WO2025128566A1 - Synthetic high-density lipoprotein and method for treating sepsis and related conditions - Google Patents

Abstract

The presently-disclosed subject matter includes a structure that comprises a shell comprising phospholipids, having an outer surface and defining an inner core; and a polypeptide bound to the outer surface of the shell, wherein the polypeptide comprises (i) the sequence of SEQ ID NO: 2, 3, 4, or 5. The phospholipids and polypeptide of the structure can self-assemble into a non-naturally-occurring high-density lipoprotein (sHDL).

Description

SYNTHETIC HIGH-DENSITY LIPOPROTEIN AND METHOD FOR TREATING SEPSIS AND RELATED CONDITIONS by

Chang-Guo Zhan Xiangan Li Fang Zheng YaxiaYuan

Ling Guo

Assignee: University of Kentucky Research Foundation, and The United States Government as Represented by the Department of Veterans Affairs

Attorney Docket No.: 13177N/2702WO

RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application Serial No. 63/609,849 filed December 13, 2023, the entire disclosure of which is incorporated herein by this reference.

GOVERNMENT INTEREST

[0002] This invention was made with government support under grant number 1I01BX004639 awarded by the U.S. Department of Veterans Affairs, and grant number R35GM141478 awarded by the National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (Zhan UKRF 2702WO.xml;

Size: 5357 bytes; and Date of Creation: December 9, 2024) is herein incorporated by reference in its entirety. TECHNICAL FIELD

[0004] The presently-disclosed subject matter generally relates to synthetic high-density lipoproteins (sHDP) and methods for treating sepsis and relate conditions. In particular, certain embodiments of the presently-disclosed subject matter relate to sHDP having a unique polypeptide bound to the outer surface of a phospholipid shell of the sHDP, and compositions and methods that make use of the sHDP.

INTRODUCTION

[0005] Sepsis is caused by a dysregulated host response to infection. It approaches 19 million per year globally¹'³. The prognosis for sepsis remains grim, with a mortality rate exceeding 30%, due to lack of an efficient therapy¹'³. So far, efforts (more than 100 clinical trials) to block one or another component of the inflammatory or coagulation pathway s have had little impact on patient survival⁴.

[0006] Sepsis is caused by a cascade of dysregulated host responses, including multiple factors/steps: 1) upon infection, bacteria release endotoxins; 2) endotoxins activate immune effector cells to produce inflammatory cytokines and chemokines; 3) inflammatory cy tokines and chemokines activate endothelial cells (EC), resulting in endothelial dy sfunction manifested by vascular leakage, increased leukocyte adhesion, altered vascular tone and a shift in the hemostatic balance towards a procoagulant phenotype; in addition, sepsis induces hemolysis. Broken red blood cells release highly toxic heme that causes cell damage; Furthermore, the high levels of inflammatory cytokines and chemokines also cause cell damage which releases damage-associated molecular pattern molecules (DAMPs) that further dysregulate immune response⁵.

[0007] All of these responses lead to irreversible multi-organ failure and septic death⁶'⁹. A great challenge therefore is that multiple factors/steps contribute to sepsis and simply targeting one of the regulatory factors/steps has shown to have limited effects. Targeting an endogenous factor with multi-protective effects against sepsis has been proposed as a unique approach for sepsis therapy¹⁰.

[0008] High-density lipoprotein (HDL) is a major component of circulating blood^{11 12}. HDL is well recognized as a protective factor against cardiovascular disease and other chronic inflammatory diseases because of its broad spectrum of activity including the regulation of immunity and vascular EC functions¹²’¹³. While most existing knowledge of HDL has been acquired in non-infectious conditions, extensive evidence suggests that HDL likely plays pivotal protective roles in all the steps of sepsis, including detoxification of endotoxin, suppression of inflammatory signaling in immune effector cells and inhibition of EC activation¹⁴'³⁰.

[0009] Upon infection, gram-negative bacteria release lipopolysaccharides (LPS) which bind to TLR4 on immune effector cells to initiate a downstream signaling cascade, leading to activation of proinflammatory genes to produce high levels of cytokines such as TNF-a and IL-6, resulting in cell damage³¹. A body of evidence indicates that HDL detoxifies LPS through two mechanisms: i) HDL neutralizes LPS¹⁵'²³. Most LPS in circulation exist in HDL- bound form²²’²³, and HDL-LPS binding attenuates LPS-TLR4 interactions ^16,24-26 ; ii) Recent studies including ours suggest that HDL acts together with its receptor, the scavenger receptor BI (SR-BI), to promote LPS clearance²⁷'²⁹. In vitro, HDL promotes SR-BI-mediated LPS uptake by 4-fold in SR-BLtransfected HEK cells and by 2-fold in primary hepatocytes²⁷. In vivo, mice deficient in SR-BI or HDL display impaired LPS clearance in LPS or sepsis animal models²⁷’²⁹’³⁰. These findings suggest that HDL neutralizes LPS and promotes LPS clearance via SR-BI-mediated LPS uptake, which presents a more efficient mechanism for LPS detoxification than simple neutralization by anti-LPS antibodies.

[0010] Lipoteichoic acid (LT A), released by gram-positive bacteria, activates the TLR2/6 pathway to generate high levels of inflammatory cytokines, causing cell damage. Similar to LPS, LTA is associated with HDL in circulation and the binding of HDL-LTA neutralizes LTA^32,33. Given the structural similarity between LPS and LTA, it is likely that HDL neutralizes LTA and promotes LTA clearance via SR-BI-mediated LTA uptake.

