Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
  • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
  • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
  • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
  • C07K14/675—Beta-endorphins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
  • C07K14/70—Enkephalins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
  • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

• G protein-coupled receptors encompass the largest class of receptors in humans (Lengger et al.2019).These receptors are characterized by seven membrane-spanning domains, with N-termini exposed outside of the cell, and C-termini at the cell cytoplasm (Zhou et al.2019). It was found that 831 GPCRs respond to a variety of external stimuli and are important drug targets-- roughly 34% of all FDA-approved drugs target a GPCR (Hauser et al.2017).
• yeast While humans and yeast had their last common ancestor over 1 billion years ago, they share several thousand genes, enabling use of the well-characterized and genetically-tractable yeast to study human proteins and systems (Kachroo et al.2022; Kachroo et al.2015). Yeast also have GPCRs, but just three, and two signaling pathways that are orthogonal to each other, the glucose sensing and pheromone response pathways (Versele et al.2001) The yeast pheromone response pathway can be coopted to express heterologous GPCRs, providing a “null” background to study and manipulate these receptors.
• the native yeast pheromone GPCR responds to agonist, stabilizing an active conformation of the receptor that enables downstream signaling (Weis et al.2018).
• This signaling is mediated by the separate G protein heterotrimer consisting of alpha, beta and gamma subunits.
• Yeast has a single G alpha, Gpa1, that signals with Ste2 (Versele et al. 2001).
• GDP guanosine diphosphate
• GTP guanosine triphosphate
• the native receptor, Ste2 is knocked out, in addition to the proteins Sst2 and Far1, which increase sensitivity of the heterologous receptor’s signaling and eliminate the cell cycle arrest response upon pheromone response Attorney Docket No.10046-513WO1 pathway activation (Apanovitch et al.1998; Alvaro et al.2016).
• a reporter under control of the transcription factor Ste12 whose translocation is activated by the pheromone response pathway’s MAP kinase cascade (Chen et al.2007), can be utilized as a readout of heterologous GPCR activity in response to an agonist.
• MAP kinase cascade Choen et al.2007
• chimeras of human G alpha proteins have been utilized to further improve signaling with heterologous receptors.
• Gpa1 The five amino acids at the C-terminus of Gpa1 are replaced with the corresponding human sub-type residues, enabling more efficient coupling of the G alpha and GPCR (Brown et al.2000). While the unmodified Gpa1 has been shown to function in some cases (Mukherjee et al.2015), usually human chimeras aid with effective signaling (Erlenbach et al.2001). Typically, a native GPCR-G alpha chimera pair is used, but sometimes this pair does not produce the highest signal, highlighting the importance of screening a panel of multiple chimeras (Shaw et al.2022; Lengger et al.2022).
• yeast have been used as a tool to study the native receptors (Ladds et al.2005) and as a chassis to study mutational effects, including the development of orthogonal GPCR-ligand pairs (Armbruster et al.2007; Dong et al.2010).
• the genetic tractability of yeast has enabled the discovery of new ligands (King et al.1990; Price et al.1995; Klein et al.1998), including the deorphanization of receptors (Yasi et al.2019).
• expression and utilization of human GPCRs in yeast remains a challenge.
• test molecule for use in a cell-based screening method, wherein the molecule comprises: a molecule of interest comprising a message/address peptide; a linker sequence; and an anchor sequence which anchors the test molecule to a plasma membrane of the cell.
• Also disclosed herein is a method of screening to determine interaction between a capture protein and a molecule of interest, the method comprising the steps of: providing the test molecule; providing a eukaryotic cell, wherein said eukaryotic cell comprises a non- endogenous capture protein; allowing the molecule of interest and the non-endogenous capture protein to interact; and determining interaction between the molecule of interest and the capture protein.
• a eukaryotic cell for use in a high throughput screening assay, wherein said cell comprises: one or more test molecules, wherein said test molecule is anchored to the cell’s plasma membrane; and a capture protein, wherein said capture protein is found in the cell’s plasma membrane, and is within proximity to the molecule of interest such that the molecule of interest and the capture protein can interact.
• an expression vector encoding a protein of interest, wherein said protein of interest comprises a) a leader sequence which targets the protein for cell secretion; b) a molecule of interest coupled to a message/address peptide; c) a linker sequence; and d) an anchor sequence which anchors the molecule to a plasma membrane of the cell.
• Figure 1A-B shows schematics of the native pheromone-response pathway and the modified pheromone response pathway for human GPCR expression.
• A To enable signaling with human GPCRs, the yeast GPCR Ste2 is knocked out in addition to Far1 and Sst2. A chimera of the yeast G alpha Gpa1 and the human subtype G alpha is used.
• B A GFP reporter is added under control of the transcription factor Ste12.
• Figure 2 shows dose responses with dynorphin A 1-8 and the human opioid receptors in different sterol backgrounds.
• the receptors were driven by pTDH3 and integrated into the yeast genome.
• Figure 3 shows tethered peptide strategy. Peptides are expressed tethered to the truncated Flo1 protein, Flo42. The C-terminus of this protein encodes for a GPI anchor that localizes the peptide-protein complex to the plasma membrane, until the anchor is cleaved and Flo42 localizes to the inside of the cell wall. Localization of peptide agonists near their cognate GPCRs enables binding and signaling, triggering the MAP kinase cascade, and the expression of the GFP reporter.
• Figure 4 shows inducible expression of tethered dynorphin A 1-8 with the glycine- serine linker. Assays utilized the ergosterol-producing strain.
• Figure 5 shows expression cassette for the tethered peptide libraries. The MF alpha leader sequence targets the protein product for cell secretion. The library is randomized in either the first or last four residues of the eight amino acid dynorphin A 1-8 wild type peptide. The addition of the glycine-serine linker leads to greater GPCR signaling and downstream GFP expression. The Flo42 truncated protein localizes the peptides to the plasma membrane and cell wall.
• FIG. 6 shows a heat map of amino acid enrichment for each position by the log proportion metric.
• Figure 7 shows library 1 variants by log proportion with the stringent nucleotide count filter.
• Figure 8 shows library 2 variants by log proportion with the stringent nucleotide count filter.
• Figure 9A-B shows enrichment of residues at each position with DOR by total nucleotide counts in the positive GFP pool. (A) shows DOR, library 1, and (B) shows DOR, library 2.
• Figure 10A-B shows enrichment of residues at each position with KOR by total nucleotide counts in the positive GFP pool.
• FIG. 11A-B shows enrichment of residues at each position with MOR by total nucleotide counts in the positive GFP pool.
• FIG. 12 shows Venn diagram of significant amino acid motifs for library 1 by the log proportion metric.
