromethyl moiety. The specific mechanism of these substitutions is currently unclear. GS-6207 was docked into the crystal structure in the native form of CA (Gres et al., 2015). The results showed that GS-6207 binds in the same binding pocket as GS-CA1 and has a slightly better Glide score (-14.362 for GS-6207 vs. -11.271 for GS-CA1), indicating better binding affinity. It was also noted that the orientation of the cyclopentadienylpyrazole ring in the docked GS-6207 was shifted by ∼180° compared to GS-CA1, resulting in the trifluoromethyl moiety being exposed to the solvent (Figure 3A). Another significant difference between the docked complexes of CA / GS-CA1 and CA / GS-6207 is the conformation of the side chains of K70 and R173. In the CA / GS-6207 complex, the side chain of K70 moves approximately 2.5% toward the binding pocket from its position in the CA / GS-CA1 complex (Figure 3B, solid arrow). It also forms a hydrogen bond with the C=O of the amide group in GS-6207 (Fig. 3B, dotted line). The other H bonds may be one of the reasons why the Glide score of GS-6207 is better than that of GS-CA1. Although the side chain conformation of R173 also changes (Fig. 3B), it does not seem to be significant.

[0067] Comparison with PF74 / CA and BI-2 / CA crystal structures

[0068] Five mutations (Q67H, K70R, T107N, L111I, and H87P) confer resistance to PF74 (Blair et al., 2010; Shi et al., 2011; Shi et al., 2015; and Zhou et al., 2015). Residues Q67, K70, T107, and L111 are located on helices 4 and 5, while H87 is part of the CypA binding loop (residues 85–93) (Gamble et al., 1996; and Ambrose and Aiken, 2014). The only common resistance mutation between GS-CA1 and PF74 is Q67H (Perrier et al., 2017), although other GS-CA1 resistance residues (L56, M66, L74, and A105) are also within interaction distance of PF74. Figure 3C shows the superposition of the CA / PF74 crystal structure (Gres et al., 2015) and the CA / GS-CA1 model. It can be clearly seen from the figure that all three rings of PF74 (two benzene rings and one indole ring) superimpose very well on three different rings of GS-CA1 (dashed circles 1, 2, and 3 in Figure 3D). The PF74 indole ring superimposes on the cyclopentadienylpyrazole ring of GS-CA1 (ring 1). One of the two benzene rings of PF74 superimposes on the difluorobenzene ring of GS-CA1 (ring 2), while the other PF74 benzene ring is located in a position that is topologically similar to the indazole ring of GS-CA1 (ring 3). In addition, the polar part of PF74 topologically matches the polar part of GS-CA1. Therefore, the acetamide part of GS-CA1 superimposes well with the corresponding part of PF74. These data suggest that there are certain common structural features and interactions between GS-CA1 and PF74.

[0069] During the entry of GS-CA1 into the IFD of the CA-hexamer, the conformations of most side chains in the GS-CA1 / PF74 binding pocket did not change significantly compared with the CA / PF74 crystal structure, with the exception of the K70 side chain (Figure 3C). The position of the K70 Nζ atom was shifted from its position in the CA / PF74 complex. (Figure 3C), indicating that there is no interaction between K70 and GS-CA1, in contrast to the interaction between K70 and PF74. The absence of this interaction may be the reason why K70 did not mutate during the in vitro resistance selection studies of GS-CA1 (Perrier et al., 2017). As mentioned above, the interaction of K70 is restored in the CA / GS-6207 complex. At present, the characteristics of the resistance mutations of GS-6207 are unknown.

[0070] BI-2 is one of two compounds in the 4,5-dihydro-1H-pyrrolo[3,4-c]pyrazol-6-one series that have been shown to bind to CA hexamer. BI-2 has been shown to stabilize CA hexamer and inhibit HIV-1 early in infection (Lamorte et al., 2013). Selection of viruses resistant to BI-2 identified mutations at residues A105 and T107 of the CA-NTD (Lamorte et al., 2013). The high-resolution structure of CA in complex with BI-2 showed that it binds in the PF74 binding site. Superposition of the three compounds (GS-CA1, PF74, and BI-2) obtained by superposition of the Cα-atoms of the CA-NTD showed that the three compounds share a common binding mode with the CA-hexamer (Figure 3E). The docking results of GS-CA1 showed that CA residue A105 is within the interaction distance of GS-CA1, and the common resistance mutation A105T between GS-CA1 and BI-2 further confirms that the two compounds share part of the binding site.

[0071] Comparison with the CPS6 / CA and NUP153 / CA crystal structures

[0072] The crystal structure of CA in complex with short peptides derived from CPSF6 and NUP153 showed that both peptides share a binding pocket occupied by PF74 and BI-2 (Price et al., 2014), although the bound peptides have other interactions. To determine whether GS-CA1, CPSF6, and NUP153 share common structural features when bound to CA, the crystal structures of CA / CPSF6 and CA / NUP153 were superimposed on a simulated CA / GS-CA1 complex. Figure 4 shows this superposition, demonstrating that the conformation of GS-CA1 docked in the CA-hexamer follows the fold of the CPSF6 peptide (Figure 4A). The F321 side chain of CPSF6 superimposes perfectly on the difluorobenzyl moiety of GS-CA1.

[0073] Similar to CPSF6, the NUP153 backbone follows the conformation of GS-CA1, with F1417 of NUP153 perfectly superimposed on the difluorobenzyl moiety of GS-CA1 (Figure 4B). In addition, there is a hydrophobic interaction between the mesyl moiety of GS-CA1 and P38 of CA (atoms of P38 not shown). Similar interactions were noted between F1415 and P38 of NUP153 (Figure 4B).