[0011] Macrophages and neutrophils are major immune effector cells responsible for inflammatory cytokine production in sepsis³⁴. The inflammatory response is required for fighting against infections. However, the dysregulation of this response produces too many cytokines, leading to sepsis. A body of evidence indicates that HDL is a key modulator of inflammatory response in macrophages/neutrophils¹¹’³⁵'³⁹: i) HDL promotes the efflux of free cholesterol from macrophages, resulting in suppression of LPS-induced inflammatory signaling⁴⁰’⁴¹; and ii) HDL upregulates the transcriptional regulator ATF3 which downregulates the expression of inflammatory molecules³⁹.

[0012] ECs are activated by endotoxin and inflammatory cytokines^8,9’⁴². As discussed above, HDL can attenuate EC activation by promoting endotoxin detoxification and suppressing inflammatory cytokine production in macrophages/neutrophils. In addition, earlier studies demonstrated that HDL has a variety of activities that modulate EC functions, including: inhibition of adhesion molecule expression and cytokine production stimulated by LPS, TNF-a, IL- ip or thrombin⁴³’⁴⁴

[0013] Numerous clinical studies have shown that the levels of HDL drop markedly in septic patients, and this is associated with a poor prognosis¹⁰’⁴⁵’⁴⁶. ApoA-I null mice were used as an HDL deficient model and the role of HDL was tested using cecal ligation and puncture (CLP) as a model of sepsis. A deficiency in HDL was found to lead to a susceptibility to CLP -induced death, as well as less LPS neutralization and LPS clearance ³⁰. Increasing HDL levels by over expression of ApoA-I was found to improve the survival of CLP³⁰. These clinical and experimental findings show that a decrease in HDL levels is a risk factor for sepsis and raising circulating HDL levels can provide an efficient therapy for sepsis.

[0014] A number of earlier studies showed that sHDL treatment improved survival in LPS-challenged animals⁴⁷'⁵⁰. Using gram-negative bacterial infection model, Queszado et al. showed that administration of sHDL suppressed inflammatory’ cytokine production, but the sHDL failed to improve survival in this study due to toxicity and impurity of the sHDL product⁶⁴. An earlier study using ApoAI mimetic peptide 4F showed that the mimetic peptide 4F treatment increases HDL-cholesterol levels and improves survival in CLP-treated rats⁵¹. Unfortunately, survival was only monitored for two days in that study which used naked ApoAI peptide with a short circulation time. Thus, it is likely that the naked ApoAI peptide cannot provide protection bey ond two days.

[0015] The efficacy of a second generation of sHDL, ETC642, (the composition of ETC642 is described in the literature⁵²’⁵³) was tested on CLP-challenged mice. ETC642 is a 22-amino acid ApoAI mimetic peptide bound to phospholipids for form sHDL. A single dose of ETC642 increased HDL-cholesterol level for up to 48h in a dose-dependent manner in human⁵⁴’⁵⁵. ETC642 was administered to B6 mice 2h post CLP and it was found that the ETC642 treatment significantly increased plasma HDL-cholesterol levels and improved 7d survival rate in CLP-treated mice¹⁰. In terms of the structure, HDL is a nanodisc stabilized by ApoA-I. It can also be stabilized by memetic peptides of ApoA-I, resulting in the sHDL nanodiscs. As a result, previously reported sHDL nanoparticles were all designed based on the ApoA-I sequence. [0016] Despite advances in the field, there remains a need in the art for improved compositions and methods for use in treating sepsis.

SUMMARY

[0017] The presently-disclosed subject matter meets some or all of the above-identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.

[0018] This Summary describes several embodiments of the presently-disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This Summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently-disclosed subject matter, whether listed in this Summary or not. To avoid excessive repetition, this Summary' does not list or suggest all possible combinations of such features.

[0019] The presently-disclosed subject matter includes non-naturally-occurring high- density lipoproteins (sHDP) and methods for treating sepsis and relate conditions. In particular, certain embodiments of the presently-disclosed subject matter relate to sHDP having a unique polypeptide bound to the outer surface of a phospholipid shell of the sHDP, and compositions and methods that make use of the sHDP. The presently-disclosed subject matter also includes method of designing a polypeptide for use in formation of a sHDL, as disclosed herein.

[0020] The presently-disclosed subject matter includes a structure that comprises a shell comprising phospholipids, having an outer surface and defining an inner core; and a polypeptide bound to the outer surface of the shell, wherein the polypeptide comprises (i) the sequence of SEQ ID NO: 2, 3, 4, or 5. The phospholipids and polypeptide of the structure can self-assemble into a non-naturally-occurring high-density lipoprotein (sHDL).

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are used, and the accompanying drawings of which:

[0022] FIG. 1A-1G. Structures of the designed YGZL peptides in comparison with ApoA-I based ETC-642. The positively and negatively charged residues in Apo A-I dimer are underlined and double underlined, respectively. Compared to ETC-642, different residues that are neutral have dotted underlining. (A) ApoA-I structure. (B) Alignment of amino acid sequences of ETC642 and YGZL sHDL peptides. Structures of (C) ETC642, (D) YGZL1, (E) YGZL2, (F) YGZL3, and (G) YGZL4 in their dimer form.