• Figure 13 shows Venn diagram of significant amino acid motifs for library 1 by the log proportion metric.
• Figure 14 shows logo map of variants in library 1 that are unique for individual receptors by the log proportion metric.
• Figure 15 shows logo map of variants in library 2 that are unique for individual receptors by the log proportion metric.
• Figure 16 shows logo map of variants in library 1 that are unique for individual receptors by the log proportion metric with the stringent read count filter.
• Figure 17 shows logo map of variants in library 2 that are unique for individual receptors by the log proportion metric with the stringent read count filter. DOR is not included because only the wild type motif, LRRI, met the read count threshold, and was not unique to this receptor.
• Figure 18 shows dose responses with exogenous dynorphin A 1-13 with KOR and DOR in both the ergosterol- and cholesterol-producing strains. Receptors were expressed on 2-micron plasmids.
• Figure 19 shows peptides had the long glycine-serine linker. There was no signaling with any of the N-termini anchored peptides, or the untethered peptides. Receptors were expressed on 2-micron plasmids, and tethered peptides constitutively on Cen6 plasmids.
• Figure 20 shows tethered peptide controls. Expression was driven by pGAL1 and integrated into the yeast genome. All data was in the cholesterol-producing strain, CJM263.
• Figure 21 shows Pearson correlation between technical replicates before filtering.
• Figure 22 shows Pearson correlation between technical replicates after filtering around a minimum log proportion of -11 and a maximum log proportion cutoff of -2.
• Figure 23 shows comparison of Pearson correlations before and after filtering.
• Figure 24 shows number of unique variant counts in each library and receptor pair for the GFP positive and GFP negative pools.
• Figure 25 shows variants with stop codons in the variable region. “False” means that the sequence has no stop codon, “true” means that it does. Within this grouping, number of variants in the negative and positive GFP populations are plotted.
• Attorney Docket No.10046-513WO1 [0045]
• Figure 26 shows the tethering strategy for various linker and tether combinations.
• Figure 27 shows two examples of isolates that signaled strongly and were unique to individual receptors. They were identified and cloned individually. The two examples shown are delta-specific peptides (YGMELRRI, SEQ ID NO: 12; and YGFDLLRI, SEQ ID NO: 13). For all of the receptors, the fifth residues appeared to be particularly important for activity and specificity.
• Biological sample as used herein is a sample of biological tissue or fluid. Such samples include, but are not limited to, tissue isolated from humans. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.
• a biological sample is typically obtained from a eukaryotic organism, such as insects, protozoa, birds, fish, reptiles, and preferably a mammal such as rat, mouse, cow, dog, guinea pig, or rabbit, and most preferably a primate such as chimpanzees or humans.
• a eukaryotic organism such as insects, protozoa, birds, fish, reptiles, and preferably a mammal such as rat, mouse, cow, dog, guinea pig, or rabbit, and most preferably a primate such as chimpanzees or humans.
• the phrase “functional effects” in the context of assays for testing compounds that modulate CB1R mediated activity includes the determination of any parameter that is indirectly or directly under the influence of the receptor, e.g., functional, physical and chemical effects.
• determining the functional effect is meant assays for a compound that increases or decreases a parameter that is indirectly or directly under the influence of a receptor, e.g., functional, physical and chemical effects.
• Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties, patch clamping, voltage-sensitive dyes, whole cell currents, radioisotope efflux, inducible markers, oocyte receptor expression; tissue culture cell receptor expression; transcriptional activation of a receptor; ligand binding assays; voltage, membrane potential and conductance changes; ion flux assays; changes in intracellular second messengers such as cAMP and inositol triphosphate (IP3); changes in intracellular calcium levels; neurotransmitter release, and the like.
• spectroscopic characteristics e.g., fluorescence, absorbance, refractive index
• hydrodynamic e.g., shape
• chromatographic, or solubility properties patch clamping, voltage-sensitive dyes, whole cell currents, radioisotope efflux,
• Inhibitors are used interchangeably to refer to inhibitory, activating, or modulating molecules identified using in vitro and in vivo assays for signal transduction, e.g., ligands, agonists, antagonists, and their homologs and mimetics.
• Inhibitors are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate activity, e.g., antagonists.
• Activators are compounds that, e.g., bind to, stimulate, increase, open, activate, facilitate, enhance activation, sensitize or up regulate activity, e.g., agonists.
• Modulators include compounds that, e.g., alter the interaction of a receptor with: extracellular proteins that bind activators or inhibitor; G-proteins; kinases; and arrestin-like proteins, which also deactivate and desensitize receptors.
• Modulators include genetically modified versions of a receptor, e.g., with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like.
• Such assays for inhibitors and activators include, e.g., expressing receptors in cells or cell membranes, applying putative modulator compounds, and then determining the functional effects of a molecule of interest on the receptor.
• Samples or assays comprising receptors that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition.
• Control samples (untreated with inhibitors) are assigned a relative receptor activity value of 100%. Inhibition of receptor is achieved when the receptor activity value relative to the control is about 80%, optionally 50% or 25-0%. Activation of a receptor is achieved when the receptor activity value relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher.
• Bioly active capture protein refers to a capture protein, such as a receptor, having activity as described above, involved in interaction with a molecule of interest, such as a protein.
• wild-type refers to a gene or gene product (e.g., protein) that has the characteristics (e.g., sequence) of that gene or gene product isolated from a naturally occurring source, and is most frequently observed in a population.
• mutant refers to a gene or gene product that displays modifications in sequence when compared to the wild-type gene or gene product.
• “naturally-occurring mutants” are genes or gene products that occur in nature, but have altered sequences when compared to the wild-type gene or gene product; they are not the most commonly occurring sequence. “Synthetic mutants” are genes or gene products that have altered sequences when compared to the wild-type gene or gene product and do not occur in nature. Mutant genes or Attorney Docket No.10046-513WO1 gene products may be naturally occurring sequences that are present in nature, but not the most common variant of the gene or gene product, or “synthetic,” produced by human or experimental intervention.
• reporter is used herein in the broadest sense to describe a molecular entity, a characteristic and/or property of which (e.g., concentration, amount, expression, activity, cellular post-translational modification, localization, etc.) can be detected and correlated with a characteristic and/or property of a system containing the reporter (e.g., cell, artificial cellular entity, etc.).
• a “reporter” may be an intrinsic (e.g., endogenous) element of the system that exhibits one or more detectable and correlatable properties, or an artificial (e.g., exogenous) element engineered or introduced into the system (e.g., artificial cellular entity), that exhibits a detectable characteristic linked to process (e.g., gene expression) or component within the system.