[0074] Comparison with CA / CAP-1, CA / BD and CA / BM complexes

[0075] CAP-1 1-(3-chloro-4-methylphenyl)-3-(2-(((5-((dimethylamino)methyl)furan-2-yl)methyl)thio)ethyl)urea is an inhibitor of assembly for which no resistance mutations have been reported (Kelly et al., 2007). The structure of CAP-1 bound to CA-NTD has been solved by NMR and X-ray crystallography (Kelly et al., 2007). Comparison of the crystal and NMR structures demonstrated that CA undergoes significant conformational changes upon CAP-1 binding. Superposition of the crystal structure of the CA / CAP-1 complex on the modeled structure of the CA / GS-CA1 complex showed that the two inhibitors do not bind at a common site ( Figure 5 ). However, two residues (M66 and L69) interacted with both GS-CA1 and CAP-1. Figure 5 The position of M66 in the CA / GS-CA1 and CA / CAP-1 complexes is shown. The benzodiazepine (BD1-BD4) and benzimidazole (BM1-BM5) series of compounds bind to CA in a site close to the CAP-1 binding site (Lemke et al., 2012). Although compounds from both series have been shown to bind in the same pocket, they have different resistance mutational profiles. Mutations V36T and G61E select for BD inhibitors, whereas K30R and S33G select for BM inhibitors. Both V36 and G61 are part of the BM3 binding pocket (PDB entry 4E91) (Sticht et al., 2005). K30 is not within interaction distance of BM4, and the main chain carbonyl of S33 forms only van der Waals interactions with BM4 (PDB entry 4E92) (Lemke et al., 2012). Therefore, the resistance mechanism of BM4 does not appear to operate through direct interactions. The crystal structures of BD-3 and BM4 bound to CA-NTD showed that both compounds were within the interaction distance of M66, similar to CAP-1. Thus, the BD and BM series compounds do not share a binding site with GS-CA1, but they both share a common interaction with M66.

[0076] Coumermycin A1 bound to CA-hexamer

[0077] Coumamycin A1 (C-A1) is a gyrase B inhibitor that also inhibits HSP90 (Vozzolo et al., 2010; reviewed in Carnes et al., 2018). The crystal structure of CA / C-A1 has not yet been solved. However, docking studies predict that C-A1 binds in a pocket formed by two adjacent capsid monomers (Chen et al., 2016). This predicted binding site is likely relevant because mutations N74D and A105S confer resistance to C-A1 and both (N74 and A105) are located at the interface of two capsid monomers.

[0078] IFD was used to assess the details of the interaction between C-A1 and CA. Of the 32 docking modes predicted for C-A1 in the same PDB file (4XZF) as used by Chen et al., 2016, none were within the interaction distance of N74 or A105. The docking data predicted that the resistance of N74D and A105S to C-A1 is likely not due to binding defects conferred by mutations at these residues.

[0079] Comparison of other small molecule inhibitors of CA and their binding to GS-CA1

[0080] Several other CA inhibitors have been reported, such as CK026, I-XW-053, compound 34 (Kortagere et al., 2012), C1 (Lemke et al., 2013), and ebselen (Lemke et al., 2013). CK026 is a large molecule and has not been shown to inhibit HIV-1 in PBMCs. However, I-XW-053 and compound 34 (derivatives of CK026) exhibited inhibitory activity in PBMCs (Kortagere et al., 2014). The crystal structures of CA in complex with these compounds have not yet been solved. However, docking results combined with binding affinity measurements by surface plasmon resonance showed that compound 34 binds near P38, S41, R173, K170, and Q179 (Kortagere et al., 2014). All of these residues are within the interaction distance of GS-CA1 in the simulated CA / GS-CA1 complex (Figure 2).

[0081] Compound C1 has been shown to bind at a unique site near the CypA binding loop and affect late steps by disrupting the proper assembly of the mature capsid (Lemke et al., 2013). However, crystal structures of CA in the presence of compound C1 and the BD series of compounds revealed that C1 induces the formation of CA dimers and binds at the interface of the dimers. The mutation R132T confers resistance to C1. In the crystal structures of C1 and the BD / BM compounds, R132 forms polar interactions with compound C1. These structures also show that C1 contacts the N-terminus of helix 2 and forms hydrophobic interactions with P34, G35, I37, and P38. The benzoic acid moiety forms a direct hydrogen bond with A139 and a water-mediated hydrogen bond with S41 (Lemke et al., 2013). Both I37 and P38 form hydrophobic interactions with GS-CA1 (Figure 2).

[0082] Ebselen is a small molecule discovered during the search for inhibitors of CA dimerization. Electrospray ionization mass spectrometry experiments showed that ebselen is covalently bound to CA-CTD most likely through a selenylsulfide linkage involving C198 and C218 (Thenin-Houssier et al, 21016). Both residues are part of CA-CTD and are not within the interaction distance of GS-CA1 in our simulated CA / GS-CA1 complex. Therefore, it is predicted that the ebselen and GS-CA1 binding sites will not overlap.

[0083] Docking of designed cyclic peptide inhibitor (Pep-2-loop)

[0084] Using the crystal structures of PF74, NUP153, CPSF6, and BI-2-bound CA and the structure of a simulation of the CA / GS-CA1 (CA / GS-6207) complex, a cyclic peptide (Pep-2-loop: Cyc(SGVFTFGPVNFPG); SEQ ID NO:4) was designed containing common structural components / groups in CA-binding small molecules or peptides derived from CPSF6 and NUP153. Docking of the Pep-2-loop showed that it binds in a pocket shared by PF74, NUP153, CPSF6, GS-CA1, and GS-6207. Table 1 lists the superimposed structural components in the different complexes.

[0085] Table 1. Common structural groups / components in different CA complexes.

[0086]

[0087] 1 A portion of G318-Q319 is topologically close to the indole ring of PF74.

[0088] The designed peptides appear to share binding sites and chemical moieties that may inhibit CA function.

[0089] Binding affinity of CA to Pep-2-ring and PF74

[0090] pep-2-cyclo was synthesized by chemical methods. The intermediate linear scaffold was prepared by solid phase synthesis starting from the very acid labile 2-ClTrt resin using the Fmoc / Tbu protocol. After the linear sequence was assembled, the fully protected peptide was removed from the resin by treatment with a mixture of 1% TFA and 5% TIS. Subsequently, the peptide was cyclized by coupling the C-terminal carboxylic acid and the N-terminal amino group. The reaction was completed using PyBOP (2.5-fold excess) in the presence of DIPEA (6-fold excess) in DMF at room temperature for 6 hours. After verification of the cyclization by LC-MS, the final crude target molecule was obtained by treating the protected cyclized intermediate with a mixture of TFA and scavengers to remove other protecting groups. Preparative HPLC purification and lyophilization gave the final purified peptide, which was characterized by LC-MS.