[0023] FIG. 2A-2 J. Modeling of sHDL (Y GZL3) binding to endotoxin and the energetics of the binding process. (A) The initial state of the lipid-only particle; all lipids are randomly distributed. The positively charged choline head and the negatively charged phosphoric acid group are shaded. The X, Y, and Z axes of the coordinate system are shown by shaded arrows, respectively. (E) The initial state ofYGZL3 particle, the peptides are depicted in cartoon style. (B) The final state of the lipid-only particle, it is self-assembled to a discoid nanodisc in 3 ps simulation. The hydrophilic surface of the nanodisc is aligned with XY plane. (F) The YGZL3 particle also forms a nanodisc, where all peptides are arranged around the lipid. (C) LTA and (D) LPS are shown in stick-ball style. It could be inserted into lipid-only particle. (G) LTA and (H) LPS also could be inserted into the YGZL3 particle, however, they favorably stay near the peptides. (I) and (J) Binding free energies of LTA/LPS binding with lipid-only or YGZL3 particle.

[0024] FIG. 3A-3E. sHDL treatment restores HDL levels, and protects CLP- and bacteria-induced animal death. A) HDL-cholesterol concentrations. Plasma was collected 18h post CLP. HDL was prepared by sequential ultracentrifugation as described⁵⁹ (1.5 ml plasma from 5 mice were used to make one HDL preparation, n=3). B to E) B6 mice were subjected to CLP (21G needle, 2/3 ligation) as previously described²⁹’³⁰. 2h post CLP, the mice were treated with/without different sHDLs at 7.5mg sHDL protein/kg body weight (i.v.), and the survival was monitored for 7 days.

[0025] FIG. 4. sHDL (Y GZL3) treatment protects bacteria-induced animal death. B6 mice were administered intranasally with lxl0⁷ cfu ofP. aeruginosa in 50 pl of PBS. Two hour later, the mice were treated with/without sHDL and survival was monitored for 7d. [0026] FIG. 5. sHDL suppresses LPS-induced activation of NF-KB. HEK-Blue cells expressing TLR4, were cultured to 70% confluency and treated with LPS in the presence of sHDL for 16 hours. Samples (100 pl of the cell culture medium) were mixed with 100 pl of HEK-Blue Detection and the activation of the NF-KB reporter was quantified by measuring absorption at 650 nm. Data are from three independent experiments and were analyzed by one-way ANOVA. Significant differences with /’ < 0.05 are denoted by different letters (that is, bars with different letters denote significant differences, whereas bars with the same letter are not statistically different).

[0027] FIG. 6. sHDL suppresses LTA-induced activation of NF-KB. HEK-Blue cells expressing TLR2 were cultured to 70% confluency and treated with LPS LTA (D) in the presence of sHDL for 16 hours. Samples (100 pl of the cell culture medium) were mixed with 100 pl of HEK-Blue Detection and the activation of the NF-KB reporter was quantified by measuring absorption at 650 nm. Data are from three independent experiments and were analyzed by one-way ANOVA. Significant differences with /^J < 0.05 are denoted by different letters (that is, bars with different letters denote significant differences, whereas bars with the same letter are not statistically different).

[0028] FIG 7. sHDL suppresses TNF-a in RAW cells. RAW 264.7 cells were cultured to 80% confluency and treated with LPS (KI 2, 2 ng/ml) in the presence of sHDL (0, 15, 30, 60, or 120 pg peptide/ml) for 18 hours. The concentrations of TNF-a in the medium were measured by ELISA. Data are from three independent experiments and were analyzed by one-way ANOVA. Significant differences with /’ < 0.05 between different concentrations of sHDL are denoted by different letters.

[0029]

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0030] SEQ ID NO: 1 is PVLDLFRELLNELLEALKQKLK, which is referred to as ETC642, a 22 ammo acid peptide that is a known ApoA-I mimetic peptide, which mimics the structure of the a-helices of ApoA-I when combined with lipids.

[0031] SEQ ID NO: 2 is PVLDLFRELLKELLEALEKKLK, which is a unique sequence disclosed herein and referred to as YGZL1, and which was obtained by strategically-modifying charged residues of ETC642. [0032] SEQ ID NO: 3 is PVLDLFRELLNKLLEALKQELK, which is a unique sequence disclosed herein and referred to as YGZL2, and which was obtained by strategically-modifying charged residues of ETC642.

[0033] SEQ ID NO: 4 is PVLEELRERLASFLEKLRERLL, which is a unique sequence disclosed herein and referred to as YGZL3, and which was designed in view of ApoE sequences.

[0034] SEQ ID NO: 5 is PVLEELRQRLNEFLELLRQRLL, which is a unique sequence disclosed herein and referred to as YGZL4, and which was designed in view of ApoE sequences.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0035] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.

[0036] The presently-disclosed subject matter includes non-naturally-occurring high- density lipoproteins (sHDP) and methods for treating sepsis and relate conditions. In particular, certain embodiments of the presently-disclosed subject matter relate to sHDP having a unique polypeptide bound to the outer surface of a phospholipid shell of the sHDP, and compositions and methods that make use of the sHDP. The presently-disclosed subject matter also includes method of designing a polypeptide for use in formation of a sHDL, as disclosed herein.