• an intrinsic element of the system that exhibits one or more detectable and correlatable properties
• an artificial element engineered or introduced into the system e.g., artificial cellular entity
• a detectable characteristic linked to process e.g., gene expression
• Suitable reporters include, but are not limited to: intrinsic genes or proteins (e.g., expression, concentration, activity, or protein-protein interactions of which may be correlated to a particular stimuli), exogenous genes or proteins (e.g., expression, concentration, activity, or protein-protein interactions of which may be correlated to a particular stimuli), luciferases, a beta lactamases, CAT, SEAP, a fluorescent proteins, etc.
• native receptor refers to a ligand-binding protein of a cellular entity (e.g., located on the cell surface) that is also expressed by a non-engineered ancestral cell of the cellular entity.
• the native receptor on the cellular entity binds a ligand recognized or bound by the native receptor of the ancestral cell.
• non-native receptor refers to a ligand-binding protein of an artificial cellular entity (e.g., located on the cell surface) that is not present in/on an ancestral cell of the artificial cellular entity.
• the non-native receptor on the artificial cellular entity typically binds a ligand not recognized or bound by native receptors of the ancestral cell.
• Non-native receptors may be receptors that are native to another cell type, a chimera of a native receptor and a receptor native to another cell type, a mutated native receptor (e.g., having various amino acid substitutions, deletions, and/or additions), an engineered receptor (e.g., a receptor that is not native to any cell), a chimera of a native receptor and an engineered receptor, etc.
• isolated purified or “biologically pure” refer to material that is substantially or essentially free from components which normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.
• a protein that is the predominant species present in a preparation is Attorney Docket No.10046-513WO1 substantially purified.
• the term “purified” denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, optionally at least 95% pure, and optionally at least 99% pure.
• Nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.
• nucleic acids containing known nucleotide analogs or modified backbone residues or linkages which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
• analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2- O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
• nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
• degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J. Biol. Chem.260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
• nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
• polypeptide peptide
• protein is used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.
• amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
• Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ - carboxyglutamate, and O-phosphoserine.
• Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
• Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
• Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
• “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
• nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
• each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
• TGG which is ordinarily the only codon for tryptophan
• amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
• Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et al., Molecular Biology of the Cell (3rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980).
• Primary structure refers to the amino acid sequence of a particular peptide.
• Secondary structure refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains. Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 50 to 350 amino acids long.
• Typical domains are made up of sections of lesser organization such as stretches of ⁇ -sheet and ⁇ - helices.
• “Tertiary structure” refers to the complete three dimensional structure of a polypeptide monomer.
• Quaternary structure refers to the three dimensional structure formed by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms.
• a “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
• useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins for which ant or 7 can be made detectable, e.g., by incorporating a radiolabel into the peptide, and used to detect antibodies specifically reactive with the peptide).
• a “labeled nucleic acid probe or oligonucleotide” is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Attorney Docket No.10046-513WO1 Waals, electrostatic, or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe.
• a “nucleic acid probe or oligonucleotide” is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation.
• a probe may include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.).
• the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization.
• probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. It will be understood by one of skill in the art that probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions.
• the probes are optionally directly labeled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence.
• the term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
• recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
• heterologous when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature.
• the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source.
• a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
• a “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid.
• a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type Attorney Docket No.10046-513WO1 promoter, a TATA element.
• a promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
• a “constitutive” promoter is a promoter that is active under most environmental and developmental conditions.
• An “inducible” promoter is a promoter that is active under environmental or developmental regulation.
• the term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
• An “expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell.
• the expression vector can be part of a plasmid, virus, or nucleic acid fragment.
• the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
• sequences are then said to be “substantially identical.” This definition also refers to the compliment of a test sequence. Optionally, the identity exists over a region that is at least about 50 amino acids or nucleotides in length, or more preferably over a region that is 75-100 amino acids or nucleotides in length.
• sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
• test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
• the sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
• a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may Attorney Docket No.10046-513WO1 be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
• Methods of alignment of sequences for comparison are well-known in the art.
• Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math.2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol.
• PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity.
• PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987). The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153 (1989).
• the program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids.
• the multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences.
• Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences.
• the final alignment is achieved by a series of progressive, pairwise alignments.
• the program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters.
• PILEUP a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
• PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res.12:387-395 (1984).
• BLAST and BLAST 2.0 algorithms are described in Altschul et al. Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol.215:403-410 (1990), respectively.
• Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (which can be found on Attorney Docket No.10046-513WO1 the World Wide Web at ncbi.nlm.nih.gov).
• This algorithm involves first identifying high scoring sequence pairs (I-ISPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.
• T is referred to as the neighborhood word score threshold (Altschul et al., supra).
• These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
• the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0).
• a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached.
• the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
• the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)).
• nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
• P(N) the smallest sum probability
• An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
• a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
• Attorney Docket No.10046-513WO1 Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
• the phrase “selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
• stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures.
• Tm thermal melting point
• Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, optionally 10 times background hybridization.
• Exemplary stringent hybridization conditions can be as following: 50% formamide, 5 ⁇ SSC, and 1% SDS, incubating at 42° C., or, 5 ⁇ SSC, 1% SDS, incubating at 65° C., with wash in 0.2 ⁇ SSC, and 0.1% SDS at 65° C.
• Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
• Exemplary “moderately stringent Attorney Docket No.10046-513WO1 hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1 ⁇ SSC at 45° C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. [0084] The phrase “selectively associates with” refers to the ability of a nucleic acid to “selectively hybridize” with another as defined above. [0085] By “host cell” is meant a cell that contains an expression vector and supports the replication or expression of the expression vector.
• Host cells may be eukaryotic cells such as yeast, insect, amphibian, or mammalian cells such as CHO, HeLa and the like, e.g., cultured cells, explants, and cells in vivo.
• eukaryotic cells such as yeast, insect, amphibian, or mammalian cells such as CHO, HeLa and the like, e.g., cultured cells, explants, and cells in vivo.
• CHO, HeLa and the like e.g., cultured cells, explants, and cells in vivo.
• Tethered agonism strategies such as those disclosed in WO2019/228362A1 (herein incorporated by reference in its entirety for its teaching concerning tethered peptides), are known in the art. Disclosed herein are using those methods in screening assays, and molecules which can be used in said screening assays.
• Tethered agonism relies on the display of a protein in the periplasmic space (inside of the cell wall) of a eukaryotic cell, such as a yeast host cell.
• the eukaryotic cell comprises one or more test molecules, and one or more capture proteins (such as a receptor) located in the periplasmic space of the yeast host cell.
• the test molecule and the capture protein are in proximity to each other, such that they can interact with each other.