[0091] The binding affinity of CA for Pep-2-ring and PF74 was determined using a microscale thermophoresis (MST) assay. MST is based on thermophoresis, the directional movement of molecules under a temperature gradient, depending on various molecular properties, including size, charge, hydration layer or conformation. It is therefore highly sensitive to almost any change in molecular properties, allowing for the precise quantification of molecular events independent of the size or properties of the sample studied (Jerabek-Willemsen, 2014). During the MST experiment, a temperature gradient is induced by an infrared laser. The directional movement of molecules through the temperature gradient is detected and quantified using a covalently attached fluorophore. Figures 6A and 6B show the binding isotherms obtained by plotting the difference in normalized fluorescence against increasing concentrations of Pep-2-ring and PF74, respectively. Pep-2-ring (K d.pep-2-环 ) and PF74(K d.PF74 The binding affinity of ) to CA was estimated by fitting the data points to a quadratic equation (Equation 1). The K d.pep-2-环 was 32±3nM, and K d.PF74 The binding affinity of CA-hexamer to PF74 was previously determined by isothermal calorimetry (ITC) to be 262 nM (Bhattacharya et al., 2014), which is consistent with the K determined by MST in this paper. d.PF74 These data indicate that Pep-2-Ring binds CA with ∼7-fold greater affinity.

[0092] Cyclic peptides

[0093] Therefore, certain aspects of the present disclosure relate to a system comprising F-X 1 –F–X 2 –P–V–X 3 -F (SEQ ID NO: 2), wherein X 1 , X 2 and X 3 Each independently is glycine (G), lysine (K) or a polar uncharged amino acid. In certain aspects of SEQ ID NO: 2:

[0094] X 2 is glycine, X 1 and X 3 each is a polar uncharged amino acid; and / or

[0095] X 1 is threonine (T) and / or X 3 It is asparagine (R).

[0096] In certain aspects of the cyclic peptides disclosed herein, the cyclic peptide comprises X 4 -X 5 -X 6 -FX 1 -FX 2 -PVX 3 -FX 7 -X 8 (SEQ ID NO: 3), wherein the sequence FX 1 -FX 2 -PVX 3 -F (SEQ ID NO: 2) is as defined above, and wherein X 4 , X 5 , X 6 , X 7 and X 8 Each is independently selected from the group consisting of glycine (G), proline (P), lysine (K), polar uncharged amino acids and hydrophobic amino acids. In certain aspects of SEQ ID NO: 3:

[0097] X 4 It is a polar uncharged amino acid;

[0098] X 4 is serine (S);

[0099] X 6 It is a hydrophobic amino acid;

[0100] X 6 is valine (V);

[0101] X 7is proline (P); and / or

[0102] X 5 and / or X 8 It is glycine (G).

[0103] In certain aspects of SEQ ID NO: 2 and / or SEQ ID NO: 3, X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 or X 8 At least one of them is lysine (K).

[0104] In some aspects, the cyclic peptides of the present disclosure comprise the amino acid sequence of SGVFTFGPVNFPG (SEQ ID NO: 4). In some aspects, the cyclic peptides of the present disclosure consist of the amino acid sequence of SGVFTFGPVNFPG (SEQ ID NO: 4). In some aspects, the cyclic peptides of the present disclosure comprise the amino acid sequence of SGVFYFWPVNFPG (SEQID NO: 5). In some aspects, the cyclic peptides of the present disclosure consist of the amino acid sequence of SGVFYFWPVNFPG (SEQID NO: 5).

[0105] In certain aspects, the cyclic peptide comprising SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and / or SEQ ID NO: 5 further comprises one or more additional amino acids. In certain aspects, at least one of the additional amino acids is lysine (K).

[0106] Certain aspects relate to cyclic peptides comprising the amino acid sequence of SGVFTFGPVNFPG (SEQ ID NO: 4) or SGVFYFWPVNFPG (SEQ ID NO: 5), except that the sequence has one or two changes selected from amino acid substitutions, single amino acid additions; single amino acid deletions, or a combination of two thereof. Certain aspects relate to cyclic peptides consisting of the amino acid sequence of SGVFTFGPVNFPG (SEQ ID NO: 4) or SGVFYFWPVNFPG (SEQ ID NO: 5), except that the sequence has one or two changes selected from amino acid substitutions, single amino acid additions; single amino acid deletions, or a combination of two thereof. That is, there may be up to two single amino acid additions, two single amino acid additions, or two single amino acid deletions, as long as the number of changes does not exceed two. That is, there may be one or any combination of a single amino acid substitution, a single amino acid addition, and a single amino acid deletion, as long as the number of changes does not exceed two. In certain aspects, the sequence has one or two amino acid substitutions, but no amino acid additions or amino acid deletions. In certain aspects, the sequence has only one amino acid substitution. In certain aspects, the amino acid substitutions are conservative amino acid substitutions recognized in the art. In some aspects, the cyclic peptide further comprises one or more other amino acids. In some aspects, at least one of the other amino acids is lysine (K).

[0107] In certain aspects of any of the cyclic peptides disclosed herein, the cyclic peptide is 50 amino acids or less, 40 amino acids or less, 30 amino acids or less, 25 amino acids or less, 20 amino acids or less, 19 amino acids or less, 18 amino acids or less, 17 amino acids or less, 16 amino acids or less, 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, or has a length of 8 amino acids. In certain aspects of any of the cyclic peptides disclosed herein, the cyclic peptide has a length of 50 amino acids or less but not less than 8, 40 amino acids or less but not less than 8, 30 amino acids or less but not less than 8, 25 amino acids or less but not less than 8, 20 amino acids or less but not less than 8, 19 amino acids or less but not less than 8, 18 amino acids or less but not less than 8, 17 amino acids or less but not less than 8, 16 amino acids or less but not less than 8, 15 amino acids or less but not less than 8, 14 amino acids or less but not less than 8, 13 amino acids or less but not less than 8, 12 amino acids or less but not less than 8, 11 amino acids or less but not less than 8, 10 amino acids or less but not less than 8, 9 amino acids or less but not less than 8 or has a length of 8 amino acids.

[0108] In certain aspects, the cyclic peptides of the present disclosure further comprise dyes, chelators, radionuclides, or any combination of dyes, chelators, and radionuclides, for example, for in vitro or in vivo imaging and / or therapeutic purposes. Examples of dyes include, but are not limited to, fluorescein, tetramethylrhodamine, and near-IR dyes (e.g., Alexafluor and Cy series, such as Cy5, Cy5.5, and Cy7). Examples of chelators include, but are not limited to, DOTA, NOTA, DFO, and TCMC. Examples of radionuclides include, but are not limited to 18 F. 111 In, 99 mTc, 64 Cu, 89 Zr, 177 Lu, 225 Ac, 212 Pb and 203 Pb.