[0037] The presently-disclosed subject matter includes a structure that comprises a shell comprising phospholipids, having an outer surface and defining an inner core; and a polypeptide bound to the outer surface of the shell, wherein the polypeptide comprises (i) the sequence of SEQ ID NO: 2, 3, 4, or 5. The phospholipids and polypeptide of the structure can self-assemble into a non-naturally-occurring high-density lipoprotein (sHDL). [0038] In some embodiments of the structure, the phospholipids comprise phingomyelins (SM) and dipalmitoylphosphatidylcholine (DPPC).

[0039] In some embodiments, the structure has enhanced binding affinity to lipopolysaccharide (LPS) and/or lipoteichoic acid (LT A), as compared to a natural high- density lipoprotein (HDL) or a sHDL comprising the polypeptide of SEQ ID NO: 1.

[0040] In some embodiments, administering the structure to a subject achieves increased plasma HDL-cholesterol levels, suppression of inflammatory response, protection against bacteria-induced death, and improved survival rates in a subject having a bacterial infection, including sepsis.

[0041] The presently-disclosed subject matter also includes a composition comprising a structure or sHDL as disclosed herein and a pharmaceutically-acceptable carrier.

[0042] The presently-disclosed subject matter also includes a structure or sHDL as disclosed herein that has been lyophilized.

[0043] The presently-disclosed subject matter also includes a method of treating a bacterial infection in a subject, comprising administering an effective amount of a structure as disclosed herein to a subject in need thereof. In some embodiments the bacterial infection is sepsis.

[0044] In some embodiments, the method further comprises determining binding of the structure to lipopolysaccharide (LPS) and/or lipoteichoic acid (LTA) in the subject. In some embodiments, the method further comprises determining clearance of lipopolysaccharide (LPS) and/or lipoteichoic acid (LTA) in the subject.

[0045] In some embodiments of the method, the structure has enhanced binding affinity to lipopolysaccharide (LPS) and/or lipoteichoic acid (LTA), as compared to a natural high- density lipoprotein (HDL) or a sHDL comprising the polypeptide of SEQ ID NO: 1.

[0046] In some embodiments of the method, administering the structure to a subject achieves increased plasma HDL-cholesterol levels, suppression of inflammatory response, protection against bacteria-induced death, and improved survival rates in a subject having a bacterial infection, including sepsis.

[0047] The presently-disclosed subject matter also includes a method of designing a polypeptide for use in formation of a non-naturally-occurring high-density lipoprotein (sHDL), as disclosed herein. In some embodiments, the method includes selecting a candidate polypeptide, and performing molecular dynamics (MD) simulations to investigate the self-assembly of the candidate polypeptide-based sHDLs. In some embodiments, the method further involves investigating the candidate polypeptide-based sHDL’s efficacy in binding a target of interest. For example, the target of interest could be lipopolysaccharide (LPS) and/or lipoteichoic acid (LTA) for sepsis, as disclosed herein. In some embodiments of the method, the MD simulations can be coarse-grained (CG) MD simulations.

[0048] While the terms used herein are believed to be well understood by those of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter.

[0049] Unless defined otherw ise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong.

[0050] All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety.

[0051] Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0052] As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUBMB Joint Commission on Biochemical Nomenclature (See, iubmb.qmul.ac.uk/).

[0053] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.

[0054] In certain instances, nucleotides and polypeptides disclosed herein are included in publicly-available databases, such as GENBANK® and SWISSPROT. Information including sequences and other information related to such nucleotides and polypeptides included in such publicly-available databases are expressly incorporated by reference. Unless otherwise indicated or apparent the references to such publicly-available databases are references to the most recent version of the database as of the fding date of this Application.

[0055] The present application can “comprise” (open ended) or “consist essentially of’ the components of the present invention as well as other ingredients or elements described herein. As used herein, “comprising” is open ended and means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms “having” and “including” are also to be construed as open ended unless the context suggests otherwise.

[0056] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.

[0057] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

[0058] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, in some embodiments ±0.1%, in some embodiments ±0.01%, and in some embodiments ±0.001% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0059] As used herein, ranges can be expressed as from “about” one particular value, and/or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. [0060] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally variant portion means that the portion is variant or non-variant.

[0061] As used herein, the terms “treatment” or “treating” relate to curing or substantially curing a condition (e.g., a bacterial infection, sepsis), as well as ameliorating at least one symptom of the condition, and are inclusive of prophylactic treatment and therapeutic treatment.

[0062] As used herein, the term “subject” refers to a target of administration. The subject of the herein disclosed methods can be a vertebrate, such as a mammal. Thus, the subject of the herein disclosed methods can be a human or non-human. Thus, veterinary therapeutic uses are provided in accordance with the presently disclosed subject matter.

[0063] As used here, a “pharmaceutically-acceptable carrier” can include aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bactenostats, bactericidal antibiotics and solutes that render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents.

[0064] The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the present invention.

EXAMPLES

[0065] Example 1: Unique Non-Naturally-Occurring High-Density Lipoproteins

[0066] As disclosed herein, with consideration to the dimer of ApoA-I protein containing many paired positively and negatively charged residues, it was contemplated that these pairs of charged residues could be important for forming a parallel dimer structure as well as the function of ApoA-I, the major component of HDL. As disclosed herein, through computational simulations, several new' types of sHDL were designed, two based on ApoAI sequence (YGZL1 (SEQ ID NO: 2) and YDZL2 (SEQ ID NO: 3)) and two based on ApoE sequence (YDZL3 (SEQ ID NO: 4) and YGZL4 (SEQ ID NO: 5)).