• the eukaryotic cell can be used to determine if the capture protein and the test molecule are able to interact.
• the test molecule to be displayed in the periplasmic space of the eukaryotic cell can be prepared by providing a molecule of interest comprising a message/address peptide; a linker sequence; and an anchor sequence which anchors the test molecule to a plasma membrane of the cell. Linkage can be covalent or noncovalent and is described in more detail below. Examples of the test molecule, and methods of using them, can be seen in Figures 3, 4, and 5.
• test Molecules Disclosed herein is a non-naturally occurring test molecule for use in a cell-based screening method, wherein the molecule comprises: a molecule of interest comprising a message/address peptide; a linker sequence; and an anchor sequence which anchors the test molecule to a plasma membrane of the cell.
• the test molecule comprises a molecule of interest, which is the actual portion of the test molecule which is to be tested for interaction with the capture protein.
• the molecule of interest can be selected from the group comprising a polypeptide, a peptide, a small molecule, a natural product, a peptidomimetic, a nucleic acid, a lipid, lipopeptide, or a carbohydrate, for example.
• the molecule of interest can be coupled to a message/address peptide, or in some circumstances, the message/address peptide can be the test molecule itself. An example of this can be seen in Figure 4.
• the message/address peptide is used to locate the test molecule to the desired area of the plasma membrane, such as near the desired capture protein.
• the “message” portion of the message/address peptide is used to make contact with the capture protein (such as a receptor) deep in a binding pocket of the receptor. This region is highly conserved between receptors, and is crucial for signaling. The “message” portion can be customized based on the desired capture protein. Due to the fact that messages are highly conserved, message portions of the peptide can be designed so that they can target the peptide to multiple receptors, for example. [0092] The remaining portion of the message/address peptide residues are referred to as the “address.” This region contacts the receptor higher up the binding pocket, and is important for receptor-ligand specificity.
• the message/address peptide can originate from, or be a derivative of, at least one of the enkephalin, dynorphin and endorphin peptides.
• the message portion of the message/address peptide can comprise the amino acids tyrosine-glycine-glycine- phenylalanine. Specific examples of such peptides can be found in SEQ ID NOS: 10-33 (Table 6).
• the message/address peptide can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids in length.
• the message/address peptide is 8 amino acids in length, wherein the first 4 amino acids are message, and the last four are the address. This is discussed in more detail in Example 1, and example motifs can be found in Table 1 and Table 6.
• Attorney Docket No.10046-513WO1 [0094]
• the test molecule is a protein
• the protein can further comprise a leader sequence which targets the test molecule for cell secretion. Examples of leader sequences include the MF alpha leader sequence, which is shown by way of example in SEQ ID NO: 7. Other leader sequences which can be used are known to those of skill in the art.
• the linker sequence plays an important role in the test molecule.
• An example of a linker which can be used is a “glycine-serine linker,” wherein all, or a majority of, the residues comprising the linker are glycine and serine residues. In other words, the linker can consist of glycine and serine residues.
• the addition of a linker can enable increased movement of the peptides, allowing greater interaction with the receptor.
• Figures 4 and 19 show the importance of using a linker, and the effectiveness of glycine-serine-based linker.
• An example of a nucleic acid encoding a glycine-serine linker can be seen in SEQ ID NO: 9.
• the amino acid sequence of the glycine-serine linker can be GGGSGGGGSGGGSGGGGS (SEQ ID NO: 34).
• the linker can be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids in length, or longer, or shorter. In a preferred embodiment, the linker is 18 amino acids in length. Glycines and serines can make up 80, 85, 90, 95, or 100% of the linker.
• the test molecule can also comprise an anchor sequence.
• the anchor sequence comprises a signal sequence that directs transport of the test molecule to the eukaryotic host cell periplasm, plasma membrane, or cell wall such that the test molecule is displayed in the periplasm.
• the anchor sequence can comprise a membrane-spanning transmembrane domain that projects the test molecule into the periplasm.
• the anchor sequence comprises a cell-membrane associated protein domain that localizes to an external face of the cell membrane such that the displayed protein variant is projected into the periplasm.
• the periplasm anchor sequence is a protein that binds to an inner face of the cell wall such that the displayed test molecule is projected into the periplasm.
• the anchor sequence comprises a signal sequence that directs transport of the fusion protein to the eukaryotic host cell periplasm, and the anchor sequence is sufficiently large that the test molecule is retained in the periplasm.
• the anchor sequence is a component of a periplasmic protein complex that is sufficiently large that formation of the complex in the periplasm results in retention of the test molecule in the periplasm.
• the anchor sequence can comprise Flo1 or a derivative thereof, such as Flo42.
• Attorney Docket No.10046-513WO1 [0098]
• the components of the test molecule described above can be attached, or linked, to each other in various manners.
• a molecule of interest may be linked covalently to an anchor sequence to form a fusion protein.
• a test molecule may form a complex with an anchor sequence, wherein the test molecule and the anchor sequence are linked noncovalently by molecular binding interactions in the complex.
• test molecule and the anchor sequence can be linked covalently by a non-peptidic bond in a complex.
• the non-peptidic bond is a disulfide bond.
• a protein to be displayed in the periplasmic space of the eukaryotic host cell can also be prepared by linking the protein to be displayed to a secretion signal.
• the test molecule for use in this method comprises a molecule of interest comprising a message/address peptide; a linker sequence; and an anchor sequence which anchors the test molecule to a plasma membrane of the cell.
• This “test molecule” is described in detail above, and examples of the test molecule can be found in Example 1.
• the eukaryotic cell which is described herein can be yeast, insect, amphibian, or mammalian cells such as CHO, HeLa and the like, e.g., cultured cells, explants, and cells in vivo.
• the host cell is a yeast cell.
• the genus of the yeast host cell can comprise Saccharomyces, Candida, Pichia, Kluyveromyces, and Yarrowia. In some embodiments, the genus of the yeast host cells can be Saccharomyces. In some embodiments, the species of the yeast host cell is Saccharomyces cerevisiae. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more genes of the host cell that encode undesirable cellular functions or responses of the host cell can be disabled or replaced. For example, the cell cycle arrest genes can be knocked out. [0101]
• the eukaryotic host cell comprises a non-endogenous capture protein.
• the non-endogenous capture protein examples include, but are not limited to, a receptor, an ion channel, and a transporter.
• the receptor can be a G-coupled protein receptor (GPCR). More specifically, the GPCR can be a cannabinoid receptor, such as CB1R or CB2R.
• the capture protein can comprise one or more mutations that affect its binding activity. [0102] In this method, the capture protein and the molecule of interest are allowed to interact, meaning that they are placed under sufficient conditions such that they are allowed to come Attorney Docket No.10046-513WO1 into contact with each other. For example, both the molecule of interest and the capture protein can both be located in the plasma membrane.