[0109] In some aspects, the cyclic peptides of the present disclosure bind to HIV-1 capsid protein. In some aspects, the amino acid sequence of HIV-1 capsid protein comprises:

[0110]

[0111] In certain aspects, the amino acids of the HIV-1 capsid protein comprise a sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 1. In certain aspects, the amino acids of the HIV-1 capsid protein comprise a sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 1, wherein the binding pocket is conserved with SEQ ID NO: 1.

[0112] In some aspects, the cyclic peptide binds to the binding pocket of the HIV-1 capsid protein located between the C-terminal domain (CTD) and the N-terminal domain (NTD) of the capsid protein. In some aspects, the cyclic peptide interacts with a residue of the HIV-1 capsid protein comprising L56 of SEQ ID NO: 1, N57 of SEQ ID NO: 1, M66 of SEQ ID NO: 1, or any combination thereof.

[0113] In some aspects, the cyclic peptides of the present disclosure have a higher binding affinity to HIV-1 capsid protein (see Examples) than the well-characterized capsid inhibitor PF74. For example, in some aspects, the cyclic peptides of the present disclosure have a higher binding affinity to HIV-1 capsid protein than the capsid inhibitor PF74 by at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times.

[0114] In certain aspects, as described elsewhere herein, the cyclic peptides of the present disclosure inhibit assembly and / or disassembly of HIV-1 capsids. In certain embodiments, the cyclic peptides inhibit HIV-1 capsid assembly and / or disassembly with an IC50 of about 2 μM to about 4 μM. In certain embodiments, the cyclic peptides inhibit HIV-1 capsid assembly and / or disassembly with an IC50 of about 3.2 μM.

[0115] In certain aspects, the cyclic peptides of the present disclosure exhibit inhibitory activity.

[0116] Some aspects provide methods for producing cyclic peptides of the present disclosure. In some aspects, the method includes producing a linear peptide of the desired sequence of the cyclic peptide, and cyclizing the linear peptide to produce the cyclic peptide. In some aspects, the linear peptide is chemically synthesized, such as but not limited to the synthesis described elsewhere herein. In some aspects, the linear peptide is translated in a host cell, such as described elsewhere herein, using isolated polynucleotides, vectors and / or host cells.

[0117] Linear peptides

[0118] Certain aspects of the present disclosure relate to linear peptides, for example, for use as precursors to the cyclic peptides of the present disclosure or as inhibitors themselves. Thus, certain aspects of the present disclosure relate to a peptide comprising F-X 1–F–X 2 –P–V–X 3 -F (SEQ ID NO: 2) amino acid sequence, wherein X 1 , X 2 and X 3 Each independently is glycine (G), lysine (K) or a polar uncharged amino acid. In certain aspects of SEQ ID NO: 2:

[0119] X 2 is glycine, X 1 and X 3 each is a polar uncharged amino acid; and / or

[0120] X 1 is threonine (T) and / or X 3 It is asparagine (R).

[0121] In certain aspects of the linear peptides disclosed herein, the linear peptide comprises a sequence X 4 -X 5 -X 6 -FX 1 -FX 2 -PVX 3 -FX 7 -X 8 (SEQ ID NO: 3), wherein the sequence FX 1 -FX 2 -PVX 3 -F (SEQ ID NO: 2) is as defined above, and wherein X 4 , X 5 , X 6 , X 7 and X 8 Each is independently selected from the group consisting of glycine (G), proline (P), lysine (K), polar uncharged amino acids and hydrophobic amino acids. In certain aspects of SEQ ID NO: 3:

[0122] X 4 It is a polar uncharged amino acid;

[0123] X 4 is serine (S);

[0124] X 6 It is a hydrophobic amino acid;

[0125] X 6 is valine (V);

[0126] X 7 is proline (P); and / or

[0127] X 5 and / or X 8 It is glycine (G).

[0128] In certain aspects of SEQ ID NO: 2 and / or SEQ ID NO: 3, X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 or X 8 At least one of them is lysine (K).

[0129] In some aspects, the linear peptides of the present disclosure comprise the amino acid sequence of SGVFTFGPVNFPG (SEQ ID NO: 4). In some aspects, the linear peptides of the present disclosure consist of the amino acid sequence of SGVFTFGPVNFPG (SEQ ID NO: 4). In some aspects, the linear peptides of the present disclosure comprise the amino acid sequence of SGVFYFWPVNFPG (SEQID NO: 5). In some aspects, the linear peptides of the present disclosure consist of the amino acid sequence of SGVFYFWPVNFPG (SEQID NO: 5).

[0130] In certain aspects, the linear peptide comprising SEQ ID NO: 2, SEQ ID NO: 3 and / or SEQ ID NO: 4 further comprises one or more additional amino acids. In certain aspects, at least one of the additional amino acids is lysine (K).

[0131] Certain aspects relate to a linear peptide comprising the amino acid sequence of SGVFTFGPVNFPG (SEQ ID NO: 4) or SGVFYFWPVNFPG (SEQ ID NO: 5), except that the sequence has one or two changes selected from amino acid substitutions, single amino acid additions; single amino acid deletions, or a combination of two thereof. Certain aspects relate to a linear peptide consisting of the amino acid sequence of SGVFTFGPVNFPG (SEQ ID NO: 4) or SGVFYFWPVNFPG (SEQ ID NO: 5), except that the sequence has one or two changes selected from amino acid substitutions, single amino acid additions; single amino acid deletions, or a combination of two thereof. That is, there may be two single amino acid additions, two single amino acid additions, or two single amino acid deletions, as long as the number of changes does not exceed two. That is, there may be one or any combination of a single amino acid substitution, a single amino acid addition, and a single amino acid deletion, as long as the number of changes does not exceed two. In certain aspects, the sequence has one or two amino acid substitutions, but no amino acid additions or amino acid deletions. In certain aspects, the sequence has only one amino acid substitution. And, in certain aspects, the amino acid substitutions are conservative amino acid substitutions recognized in the art. In certain aspects, such linear peptides further comprise one or more other amino acids. In certain aspects, at least one of the other amino acids is lysine (K).