[0067] Example 2: Computational Modeling and Simulations

[0068] Concerning the general computational strategy, it is known that amphipathic a- helical proteins, like ApoA-I, have the unique ability to self-assemble around a cylindrical lipid bilayer, forming discoidal lipid-protein particles known as nanodiscs.⁶⁰’⁶¹ The most widely accepted structural model for nanodiscs proposes that two amphipathic a-helical proteins wrap around the lipid bilay er in a double-belt configuration.⁶² This model has gained support from various experimental methods.⁶³'⁶⁵ Intriguingly, altering the sequence length of the amphipathic a-helical proteins encircling the lipid bilayer can change the size of the nanodiscs without affecting their discoidal shape.⁶⁰ Moreover, short a-helical analog peptides derived from the native amphipathic sequence of ApoA-I can also form a discoidal peptidelipid complex,⁶⁶ similar in size to nanodiscs composed of native ApoA-I and lipids.⁶⁷ This suggests that synthetic high-density lipoproteins (sHDL), made from short amphipathic a- helical peptides and lipids, are likely to form nanodisc-like structures in aqueous solutions. The nanodisc structure of ETC-642, an ApoA-I consensus sequence based sHDL particle, was confirmed in previous work with transmission electron microscopy (TEM) and molecular dynamics (MD) simulations⁶⁸.

[0069] Based on the previously reported model of ETC-642 particle,⁶⁸ as well as the observed electrostatic interaction pattern observed in ApoA-I protein, novel peptides YGZL1 and YGZL2 were designed for formation of sHDL with enhanced binding affinity to lipopolysaccharide (LPS) and lipoteichoic acid (LTA), key targets in anti-sepsis therapy. In addition, attempts were made to further modify ETC-642 by designing novel peptides YGZL3 and YGZL4 with reference to another less studied HDL constitutive apolipoprotein, ApoE. In this study, MD simulations were performed to investigate the self-assembly of the YGZL peptides-based nanoparticles, as well as their effectiveness in neutralizing LTA and LPS, to demonstrate the molecular mechanisms of the designed anti-sepsis sHDL.

[0070] For the simulation of synthetic high-density lipoprotein (sHDL) particles, all- atomistic simulations are constrained to the nanosecond timescale due to the large system size, which is inadequate for studying nanoparticle self-assembly.⁶⁹ Therefore, coarse-grained (CG) molecular dynamics (MD) simulations were selected. This method has been effectively used in previous studies to investigate the assembly of lipoprotein particles and permits simulations on the microsecond timescale,⁶⁹'⁷² offering a more practical and cost-effective approach for simulating the sHDL systems. Consequently, the CG models are employed in subsequent MD simulations.

[0071] Example 3: CG model of YGZL peptides

[0072] In constructing the CG model of YGZL peptides, the process begins by mimicking the repeated a-helical fragment of Apo A-I. PyMol⁷³ was used to construct an initial atomistic model with an a-helical secondary structure. This atomistic model is then transformed into a CG model, utilizing the MARTINI force field designed specifically CG system.⁷⁴’⁷⁵

[0073] Example 4: CG model of solvent molecules, ions, sphingomyelin, and 1,2- dipalmitoyllecithin

[0074] The CG model for solvent molecules represents four water molecules as a single CG bead. In the case of ions, they are modeled as charged CG beads, with their first hydration shell being implicitly included ⁷⁵ For the lipids, specifically sphingomyelin (PPCS) and 1,2-dipalmitoyllecithin (DPPC), parameters from the MARTINI force field for lipids were adopted.⁷⁶

[0075] Example 5: CG model for the LTA and LPS

[0076] Parameterization for LTA is based on the parameters of galactosyldiacylglycerol (DGDG) from the MARTINI force field for glycolipids. ²⁰’ ²¹ An additional Q_a bead was added to represent the extra phosphate group in LTA, with bond and angle parameters sourced from the phosphatidylinositol (PI) parameters in the same force field. For LPS, the parameterization process involves iteratively fitting the CG model to the all-atomistic model. This approach aligns with the protocol used in Lopez's research, ensuring a rigorous and accurate modeling process.⁷⁷

[0077] Example 6: Assembly of sHDL particle

[0078] To start the assembly of sHDL particles, the initial model of the simulation system for YGZL peptides was constructed. This involves a random distribution of 20 YGZL peptides, 75 PPCS molecules, and 75 DPPC molecules, following a 1 :3.75:3.75 molar ratio, which aligns with the composition of the reported HDL-like ETC-642 particle. Each system contains 20 a-helical peptides, equivalent to the number of a-helices in two Apo A-I lipoproteins according to the double-belt model, along with 150 lipids. This composition approximates the reported model of discoidal HDL particles.⁶⁰’⁶²’⁷¹’⁷⁸ For a control, a lipid- only particle simulation system is also constructed. This system is comprised of a random distribution of 75 PPCS molecules and 75 DPPC molecules, without the addition of peptides. Subsequently, both systems are supplemented with CG beads representing water and ions (0. 15 M NaCl), creating a physiological environment. The simulations are performed for a total duration of 3 microseconds with a 30 fs integration time step at a temperature of 323K, using the Gromacs software.⁷⁹ This elevated temperature in the CG simulation is chosen to replicate the results of all-atom simulations typically conducted at 300-315K.^{/2> 13}- ¹⁹