• Polynucleotides encoding the test molecule, and/or the polynucleotides encoding the capture protein can be provided by expression vectors. In other embodiments, they can be integrated into the yeast host cell genome at a target locus.
• a "vector" is a composition of matter which can be used to deliver a nucleic acid of interest to the interior of a cell.
• vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
• vector includes an autonomously replicating plasmid or a virus.
• An expression construct can be replicated in a living cell, or it can be made synthetically.
• expression construct is used interchangeably to demonstrate the application of the invention in a general, illustrative sense, and are not intended to limit the invention.
• the nucleic acid encoding a polynucleotide of interest is under transcriptional control of a promoter.
• promoter refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
• the term promoter is used herein to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase I, II, or III. Enhancer elements may be used in association with the promoter to increase expression levels of the constructs.
• an expression vector comprises a promoter "operably linked" to a polynucleotide encoding a test molecule or a capture protein.
• operably linked or "under transcriptional control” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the test molecule or the capture protein.
• transcription terminator/polyadenylation signals will also be present in the expression construct. Examples of such sequences include, but are not limited to, those derived from SV40, as described in Sambrook et al, supra, as well as a bovine growth hormone terminator sequence (see, e.g., U.S. Patent No.5,122,458).
• UTR sequences can be placed adjacent to the coding sequence in order to enhance expression of Attorney Docket No.10046-513WO1 the same.
• Such sequences may include UTRs comprising an internal ribosome entry site (IRES).
• IRES internal ribosome entry site
• IRES permits the translation of one or more open reading frames from a vector.
• the IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation. See, e.g., Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm.
• IRES sequences are known and include sequences derived from a wide variety of viruses, such as from leader sequences of picomaviruses such as the encephalomyocarditis virus (EMCV) UTR (Jang et al. J. Virol.
• EMCV encephalomyocarditis virus
• IRES sequences will also find use herein, including, but not limited to IRES sequences from yeast, as well as the human angiotensin II type 1 receptor IRES (Martin et al ,Mol. Cell Endocrinol. (2003) 212:51-61), fibroblast growth factor IRESs (FGF-l IRES and FGF-2 IRES, Martineau et al. (2004) Mol. Cell. Biol.24(l7):7622-7635), vascular endothelial growth factor IRES (Baranick et al. (2008) Proc. Natl. Acad. Sci. U.S.A.105(12):4733-4738, Stein et al. (l998)Mo/. Cell.
• IRES insulin-like growth factor 2
• Clontech Mountain View, CA
• Invivogen San Diego, CA
• Addgene Cambridge, MA
• GeneCopoeia Rockville, MD
• IRESite The database of experimentally verified IRES structures (iresite.org).
• An IRES sequence may be included in a vector, for example, to express a test molecule for display in combination with capture protein from an expression cassette.
• a polynucleotide encoding a viral T2A peptide can be used to allow production of multiple protein products from a single vector.2A linker peptides are inserted between the coding sequences in the multicistronic construct.
• the 2A peptide which is self cleaving, allows co-expressed proteins from the multicistronic construct to be produced at equimolar levels.2A peptides from various viruses may be used, including, but not limited to 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus- 1. See, e.g., Kim et al.
• the expression construct comprises a plasmid suitable for transforming a yeast cell.
• Yeast expression plasmids typically contain a yeast-specific origin of replication (ORI) and nutritional selection markers (e.g., HIS3, URA3, LYS2, LEU2, TRP1, MET15, ura4+, leul+, ade6+), antibiotic selection markers (e.g., aphAl or ble), fluorescent markers (e.g., mCherry, green fluorescent protein), bioluminescent markers (e.g., luciferase), or other markers for selection of transformed yeast cells.
• the yeast plasmid may further contain components to allow shuttling between a bacterial host (e.g., E. coli) and yeast cells.
• yeast plasmids A number of different types are available including yeast integrating plasmids (Yip), which lack an ORI and are integrated into host chromosomes by homologous recombination; yeast replicating plasmids (YRp), which contain an autonomously replicating sequence (ARS) and can replicate independently; yeast centromere plasmids (YCp), which are low copy vectors containing a part of an ARS and part of a centromere sequence (CEN); and yeast episomal plasmids (YEp), which are high copy number plasmids comprising a fragment from a 2 micron circle (a natural yeast plasmid) that allows for 50 or more copies to be stably propagated per cell.
• Yip yeast integrating plasmids
• ARS autonomously replicating sequence
• YCp yeast centromere plasmids
• CEN yeast episomal plasmids
• yeast episomal plasmids YEp
• regulatory sequences may also be desirable, which allow for regulation of expression of the protein sequences relative to the growth of the host cell.
• Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
• a pheromone-inducible promoter such as a PRM1 or FUS2 promoter can be used to make transcription dependent on activation of the pheromone signaling pathway.
• the control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector.
• the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.
• Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site- directed mutagenesis, are well known to those skilled in the art. See, e.g., Sambrook et ah, supra; DNA Cloning, Vols.
• recombinant polynucleotides encoding protein variants are cloned into a periplasm-targeting expression vector comprising: a) a polynucleotide encoding a signal peptide; b) a cloning site suitable for in-frame insertion of a polynucleotide encoding a test molecule after the polynucleotide encoding the signal peptide; c) a polynucleotide encoding a glycophosphatidylinositol (GPI) plasma membrane anchoring domain, positioned such that the vector is capable of producing a fusion protein comprising the signal peptide and the test molecule fused to the GPI plasma membrane anchoring domain; and d) a promoter operably linked to sequences encoding the fusion protein.
• a periplasm-targeting expression vector comprising: a) a polynucleotide encoding a signal peptide; b) a
• an affinity tag refers to a biomolecule, such as a polypeptide segment, that can be attached to a second biomolecule to provide for purification or detection of the second biomolecule or provide sites for attachment of the second biomolecule to a substrate.
• affinity tags include a poly-histidine tract, protein A (Nilsson et al. (1985) EMBO J.4: 1075; Nilsson et al.
• a "label” is a molecule or atom which can be conjugated to a biomolecule to render the biomolecule or a form of the biomolecule, such as a conjugate, detectable or measurable.
• labels include fluorescent agents, bioluminescent proteins, photoactive agents, radioisotopes, paramagnetic ions, chelators, and the like.