[0132] In certain aspects of any of the linear peptides disclosed herein, the linear peptide is 50 amino acids or less, 40 amino acids or less, 30 amino acids or less, 25 amino acids or less, 20 amino acids or less, 19 amino acids or less, 18 amino acids or less, 17 amino acids or less, 16 amino acids or less, 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, or has a length of 8 amino acids. In certain aspects of any of the linear peptides disclosed herein, the linear peptide is 50 amino acids or less but not less than 8, 40 amino acids or less but not less than 8, 30 amino acids or less but not less than 8, 25 amino acids or less but not less than 8, 20 amino acids or less but not less than 8, 19 amino acids or less but not less than 8, 18 amino acids or less but not less than 8, 17 amino acids or less but not less than 8, 16 amino acids or less but not less than 8, 15 amino acids or less but not less than 8, 14 amino acids or less but not less than 8, 13 amino acids or less but not less than 8, 12 amino acids or less but not less than 8, 11 amino acids or less but not less than 8, 10 amino acids or less but not less than 8, 9 amino acids or less but not less than 8, or has a length of 8 amino acids.

[0133] In certain aspects, the linear peptide comprises a C-terminal acid. In certain aspects, the linear peptide comprises a C-terminal amide.

[0134] In certain aspects, the linear peptides of the present disclosure further comprise dyes, chelators, radionuclides, or any combination of dyes, chelators, and radionuclides, e.g., for in vitro or in vivo imaging and / or therapeutic purposes. Examples of dyes include, but are not limited to, fluorescein, tetramethylrhodamine, and near-IR dyes (e.g., Alexafluor and the Cy series, e.g., Cy5, Cy5.5, and Cy7). Examples of chelators include, but are not limited to, DOTA, NOTA, DFO, and TCMC. Examples of radionuclides include, but are not limited to 18 F. 111 In, 99 mTc, 64 Cu, 89 Zr, 177 Lu, 225 Ac, 212 Pb and 203 Pb.

[0135] In some aspects, the linear peptides of the present disclosure bind to HIV-1 capsid protein. In some aspects, the amino acid sequence of HIV-1 capsid protein comprises:

[0136]

[0137] In certain aspects, the amino acids of the HIV-1 capsid protein comprise a sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 1. In certain aspects, the amino acids of the HIV-1 capsid protein comprise a sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 1, wherein the binding pocket is conserved with SEQ ID NO: 1.

[0138] In some aspects, the linear peptide binds to a binding pocket of the HIV-1 capsid protein located between the C-terminal domain (CTD) and the N-terminal domain (NTD) of the capsid protein. In some aspects, the linear peptide interacts with a residue of the HIV-1 capsid protein comprising L56 of SEQ ID NO: 1, N57 of SEQ ID NO: 1, M66 of SEQ ID NO: 1, or any combination thereof.

[0139] In certain aspects, the binding affinity of the linear peptides of the present disclosure to HIV-1 capsid protein (see Examples) is higher than that of the well-characterized capsid inhibitor PF74. For example, in certain aspects, the binding affinity of the linear peptides of the present disclosure to HIV-1 capsid protein is at least about 2 times higher, at least about 3 times higher, at least about 4 times higher, at least about 5 times higher, at least about 6 times higher, at least about 7 times higher, at least about 8 times higher, at least about 9 times higher, or at least about 10 times higher than that of the capsid inhibitor PF74.

[0140] In certain aspects, as described elsewhere herein, the linear peptides disclosed herein inhibit assembly and / or disassembly of HIV-1 capsids. In certain embodiments, the linear peptides inhibit ICs of HIV-1 capsid assembly and / or disassembly. 50 In some embodiments, the linear peptide inhibits HIV-1 capsid assembly and / or disassembly at an IC of about 2 μM to about 4 μM. 50 It is about 3.2μM.

[0141] In certain aspects, the linear peptides of the disclosure exhibit inhibitory activity.

[0142] Certain aspects provide methods for producing linear peptides of the present disclosure. In certain aspects, the linear peptides are chemically synthesized, such as, but not limited to, the synthesis described elsewhere herein. In certain aspects, the linear peptides are translated in a host cell, such as, for example, described elsewhere herein, using isolated polynucleotides, vectors and / or host cells.

[0143] Certain aspects of the present disclosure relate to pharmaceutical compositions comprising cyclic peptides or linear peptides described anywhere herein. In certain aspects, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Certain aspects of the present disclosure relate to methods for treating HIV infection in a subject in need of treatment. In certain aspects, the subject is a mammal. In certain aspects, the subject is a primate. In certain aspects, the subject is a human. In certain aspects, the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition disclosed herein. Examples of methods of administration include, but are not limited to, intravenous, intraperitoneal, inhalation, oral, or topical administration.

[0144] Some aspects of the present disclosure relate to isolated polynucleotides comprising nucleic acids encoding any peptide sequence described herein. In some aspects, the polynucleotides also comprise heterologous nucleic acids. In some aspects, this heterologous nucleic acid comprises a promoter operably connected to a nucleic acid encoding a therapeutic polypeptide. Some aspects provide vectors comprising polynucleotides of the present disclosure. In some aspects, the vector is a plasmid, such as a pET24 plasmid. Some aspects also provide host cells comprising the vector. In some aspects, the host cell is a bacterium, an insect cell, a mammalian cell, or a plant cell. For example, in some aspects, the host cell is a bacterial Escherichia coli.

[0145] Example

[0146] Example 1.

[0147] HIV-1CA structure preparation

[0148] The X-ray crystal structure of the native HIV-1 capsid protein bound to PF74 (PDB entry 4XZF) (Gres et al., 2015) was used to dock GS-CA1 with coumarins A1 (C-A1). The initial structures of GS-CA1 and C-A1 were generated using Chemsketch (Advanced Chemistry Development, Inc., Toronto, Ontario, Canada). Subsequently, these structures were minimized using MACROMODEL and then LIGPREP (Schrödinger, LLC, New York, NY). Inc.). PREPWIZARD (Schrödinger, LLC, New York, NY), which adds hydrogens, assigns bond orders, generates heteroatom states, and samples water molecule conformations, was used to prepare CA-hexamers for docking of GS-CA1 and C-A1.