[0079] Example 7: Binding model of sHDL particles with LTA/LPS

[0080] To simulate the binding conformation of LTA/LPS with sHDL particles, the approach involves randomly adding a single molecule of LTA or LPS into the solvent region of the simulation system, either with the assembled sHDL particle or the lipid-only particle (serving as a control). In this process, any solvent beads overlapping with the LTA/LPS molecules are removed. Additionally, the introduction of the LTA/LPS charge is counterbalanced by adding Na⁺ ions. These systems are then subjected to simulations for a total duration of 1 microsecond each, with a 30-fs integration time step at 323K, conducted using the Gromacs.

[0081] Example 8: Binding free energy of LTA/LPS with sHDL particles

[0082] The binding free energy between LTA/LPS and sHDL particles is estimated through Potential of Mean Force (PMF) calculations. This process is executed by utilizing the pull code and the Weighted Histogram Analysis Method (WHAM)⁸⁰’⁸¹ as implemented in Gromacs. The procedure starts with the extraction of reaction coordinates from the previously equilibrated LTA/LPS binding simulation systems. This involves pulling the center of mass (COM) of the LTA/LPS molecule 8 nm away from the COM of the sHDL particle across an 80 ns simulation with a 30-fs time step. Following this, 40 sampling windows are set for an additional 10 ns of equilibration with a 30-fs time step and another 10 ns of umbrella sampling at a finer 5 fs time step. These steps are based on the reaction coordinates, spaced at 0.2 nm intervals. To ensure the reliability of the results, the binding free energy of LTA/LPS with the lipid-only particle is also estimated, following the same methodological approach for control purposes. Furthermore, to average out any potential fluctuations and enhance the accuracy of the findings, three independent PMF calculations are conducted for each system. [0083] Example 9: Reagents

[0084] The peptide was synthesized by Genscript and purity was determined to be >95% by HPLC analysis. Egg sphingomyelin (SM) and l .2-dipalmitoyl-s«-glycero-3- phosphocholine (DPPC) were purchased from Sigma Aldrich. LPS (A. coll KI 2) was purchased from InvivoGen. All other reagents were obtained from commercial suppliers and were of analytical grade or higher.

[0085] Example 10: sHDL preparation

[0086] sHDL nanoparticles were made by co-lyophilization followed by thermal cycling as described previously¹⁰. Briefly, the peptide and phospholipids were combined and dissolved in glacial acetic acid or chloroform at a peptide: SM:DPPC ratio of 1 : 1 : 1 by weight. The resulting solution underwent rapid freezing in liquid nitrogen, which was followed by freeze-drying overnight to remove the acid. The lyophilized powder was reconstituted in IX phosphate-buffered saline (PBS) to the desired final peptide concentration and vortexed to completely dissolve, forming a cloudy white suspension. The resulting solution was subjected to three heat-cool cycles, each cycle consisting of 10 min of heating at 55°C and 10 min of cooling at room temperature, at which point, a clear solution was formed. The pH of the final sHDL solution was adjusted to 7.4 withNaOH and was then passed through a 0.2- pm sterile filter.

[0087] Example 11: In Vivo Efficacy Analysis

[0088] The cecal ligation and puncture model (CLP) and cecal slurry injection (i.p.) as sepsis model was used. CLP (21G needle, 2/3 ligation) was performed on 10- to 12-week-old male C57BL/6J mice as described previously¹⁰. Two hours after CLP, the mice were treated with 100 pl of PBS or sHDL at 7.5 mg peptide/kg body weight (i.v.). Survival was monitored for a 7-day period. Eighteen hours after CLP, HDL was isolated from the plasma by sequential ultracentrifugation as previously described ^59,82 (1.5 ml of plasma from 5 mice was used to make one HDL preparation), and the total HDL cholesterol was measured with a Wako Diagnostics kit.

[0089] Example 12: Analysis of NF-KB expression in HEK-Blue cells

[0090] HEK-Blue cells expressing TLR4 or TLR2 and an NF-KB reporter were used to assess ligand-stimulated NF-KB activation. The cells were cultured to 70% confluency and treated with LPS (KI 2, 1 ng/ml) or LTA (40 ng/ml), in the presence/absence of sHDL for 16 hours. The culture medium (100 tl) was mixed with 100 [il of HEK-Blue Detection, and the activation of the NF-KB reporter was quantified by measuring absorption at 650 nm.

[0091] Example 13: Analysis of cytokine production by RAW264.7 cells

[0092] RAW 264.7 cells were cultured to 80% confluency and treated with LPS (K12, 2 ng/ml) in the presence of sHDL (0, 15, 30, 60, or 120 pg peptide/ml) for 18 hours. The concentrations of TNF-a secreted by the cells into the cell culture medium were measured by ELISA.

[0093] Example 14: Statistical analysis

[0094] Data are presented as means ± SEM or means ± SD as indicated in the figure legends. Statistical significance in experiments comparing two groups was determined by two-tailed Student’s /-test. Companson of more than two groups was evaluated by one-way ANOVA, which was followed by Tukey’s post-hoc analysis. Means were considered to be statistically significantly different where P <0.05. Survival was analyzed by the Log-Rank test and Kaplan-Meier plots. Experimental data were statistically evaluated with GraphPad Prism Software.