• This delivery may be accomplished in vitro using laboratory procedures for transforming yeast cells well-known in the art, such as spheroplast transformation, alkaline ion treatment (e.g., Cs + or Li + ), electroporation, trans-kingdom conjugation, electroporation, and biolistic and glass bead methods (see, e.g., Kawai et al. (2010) Bioeng. Bugs. l(6):395-403, Gietz et al. (1995) Yeast 11(4):355-360, Gietz et al. (2007) Nat. Protoc.2(l):38-4l, Hinnen et al. (1978) Proc. Natl. Acad. Sci.
• the nucleic acid encoding the gene of interest may be positioned and expressed at different sites.
• the nucleic acid encoding the gene may be stably integrated into the genome of the cell via homologous recombination. This integration may be in the cognate location and orientation (gene replacement), within a gene (gene disruption), or in a random, non-specific location (gene augmentation). Integration of a construct at a target locus that disrupts a gene may be acceptable as long as the gene disruption does not interfere with cell growth or screening of the yeast periplasmic display library (e.g., avoid disruption of pheromone response if used in screening).
• the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA.
• Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
• the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane.
• a naked DNA expression construct may be transferred into cells by particle bombardment.
• the host cells are transformed with an empty vector together with the collection of linear DNA molecules encoding the test molecules, which subsequently integrate into the vector in vivo, e.g., by homologous recombination in the yeast host cells.
• the capture protein is a GPCR, such as a cannabinoid receptor (for example, CB1R or CB2R)
• the native Ste2 and/or Ste3 of the host yeast cell can be replaced with a heterologous GPCR, wherein said heterologous GPCR has a truncated N terminus of at least 5 residues; and further wherein native G alpha protein (Gpa1) has been replaced with a gene encoding a chimeric Gpa1.
• Gpa1 native G alpha protein
• the methods disclosed herein can be used as a high-throughput screen to identify test compounds which interact with the capture protein. In this case, the test molecule can be labeled.
• label and “detectable label” refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, stable (non-radioactive) heavy isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens) and the like.
• fluorescer refers to a substance or a portion thereof that is capable of exhibiting fluorescence in the detectable range.
• labels include, but are not limited to radiolabels (e.g., H, I, S, C, or P), stable (non radioactive) heavy isotopes (e.g., 13C or 15N), phycoerythrin, fluorescein, 7- nitrobenzo-2-oxa-l,3- diazole (NBD), YPet, CyPet, Cascade blue, allophycocyanin, Alexa dyes (e.g., Alexa 350, Alexa 430, Alexa 488, Alexa 532, Alexa 546, Alexa 555, Alexa 594, Alexa 647, Alexa 660, Alexa 680, and Alexa 750), Atto dyes (e.g., Atto 488, Atto 532, Atto 550, Atto 565, Atto 590, Atto 610, Atto 620, Atto 635, Atto 647, Atto 655, and Atto 680),
• radiolabels e.g
• Enzyme tags are used with their cognate substrate. As with many of the standard procedures associated with the practice of the invention, skilled artisans will be aware of additional labels that can be used. [0119] For example, interaction of the test molecule with the capture protein can trigger expression of the reporter. This can be done by way of a signaling cascade, such as the MAP Attorney Docket No.10046-513WO1 kinase cascade, which is pictured in Figure 3. In this case, test molecules that elicit a signaling pathway can be selected for expression of a reporter gene placed under the control of a specific promoter that is activated by the signaling pathway.
• a signaling cascade such as the MAP Attorney Docket No.10046-513WO1 kinase cascade
• Reporter genes used in this type of selection can be, e.g., genes conferring a detectable physical attribute (e.g., GFP and beta-lactamase) or antibiotic resistance (e.g., aminoglycoside phosphotransferase) to the cell.
• the signal can be a fluorescent one.
• some of the reporting systems that can be used with the biosensors disclosed herein include, but not limited to, fluorescence (GFP, RFP, YFP, ZsGreen), luminescence (Lux, Luc), colorimetric (beta-galactosidase), electrical, and growth (His3, Trp1, and Leu2).
• eukaryotic host cell can be engineered to express a reporter of expression of the capture protein.
• exemplary methods for evaluating phenotypes of cells include microscopy (e.g., light, - confocal, fluorescence, scanning electron, and transmission electron), fluorescence based cell sorting, differential centrifugation, differential binding, immunoassays, enzymatic assays, growth assays, and in vivo assays.
• phenotypic behaviors of the cell such as chemotaxis, morphological changes, or apoptosis can be monitored via visual inspection or microscope examination.
• HCS high-content screens
• High-content screens automate the extraction of multicolor fluorescence information derived from specific fluorescence- based reagents incorporated into cells (see, e.g., Giuliano and Taylor, Curr. Op. Cell Biol. 7:4, 1995).
• Cells can be analyzed using an optical system that can measure spatial, as well as temporal dynamics.
• fluorescent physiological indicators and “biosensors” are available to monitor changes in biochemical and molecular activities within cells (see, e.g., Giuliano et al., Ann.
• test molecules disclosed herein can comprise a library of test molecules, such that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1x10 6 , 1x10 7 , 1x10 8 , 1x10 9 , 1x10 10 or more test molecules are present in a given assay.
• test molecules can be assayed to determine their function, meaning whether or not they are capable of binding the capture protein. This can be done in one round, or over multiple rounds of selection, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more rounds. The multiple rounds of selection can be done using FACS or culture growth. In each round, a cell population can be re-started following a previous round of FACS, growth, or other means of selection known to those of skill in the art.
• Biosensors and Platforms [0124] Disclosed herein is a biosensor comprising the engineered eukaryotic cell described herein. The unique difficulties of engineering biosensors in yeast is described in Adeniran et al.
• the capture protein-expressing cells disclosed herein are used as biosensors within a system or device (e.g., POC system/device) configured for the detection of one or more analytes (test molecules) in a sample.
• a system or device e.g., POC system/device
• Contemplated herein are one or more of: reagents (e.g., buffers, etc.) storage, sample purification, introduction of the sample and biosensors, mixing, reaction, signal detection, signal quantification, communication of results (e.g., on a screen, on a printer report, etc.), etc.
• a system/device may be of any suitable configuration for carrying out the particular detection/quantification assay.
• a system/device may comprise a single unit, or multiple modules (e.g., regent module, mixing module, reaction module, detection module, etc.).
• a system/device is configured for point-of-care applications or research applications.
• Exemplary systems/devices, all or portions of which may find use in embodiments herein, are Attorney Docket No.10046-513WO1 described, for example, in: U.S. Pat. No.8,697,377; WO 2014/134537; U.S. Pat. No. 7,604,592; U.S. Pat. Pub.2013/0210652; U.S. Pat.
• the biosensors described herein may find use is any suitable field.