[0149] Example 2.

[0150] Docking of GS-CA1, GS-6207 and CA-1

[0151] All docking simulations were performed by Schrodinger Suite Master ( Suite) (Schrödinger, LLC, New York, NY). IFD was performed using Glide (Schrödinger, LLC) and the Refinement module in Prime (Schrödinger, LLC, New York, NY) to accurately predict ligand binding modes and accompanying structural changes in the receptor. The receptor mesh generation utility of Glide generated a mesh centered on PF74 in the native form crystal structure of the CA hexamer (PDB file 4XZF). Grids for docking of GS-CA1, GS6207, and C-A1. IFD optimized the side chain conformations to best determine the docking regime. The regime with the best IFD score was selected for comparison purposes.

[0152] Example 3.

[0153] Docking of designed peptide Pep-2-loop

[0154] The structure of the peptide Pep-2-ring was generated by Prime and energy minimized using the MM / GBSA (molecular mechanics-generalized Born surface area) method (Genheden and Ryde, 2010). Peptide docking into the CA-hexamer crystal structure was performed by IFD (Schrödinger LLC, New York, NY). The best scoring complex of CA / Pep-2-ring peptide was selected for analysis. PATCHDOCK (Schneidman-Duhovny et al., 2005) was also used by accessing the PatchDock web server at the World Wide Web address https: / / bioinfo3d.cs.tau.ac.il / PatchDock / to evaluate whether the two software predict different docking conformations of the Pep-2-ring.

[0155] Example 4.

[0156] Microscale thermophoresis (MST) assay

[0157] The binding affinity of CA to Pep-2-ring and PF74 was then determined by measuring thermophoresis of fluorescently labeled CA-hexamers in the presence of increasing concentrations of Pep-2-ring or PF74. The peptide Pep-2-ring was synthesized at MoleculareInteraction Core (University of Missouri), and PF74 was purchased from Sigma-Aldrich (St. Louis, MO, USA). CA was fluorescently labeled with the Alexa Fluor 647 analog NT647 according to the manufacturer's instructions (MO-L004 Whole Protein Labeling Kit; NanoTemper Technologies GmbH, Munich, Germany). 20 μM protein was incubated overnight at room temperature with a 3 molar excess of dye in the conjugation buffer provided with the labeling kit. Unreacted dye was removed by filtration through a gravity flow column provided with the kit. The eluted fractions were collected in 2x MST buffer (100 mM MOPS, pH 7.0, 200 mM NaCl, and 0.2% Pluronic F-127). The fluorescence intensity of each component was evaluated by MST (NanoTemper Technologies GmbH, Munich, Germany, Monolith NT.115), and the components containing the labeled protein were pooled. Protein concentration was determined by NANODROP (Thermo Scientific, Waltham, Massachusetts) spectrophotometer. Aliquots were stored at -80°C until use. The reaction mixture containing 200nM labeled CA and increasing concentrations of Pep-2-ring (1nM to 2000nM) was loaded into a capillary and thermophoresis was monitored at 20% LED power and high MST power with 20 seconds MST on time. MO was used to analyze the data. Affinity software (version 2.3) (NanoTempet Technologies, Canada) was plotted by fitting the data points to a quadratic equation (Equation 1) and by PRISM (version 6.0) (GraphPad Inc. La Jolla, Canada).

[0158]

[0159] Where A is an arbitrary parameter, Kd = [P][CA] / [P-CA], [P] is the concentration of free Pep-2-ring or PF74, [CA] is the concentration of free CA, [CA0] is the concentration of added CA, and P0 is the concentration of added Pep-2-ring or PF74.

[0160] *****

[0161] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following paragraphs, claims and their equivalents.

[0162] paragraph

[0163] 1. A cyclic peptide or a linear peptide, wherein the cyclic peptide or the linear peptide comprises F-X 1 –F–X 2 –P–V–X 3 -F (SEQ ID NO: 2) amino acid sequence, wherein X 1 , X 2 and X 3 Each is independently glycine (G), lysine (K) or a polar uncharged amino acid.

[0164] 2. The cyclic peptide or linear peptide according to paragraph 1, wherein X 2 is glycine, X 1 and X 3 Each is a polar uncharged amino acid.

[0165] 3. A cyclic peptide or a linear peptide according to paragraph 1 or 2, wherein X 1 is threonine (T) and / or X 3 It is asparagine (R).

[0166] 4. A cyclic peptide or a linear peptide according to any one of paragraphs 1 to 3, wherein the peptide comprises X 4 –X 5 –X 6 –F–X 1 –F–X 2 –P–V–X 3 –F–X 7 –X 8 (SEQ ID NO: 3), and wherein X 4 , X 5 , X 6 , X 7 and X 8 Each is independently selected from the group consisting of glycine (G), proline (P), lysine (K), polar uncharged amino acids and hydrophobic amino acids.

[0167] 5. The cyclic peptide or linear peptide according to paragraph 4, wherein X 4 It is a polar uncharged amino acid.

[0168] 6. A cyclic peptide or a linear peptide according to paragraph 4 or 5, wherein X 4 It is serine (S).

[0169] 7. A cyclic peptide or a linear peptide according to any one of paragraphs 4 to 6, wherein X 6 It is a hydrophobic amino acid.

[0170] 8. A cyclic peptide or a linear peptide according to any one of paragraphs 4 to 7, wherein X 6 It is valine (V).

[0171] 9. A cyclic peptide or a linear peptide according to any one of paragraphs 4 to 8, wherein X 7 It is proline (P).

[0172] 10. A cyclic peptide or a linear peptide according to any one of paragraphs 4 to 9, wherein X 5 and / or X 8 It is glycine (G).

[0173] 11. A cyclic peptide or linear peptide according to paragraph 1, wherein the peptide comprises or consists of the amino acid sequence SGVFTFGPVNFPG (SEQ ID NO: 4) or SGVFYFWPVNFPG (SEQ ID NO: 5),

[0174] Optionally, wherein the peptide comprises or consists of the amino acid sequence SGVFTFGPVNFPG (SEQ ID NO: 4),

[0175] Optionally, wherein the peptide comprises or consists of the amino acid sequence SGVFYFWPVNFPG (SEQ ID NO: 5).

[0176] 12. A cyclic peptide or a linear peptide according to any one of paragraphs 1 to 10, wherein X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 or X 8 At least one of them is lysine (K).