[0095] Example 15: Design of YGZL peptides

[0096] As depicted in Fig. 1A, the dimer of Apo A-I protein contains a lot of paired positively and negatively charged residues. These pairs of charged residues were contemplated to be important for forming a parallel dimer structure as well as the function of ApoA-I. ETC-642 (SEQ ID NO: 1) is a 22 amino acid peptide that is a known ApoA-I mimetic peptide, which mimics the structure of the a-helices of ApoA-I when combined with lipids. However, the ESP-24218 peptide of ETC-642 (Fig. 1C), derived from a consensus sequence of ApoA-I (Fig. IB), did not perform as well as expected considering the interaction of charged residue pairs.

[0097] Therefore, based on the sequence of ESP-24218, the charged residues were modified to obtain YGZL1 (FIG. IB, SEQ ID NO: 2; Fig. ID) and YGZL2 (FIG. IB, SEQ ID NO: 3; Fig. IE). Further, another Apolipoprotein of HDL, ApoE, was used as a model to adjust the ESP 24218 more substantially, aiming to explore analogues based on different sequences of ApoA-I and ApoE. After optimizing the matching of charged residue pairs, YGZL3 (FIG. IB, SEQ ID NO: 4; Fig. IF) and YGZL4 (FIG. IB, SEQ ID NO: 5; Fig. 1G) were designed based on the ApoE sequences. [0098] Further, the coarse-grained (CG) molecular dynamics (MD) simulations on these peptides-based sHDL nanoparticles reveal that both YGZL-based sHDL nanoparticles and lipid-only particles form similar nanodisc structures after self-assembly, as illustrated in Fig. 2. In the sHDL structure, the helical peptides are parallelly aligned around the edge of the lipid bilayer. This arrangement is akin to the parallel alignment of amphipathic ApoA-I proteins in the double-belt conformation. This results in the hydrophilic groups on the phospholipids being more tightly packed on both sides of the sHDL nanodisc. In contrast, lipid-only particles have hydrophilic groups on the phospholipids that are more loosely packed. Although both sHDL and lipid-only particles captured LTA/LPS during the selfassembly, the potential of mean force (PMF) calculations demonstrate increasing of the binding free energy of LTA and LPS to the YGZL-based sHDL particles, compared to the corresponding lipid-only particles (Table 1). Specifically, YGZL3- (SEQ ID NO: 4) based sHDL shows the most significant effects in increasing LTA/LPS binding to sHDL, which is consistent with its higher anti-sepsis efficacy observed in sHDL treatment study. This finding supports the assumed LTA/LPS neutralization mechanism of sHDL, which also reveals a rational strategy for improving efficacy of anti-sepsis sHDL particle.

[0099] Lipopolysaccharide (LPS) and Lipoteichoic Acid (LTA) are complex glycophospholipids with distinct structures that play important roles in sepsis. LPS consists of three main components: a polysaccharide O-antigen, a core oligosaccharide, and a glycolipid moiety known as lipid A.⁵⁶ The lipid A component is critical as it mediates the proinflammatory and cytotoxic effects of LPS,⁵⁷ effectively making it the core moiety of LPS. On the other hand, the structure of LTA varies among different Gram-positive bacteria species. It typically includes long chains of ribitol or glycerol phosphate, but a glycolipid moiety is a common feature for membrane anchoring,⁵⁸ serving as the core moiety of LTA. So, the focus was solely on the glycolipid moieties of LPS and LTA, disregarding their more variable components, which allows for a more streamlined analysis. Based on the GC MD simulations and the PMF binding free energy simulations, the binding free energies of the sHDL nanoparticles with LPS and LTA could be predicted (see Table 1 for the predicted binding free energies). Table 1. The PMF binding free energies for YGZL sHDL particle, ETC-642 sHDL particle, and lipid-only particle binding with LTA and LPS.

[Binding Energy (kcal/mol) Binding Energy Improvement (kcal/mol) Particle !

p y [ .59 9 .20.7 N/A

™-⁶42 _ ; _ -77.6 _ -2_5_1_ _ -17.7 _ '⁴_⁴_ _

YGZLl sHDL 2.3 -2.5 -12.3 ’²'⁵

YGZL2 SHDL _₇₆4 .₂₄.4 46.2 '³-⁷

YGZL3 sHDL .₈₀.7 -27.1 -20.8 '⁶'⁴

YGZL4 sHDL[ __{76 2} _₂₅ _₁₆ 3 -4-3

[00100] Example 16: Targeting HDL with synthetic HDL (sHDL) for sepsis therapy

[00101] The effect of sHDLs was tested in a CLP -induced sepsis model. As shown in Fig 3A, sHDL (YGZL3) treatment increased plasma HDL-cholesterol levels. Sepsis survival upon sHDL treatment was then tested. As shown in Fig 3B-3E, sHDLs displaced different protection against CLP -induced death. The YGZL2 and YGZL3 significantly improved 7d survival in CLP -treated mice and the YGZL3 showed the best protection. sHDL (Y GZL3) was also tested in P. aeruginosa treated mice - a bacterial infection induced pneumonia - sepsis model. The sHDL (YGZL3) treatment significantly protected the mice from /< aeruginosa-induced death (50% survival in YGZL3 treated mice compared to 0% survival in PBS treated mice) (Fig 4).