• devices/systems incorporating the capture proteins described herein find use, for example: in hospitals and medical clinics for bedside/in-room detection of biomarkers (e.g., for quick and reliable detection/diagnosis of disease, pathogen, condition, etc.); for in-the-field detection of pathogens or diagnosis; etc.
• biomarkers e.g., for quick and reliable detection/diagnosis of disease, pathogen, condition, etc.
• the biosensors herein find use, for example, in high throughput screening to search libraries of mutant proteins.
• biosensors for capture proteins.
• the structure of the receptor is highly evolvable, making it amenable to detecting multiple ligands, both naturally and non-naturally occurring.
• biosensor for the detection of compounds which interact with one or more capture proteins. Either one or more capture proteins may be expressed in a cell. Either one or both of these receptors may be genetically engineered, as described elsewhere herein.
• the analyte, or test molecule can be labeled.
• the biosensor can be high-throughput.
• yeast biosensors Examples of high throughput systems using yeast biosensors can be found in Qiu et al. (Qiu C, Zhai H, Hou J. Biosensors design in yeast and applications in metabolic engineering. FEMS Yeast Res.2019 Dec 1;19(8):foz082, herein incorporated in its entirety for its teaching regarding yeast biosensors).
• a library of analytes can be used to screen for analyte-receptor interaction.
• a differential binding assay can be used to determine if a test molecule preferentially binds one capture protein over the other.
• bind By “preferentially bind” is meant that a certain compound binds to one receptor or the other with one receptor over the other by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 7
• the methods disclosed herein can be used to quantify the expression of a target molecule.
• a quantification assay via cell sorting and flow cytometry.
• the cells can first be sorted into two pools, "off” and "on” via gating around a negative control, so any cell with a true green fluorescent signal would be called as positive.
• the more true quantification happens via testing of individual isolates identified via this cell sorting and NGS.
• Individual test molecules can be cloned and tested on a flow cytometer.
• the fluorescent signal can be measured for 10,000 or more cells, and fold change can be calculated via signal over the background of the negative control. With this strategy one can quantify test molecules in relations to other test molecules.
• the amount or sequence of an expressed mRNA of the test molecule can be determined.
• Cells, Proteins, and Nucleic Acids [0132] Disclosed herein are peptides comprising any of SEQ ID NOS: 10-33. Also disclosed are peptides wherein 1, 2, 3, or 4 amino acids of any one of SEQ ID NOS: 10-33 have been replaced.
• SEQ ID NOS: 1-9 are nucleic acids encoding various components of an engineered opioid receptor, as well as various components of a test molecule. Also disclosed are those proteins encoded by SEQ ID NOS: 1-9. Also disclosed are nucleic acids with 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOS: 1-9, or to the proteins which are encoded by these nucleic acids.
• the anchor sequence can comprise a membrane-spanning transmembrane domain or a membrane associated protein domain that projects the test molecule into the cell’s periplasm.
• the anchor sequence can be a protein that binds to an inner face of the cell wall such that the molecule of interest is projected into the cell’s periplasm.
• an expression vector encoding a protein of interest, wherein said protein of interest comprises a) a leader sequence which targets the protein for cell secretion; b) a molecule of interest coupled to a message/address peptide; c) a linker sequence; and d) an anchor sequence which anchors the molecule to a plasma membrane of the cell.
• a nucleic acid encoding this expression vector, and a cell comprising the expression vector.
• G protein-coupled receptors are a large class of membrane proteins that encompass a large percentage of drug targets. Signaling networks from GPCR activation are complex and often drug and context specific, making development of drug discovery screens challenging.
• the “null” background of the yeast Saccharomyces cerevisiae provides a simplified platform to screen human receptors. In addition to simplified signaling, the genetic tractability and cellular physiology of yeast enable tethering of peptides to the inside of the yeast cell wall in an orientation able to activate heterologous GPCRs.
• GPCRs G protein-coupled receptors
• Tethered peptide agonism has been used with individual peptide agonists, but not to screen libraries of peptides against GPCRs.
• a common strategy for the development of peptide therapeutics that target GPCRs is modification of the endogenous peptide sequence (Davenport et al.2020).
• Variant screening of endogenous peptide agonists in the tethered format could enable high-throughput analysis of many variants at a low-cost.
• Receptors of particular importance for drug discovery are the opioid receptors, ⁇ (MOR), ⁇ (KOR), and ⁇ (DOR).
• MOR opioid receptor
• KOR ⁇
• DOR ⁇
• natural, semi-synthetic, and synthetic opioids have been utilized with increasing efficacy and precision to treat pain. All three receptors regulate pain and pleasure broadly, with MOR regulating acute pain, and DOR chronic pain (Quirion et al.2020).
• KOR agonists In addition to pain, KOR agonists have the potential to regulate mood disorders and addiction (Bruchas et al.2010; Bruchas et al.2016). Development of opioid agonists that provide pain relief, and potentially treatment of other conditions, without negative side effects could be a significant way to address the current epidemic of death and overdose from opioid abuse (Manchikanti et. Al 2012). [0142] In addition to exogenous opioids used to treat pain, the opioid receptors are regulated by endogenous opioid peptides. These peptides are all cleaved from three precursors-- proenkephalin, prodynorphin, and proopiomelanocortin (Fricker et al.2020).
• the latter peptide residues are referred to as the “address.” This region contacts the receptor higher up the binding pocket, and is important for receptor-ligand specificity (Portoghese et al.1989; Cheng et al.2018). [0143] While the message-address concept of endogenous peptide activation has been paradigm for decades, evidence has accumulated that there is more flexibility in these definitions than previously thought. The phenolic hydroxyl of the tyrosine in position 1 Attorney Docket No.10046-513WO1 appears to be crucial for receptor activation (Lai et al.2006), and similar moieties are present in non-peptide opioid agonists and antagonists.
• Sst2 deletion increases the signal to noise response by decreasing receptor desensitization, and the Far1 deletion disables the cell cycle arrest response upon GPCR activation.
• ZsGreen was added behind the Fig2-Ste12 inducible promoter for GFP-expression upon heterologous GPCR-activation.
• the strains were additionally modified with the replacement of the five residues of the C-terminal native yeast G alpha, Gpa1, with the human G i ⁇ 3 residues. This G alpha sub-type is known to couple with the human opioid receptors and has been demonstrated previously in yeast (Bean et al.2022) ( Figure 1). [0146] Two of the three endogenous opioid peptide types were previously tested against the three opioid receptors in yeast (Uddin et al.2016).
• the length of the endorphins 16 residues at the shortest and 31 at the longest (Kimura et al.1980), provides more mutational space than the ability to fully cover in yeast, necessitating a library of 2.56x10 10 variants.