[0177] 13. The cyclic peptide or linear peptide according to any one of paragraphs 1 to 12, wherein the peptide further comprises one or more other amino acids, wherein at least one other amino acid is lysine (K).

[0178] 14. A cyclic peptide or a linear peptide comprising or consisting of the amino acid sequence of SGVFTFGPVNFPG (SEQ ID NO: 4) or SGVFYFWPVNFPG (SEQ ID NO: 5), except that the sequence has one or two changes selected from amino acid substitution, single amino acid addition; single amino acid deletion or a combination thereof.

[0179] 15. The cyclic peptide or linear peptide according to paragraph 14, wherein the sequence has one or two amino acid substitutions but no amino acid additions or amino acid deletions.

[0180] 16. The cyclic or linear peptide according to paragraph 15, wherein the sequence has only one amino acid substitution.

[0181] 17. The cyclic peptide or linear peptide according to any one of paragraphs 14 to 16, wherein the amino acid substitution is a conservative amino acid substitution.

[0182] 18. The cyclic peptide or linear peptide of any one of paragraphs 1 to 17, wherein the peptide further comprises a dye, a chelator, a radionuclide, or any combination of a dye, a chelator, and a radionuclide.

[0183] 19. The cyclic peptide or linear peptide according to any one of paragraphs 1 to 18, wherein the peptide binds to HIV-1 capsid protein.

[0184] 20. The cyclic peptide or linear peptide according to paragraph 19, wherein the peptide binds to a binding pocket of HIV-1 capsid protein located between the C-terminal domain (CTD) and the N-terminal domain (NTD) of the capsid protein.

[0185] 21. The cyclic peptide or linear peptide according to paragraph 19 or 20, wherein the amino acid sequence of the HIV-1 capsid protein comprises:

[0186]

[0187] And the peptide interacts with residues of the capsid protein, the capsid protein comprising L56 of SEQ ID NO: 1, N57 of SEQ ID NO: 1, M66 of SEQ ID NO: 1 or any combination thereof.

[0188] 22. A cyclic peptide or linear peptide according to any one of paragraphs 1 to 21, wherein the peptide is 50 amino acids or less, 40 amino acids or less, 30 amino acids or less, 25 amino acids or less, 20 amino acids or less, 19 amino acids or less, 18 amino acids or less, 17 amino acids or less, 16 amino acids or less, 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, or has a length of 8 amino acids.

[0189] 23. A cyclic peptide or linear peptide according to any one of paragraphs 19 to 22, wherein the peptide has a binding affinity for HIV-1 capsid protein that is at least about 2 times higher, at least about 3 times higher, at least about 4 times higher, at least about 5 times higher, at least about 6 times higher, at least about 7 times higher, at least about 8 times higher, at least about 9 times higher, or at least about 10 times higher than that of the capsid inhibitor PF74.

[0190] 24. A cyclic peptide or linear peptide according to any one of paragraphs 1 to 23, wherein the peptide inhibits the assembly and / or disassembly of HIV-1 capsid.

[0191] 25. A cyclic peptide or a linear peptide according to paragraph 24, wherein the IC 50 From about 2 μM to about 4 μM.

[0192] 26. A cyclic peptide or a linear peptide according to paragraph 25, wherein the IC 50 It is about 3.2μM.

[0193] 27. The peptide according to any one of paragraphs 1 to 26, wherein the peptide is a linear peptide.

[0194] 28. The peptide according to any one of paragraphs 1 to 26, wherein the peptide is a cyclic peptide.

[0195] 29. A method for producing the cyclic peptide of paragraph 28, the method comprising producing a linear peptide of a desired sequence for the cyclic peptide, and cyclizing the linear peptide to produce the cyclic peptide,

[0196] Optionally, wherein the linear peptide is chemically synthesized, or

[0197] Optionally, wherein the linear peptide is translated in a host cell.

[0198] 30. An isolated polynucleotide comprising a nucleic acid encoding the peptide of any of paragraphs 1 to 28.

[0199] 31. The isolated polynucleotide of paragraph 30, further comprising a heterologous nucleic acid.

[0200] 32. The isolated polynucleotide of paragraph 31, wherein the heterologous nucleic acid comprises a promoter operably linked to the nucleic acid encoding the peptide.

[0201] 33. A vector comprising the polynucleotide of any one of paragraphs 30 to 32.

[0202] 34. The vector of paragraph 33, wherein the vector is a plasmid.

[0203] 35. A host cell comprising the vector of paragraph 33 or 34.

[0204] 36. The host cell of paragraph 35, which is a bacterium, an insect cell, a mammalian cell or a plant cell.

[0205] 37. A pharmaceutical composition comprising the peptide of any one of paragraphs 1 to 28 and a pharmaceutically acceptable carrier.

[0206] 38. A method of treating HIV infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of paragraph 37.