[00102] Example 17: sHDL suppresses inflammatory response

[00103] Next, the mechanism underlying sHDL protection against sepsis was elucidated. To do this, the regulation of inflammatory signaling was investigated using HEK- Blue cells that were stably transfected to express either human TLR4 or TLR2 and an NF-KB reporter. Cells were challenged with the corresponding receptor ligands (LPS/TLR4, Fig 5; LTA/TLR2, Fig. 6) in the presence or absence of various concentrations of sHDL. In all cases, a dose-dependent decrease in NF-KB activation with increasing sHDL concentrations was observed. The effect of sHDL in microphages (RAW-276 cells) was also tested. As shown in Fig. 7, sHDLs effectively suppressed TNF-a production included by LPS. Of note, compared to YGZL1, YGZL3 displaces less efficiency in suppressing LPS-TLR4 induced NF-KB activation but more efficiency in suppressing LTA-TLR2 induced NF-KB activation and LPS induced TNF-a production in macrophages. Further study will help determine if these differences contribute to more effective rescue of YDZL3.

[00104] Example 18: Discussion

[00105] Currently there are no successful drug products in sepsis, which is a critical cause of death and healthcare burden. HDL plays critical roles in protection against sepsis, but HDL levels are often markedly decreased in septic patients, resulting in loss of protection. In this study, computational simulations were employed to design a novel type of sHDL nanoparticles based on contemplation and simulations that the pairs of charged residues would be important for forming a parallel dimer structure as well as the function of ApoA-I. Through this new approach, a new type of sHDL was designed based on ApoE sequence. The 7d survival evidence was provided, demonstrating that sHDLYGZL3 protects against sepsis in two clinically relevant sepsis models. sHDL YGZL3 was administered after induction of sepsis and not as a preventive measure. Thus, sHDL YGZL3 is a potentially effective therapy for sepsis.

[00106] Distinctly from the earlier sHDL that is made of ApoAl mimetic peptide, here computer modeling was used to generate a novel type of sHDL which is based on ApoE - another major component in HDL, and it was demonstrated for the first time that the ApoE- based novel sHDL (YGZL3) efficiently protected against septic death in two clinically relevant sepsis models. The computer modeling indicates that the ApoE mimetic peptide binds with phospholipids to form stable nanoparticles so that the sHDL (Y GZL3) will be much more effective than the first generation of sHDL made from the naked peptide.

[00107] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference, including the references set forth in the following list:

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[00108] It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

CLAIMS What is claimed is :

1. A structure, comprising: a shell comprising phospholipids, having an outer surface and defining an inner core; and a polypeptide bound to the outer surface of the shell, wherein the polypeptide comprises (i) the sequence of SEQ ID NO: 2, 3, 4, or 5.

2. The structure of claim 1, wherein the phospholipids and polypeptide self-assemble into a non-naturally-occurring high-density lipoprotein (sHDL).

3. The structure of claim 1, wherein the phospholipids comprise phingomyelins (SM) and dipalmitoylphosphatidylcholine (DPPC).

4. The structure of claim 1, wherein the structure has enhanced binding affinity to lipopolysaccharide (LPS) and/or lipoteichoic acid (LTA), as compared to a natural high- density lipoprotein (HDL) or a sHDL comprising the polypeptide of SEQ ID NO: 1.

5. The structure of claim 1, wherein administering the structure to a subject achieves increased plasma HDL-cholesterol levels, suppression of inflammatory response, protection against bacteria-induced death, and improved survival rates in a subject having a bacterial infection, including sepsis.

6. A composition comprising the structure of any one of claims 1-5 and a pharmaceutically-acceptable carrier.

7. A method of treating a bacterial infection in a subject, comprising administering an effective amount of the structure of claim 1 to a subject in need thereof.

8. The method of claim 7, wherein the bacterial infection is sepsis.

9. The method of claim 7, and further comprising determining binding of the structure to lipopolysaccharide (LPS) and/or lipoteichoic acid (LTA) in the subject.

10. The method of claim 7, and further comprising determining clearance of lipopolysaccharide (LPS) and/or lipoteichoic acid (LTA) in the subject.

11 . The method of claim 7, wherein the structure has enhanced binding affinity to lipopolysaccharide (LPS) and/or lipoteichoic acid (LTA), as compared to a natural high- density lipoprotein (HDL) or a sHDL comprising the polypeptide of SEQ ID NO: 1.

12. The method of claim 7, wherein administering the structure to a subject achieves increased plasma HDL-cholesterol levels, suppression of inflammatory response, protection against bacteria-induced death, and improved survival rates in a subject having a bacterial infection, including sepsis.

13. A method of designing a polypeptide for use in formation of a non-naturally- occurring high-density lipoprotein (sHDL), comprising: selecting a candidate polypeptide; and performing molecular dynamics (MD) simulations to investigate the self-assembly of the candidate polypeptide-based sHDLs, and optionally to investigate their efficacy in binding a target of interest (e.g., lipopolysaccharide (LPS) and lipoteichoic acid (LTA) for sepsis).

14. The method of claim 13, wherein the MD simulations are coarse-grained (CG) MD simulations.