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[0257] ·Zhou, J., Price, AJ, Halambage, UD, James, LC, and Aiken, C. (2015). HIV-1 Resistance to the Capsid-Targeting Inhibitor PF74 Results in AlteredDependence on Host Factors Required for Virus Nuclear Entry.J Virol 89, 9068-9079. Sequence Listing <110> University of Missouri Administrator K. Singh T.P. Quinn F. Galazi U. Neogi A. Sonnerberg <120> Peptide inhibitors that inhibit HIV capsid <130> 52553-196127 <150> 62 / 858,666 <151> 2019-06-07 <160> 6 <170> PatentIn Version 3.5 <210> 1 <211> 230 <212> PRT <213> Homo sapiens <400> 1 Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Gln Ala Ile Ser 1 5 10 15 Pro Arg Thr Leu Asn Ala Trp Val Lys Val Ile Glu Glu Lys Ala Phe 20 25 30 Ser Pro Glu Val Ile Pro Met Phe Thr Ala Leu Ser Glu Gly Ala Thr 35 40 45 Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His Gln Ala 50 55 60 Ala Met Gln Met Leu Lys Asp Thr Ile Asn Glu Glu Ala Ala Glu Trp 65 70 75 80 Asp Arg Leu His Pro Val His Ala Gly Pro Ile Ala Pro Gly Gln Met 85 90 95 Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln 100 105 110 Glu Gln Ile Ala Trp Met Thr Ser Asn Pro Pro Ile Pro Val Gly Asp 115 120 125 Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met 130 135 140 Tyr Ser Pro Val Ser Ile Leu Asp Ile Lys Gln Gly Pro Lys Glu Pro 145 150 155 160 Phe Arg Asp Tyr Val Asp Arg Phe Phe Lys Thr Leu Arg Ala Glu Gln 165 170 175 Ala Thr Gln Asp Val Lys Asn Trp Met Thr Asp Thr Leu Leu Val Gln 180 185 190 Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Arg Ala Leu Gly Pro Gly 195 200 205 Ala Thr Leu Glu Glu Met Thr Ala Cys Gln Gly Val Gly Gly Pro Ser 210 215 220 His Lys Ala Arg Val Leu 225 230 <210> 2 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> MISC_Features <222> (2) <223> Xaa is glycine (G), lysine (K), or a polar uncharged amino acid. <220> <221> MISC_Features <222> (4) <223> Xaa is glycine (G), lysine (K), or a polar uncharged amino acid. <220> <221> MISC_Features <222> (7) <223> Xaa is glycine (G), lysine (K), or a polar uncharged amino acid. <400> 2 Phe Xaa Phe Xaa Pro Val Xaa Phe 1 5 <210> 3 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthesis <220> <221> MISC_Features <222> (1) <223> Xaa is glycine (G), proline (P), lysine (K), polar and uncharged Amino acids or hydrophobic amino acids. <220> <221> MISC_Features <222> (2) <223> Xaa is glycine (G), proline (P), lysine (K), polar and uncharged Amino acids or hydrophobic amino acids. <220> <221> MISC_Features <222> (3) <223> Xaa is glycine (G), proline (P), lysine (K), polar and uncharged Amino acids or hydrophobic amino acids. <220> <221> MISC_Features <222> (5) <223> Xaa is glycine (G), lysine (K), or a polar uncharged amino acid. <220> <221> MISC_Features <222> (7) <223> Xaa is glycine (G), lysine (K), or a polar uncharged amino acid. <220> <221> MISC_Features <222> (10)..(10) <223> Xaa is glycine (G), lysine (K), or a polar uncharged amino acid. <220> <221> MISC_Features <222> (12)..(12) <223> Xaa is glycine (G), proline (P), lysine (K), polar and uncharged Amino acids or hydrophobic amino acids. <220> <221> MISC_Features <222> (13) <223> Xaa is glycine (G), proline (P), lysine (K), polar and uncharged Amino acids or hydrophobic amino acids. <400> 3 Xaa Xaa Xaa Phe Xaa Phe Xaa Pro Val Xaa Phe Xaa Xaa 1 5 10 <210> 4 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 4 Ser Gly Val Phe Thr Phe Gly Pro Val Asn Phe Pro Gly 1 5 10 <210> 5 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 5 Ser Gly Val Phe Tyr Phe Trp Pro Val Asn Phe Pro Gly 1 5 10 <210> 6 <211> 14 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 6 Pro Val Leu Phe Pro Gly Gln Pro Phe Gly Gln Pro Pro Leu 1 5 10

Claims

1. A cyclic peptide as shown in the amino acid sequence SGVFTFGPVNFPG (SEQ ID NO: 4), wherein the cyclic peptide is formed by a lactam bond between the carboxyl C-terminus and the amine N-terminus.

2. The cyclic peptide according to claim 1, wherein the cyclic peptide further comprises a dye, a chelating agent, a radionuclide, or any combination of a dye, a chelating agent and a radionuclide. The cyclic peptide according to claim 1 , wherein the cyclic peptide binds to HIV-1 capsid protein.

4. The cyclic peptide according to claim 3, wherein the cyclic peptide binds to a binding pocket of the HIV-1 capsid protein located between the C-terminal domain (CTD) and the N-terminal domain (NTD) of the capsid protein.

5. The cyclic peptide according to claim 3, wherein the amino acid sequence of the HIV-1 capsid protein comprises: PIVQNLQGQMVHQAISPRTLNAWVKVIEEKAFSPEVIPMFTALSEGATPQDLNTMLNTVGGHQAAMQMLKDTINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIAW MTSNPPIPVGDIYKRWIILGLNKIVRMYSPVSILDIKQGPKEPFRDYVDRFFKTLRAEQATQDVKNWMTDTLLVQNANPDCKTILRALGPGATLEEMTACQGVGGPSHKARVL(SEQ ID NO:1), And the cyclic peptide interacts with residues of a capsid protein, wherein the capsid protein comprises L56 of SEQ ID NO: 1, N57 of SEQ ID NO: 1, M66 of SEQ ID NO: 1, or any combination thereof.

6. The cyclic peptide according to claim 3, wherein the binding affinity of the cyclic peptide to HIV-1 capsid protein is at least 2 times higher than that of the capsid inhibitor PF74.

7. The cyclic peptide according to claim 1, wherein the cyclic peptide inhibits the assembly and / or disassembly of HIV-1 capsid.

8. The cyclic peptide according to claim 7, wherein the IC of the cyclic peptide is 50 2μM to 4μM.

9. The cyclic peptide according to claim 8, wherein the IC of the cyclic peptide is 50 is 3.2μM.

10. A method for producing the cyclic peptide of claim 1, the method comprising producing a linear peptide of a desired sequence for the cyclic peptide of claim 1, and cyclizing the linear peptide to produce the cyclic peptide. The method according to claim 10 , wherein the linear peptide is chemically synthesized.

12. The method of claim 10, wherein the linear peptide is translated in a host cell.

13. An isolated polynucleotide comprising a nucleic acid encoding the cyclic peptide of claim 1.

14. The isolated polynucleotide of claim 13, further comprising a heterologous nucleic acid.

15. The isolated polynucleotide of claim 13, wherein the heterologous nucleic acid comprises a promoter operably linked to the nucleic acid encoding the peptide.

16. A vector comprising the polynucleotide according to any one of claims 13 to 15.

17. The vector according to claim 16, wherein the vector is a plasmid.

18. A host cell comprising the vector of claim 16 or 17. The host cell according to claim 18 , which is a bacterium, an insect cell, a mammalian cell or a plant cell.

20. A pharmaceutical composition comprising the cyclic peptide according to claim 1 and a pharmaceutically acceptable carrier.

21. Use of the pharmaceutical composition according to claim 20 in the preparation of a medicament for treating HIV infection.

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