• the dynorphin A peptides are of a more moderate length between eight and seventeen amino acids.
• the shortest, dynorphin A 1-8, is less sub-type specific than the longer versions (Schwarzer et al.2009), potentially providing a good scaffold.
• the eight amino acids are a more manageable mutational space with the potential for saturation in a yeast-based library.
• the human opioid receptors have been expressed previously in a cholesterol- producing background.
• the receptors were assayed in both wild type ergosterol- and cholesterol-producing backgrounds with the peptides dynorphin A 1-8 and dynorphin A 1-13.
• DOR and KOR showed robust signaling with dynorphin A 1-8 in both backgrounds, while MOR showed activity at only the highest concentrations in both strains ( Figure 2). This result was surprising, as the longer and more KOR-biased peptide, dynorphin A 1-17, had a higher fold change and a pEC50 of 5.8 in the cholesterol-producing strain.
• Flo1 thought to be involved with flocculation, has a glycosyl-phosphatidylinositol (GPI) anchor at its C-terminus (Sato et al.2002). This anchor attaches to the plasma membrane until it is cleaved by phosphatidylinositol-specific phospholipase C (PI-PLC), when it then fixes to the inside of the yeast cell wall 243 ( Figure 3).
• PI-PLC phosphatidylinositol-specific phospholipase C
• Expression was driven by a galactose-inducible promoter, pGal1, and the construct was integrated into the yeast genome.
• the inducible promoter was used to control the length of peptide expression and minimize potential for unwanted selection from long-term constitutive expression.
• receptors were Attorney Docket No.10046-513WO1 integrated into the genome to avoid downstream issues with variable plasmid copy number and sorting (Bean et al.2022).
• the negative pools were enriched with variants with stop codons compared to the positive pools ( Figure 25).
• Figure 25 The negative pools were enriched with variants with stop codons compared to the positive pools.
• Several strategies were used to both identify unique binders for receptor sub-type and identify generally preferred residues and motifs. Log proportions between the positive and negative pools were determined with a filter of -11 ( Figure 6). While useful for highlighting potentially significant low count variants, this strategy could enrich for noise resulting from sorting errors. Because the libraries were sorted twice, and the fraction of cells with a GFP signal small, strong signalers were expected to be present in relatively high numbers in the positive pool. This expectation held true for the library 1 wild type sequences with KOR and DOR.
• Strains and plasmids are listed in Tables 4 and 5. Strains were derived from BY4741 using CRISPR-Cas9 or homology-based integration using auxotrophic markers. For CRISPR-Cas9 edits, the Yeast Toolkit was used to create Cas9 vectors as previously described (Lee et al.2015). Yeast were transformed with this vector and appropriate repair templates, and confirmed via sequencing and colony PCR. Non-CRISPR based edits utilized parts from the Yeast Toolkit.
• Non-peptide library yeast transformations were performed using the Zymo Research EZ Yeast Transformation II Kit. Peptide libraries were transformed first into Electromax DH10 ⁇ competent cells (Thermo Fisher) via electoporation after plasmid assembly. Transformed cultures were grown overnight and then mini-prepped. Transformation efficiency was determined via serial dilution plating after one day. Plasmid was digested via NotI-HF (NEB) to linearize the vector for genomic integration in yeast. After digestion, DNA was purified using a Zymo Clean and Concentrator kit. Roughly 75 ⁇ g of DNA was transformed into yeast in iterative transformations for each library using a protocol for large scale yeast transformations previously described (Benatuil et al.2010).
• Plasmids were constructed using Golden Gate assembly of components from the Yeast Toolkit with some adaptations. Receptors were expressed on either 2 ⁇ URA3 backbones driven by the CCW12 promoter or integrated into the yeast genome using a URA3 auxotrophic marker and driven by the TDH3 promoter. Tethered peptide cassettes were expressed on either a Cen6 HIS3 backbone or integrated into the yeast genome using HIS3 auxotrophic markers and were driven by pHHF2 for plasmid-based expression, and pGal1 for integrated expression. Oligos were ordered as gblocks or single stranded DNA from IDT.
• yeast colonies were picked and grown overnight to saturation in SD-URA or SD-URA/His media at pH 5.8 in a 2.2 mL deep well plate or 15mL tubes in either a plate-shaking incubator (30° C, 1000 rpm, 3 mm orbital) or a floor shaking incubator (30° C, 300 rpm). Cultures were diluted 1:25 the next day in either SD-URA or SD-URA/His media at pH 7.1 buffered with 100mM MOPS.
• Cultures were then diluted 1:10 their initial volume in SD-URA/His pH 7.1100mM MOPS 2% galactose 0.2% glucose media and grown for 24 hours. Cells were then resuspended in an equal volume of TE pH 8.0 and 14-15 million events were sorted using a SH800S Cell Sorter (Sony) on GFP fluorescence, gated for singlets. Cells were sorted into SD-His/URA pH 5.8 media with pen/strep and grown for two days post sorting. Cultures were again washed and induced with galactose and sorted via the same parameters into positive and negative pools and grown up for 2 days. Genome extractions were performed using a Zymo Yeast Star kit.
• Alignment options were adjusted to minimize indel calls (-B 0 -O 12,12 -L 4,4 -T 25 -k 17). All resulting alignments had less than 6% indels reported (I or D in the alignment CIGAR string). [0173] Alignment records were filtered using samtools (version 1.7) (Danecek et al.2021) to retain only mapped, properly paired, plus strand reads, without indels, that overlapped the 8- AA/24-nucleotide (“NT”) target region. Combined with knowledge of the plasmid structure, this allowed the isolation of each of the two (Area1, Area2) 4 AA/12 NT sequences.
• Gut hormone GPCRs structure, function, drug discovery. Curr. Opin. Pharmacol.31, 63–67 (2016). 71. Patriarchi, T. et al. Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors. Science 360, (2016). 72. Billerbeck, S. et al. A scalable peptide-GPCR language for engineering multicellular communication. Nat. Commun.9, 5057 (2016). 73. Pierce, K. L., Premont, R. T. & Lefkowitz, R. J. Seven-transmembrane receptors. Nat. Rev. Mol. Cell Biol.3, 639–650 (2002). 74. Elion, E. A. Pheromone response, mating and cell biology. Curr.
• mu Opioid receptor role for the amino terminus as a determinant of ligand binding affinity. Brain Res. Mol. Brain Res.76, 64–72 (2000). 128. Deng, H. B. et al. Role for the C-terminus in agonist-induced mu opioid receptor phosphorylation and desensitization. Biochemistry 39, 5492–5499 (2000). 129. Manglik, A. et al. Crystal structure of the ⁇ -opioid receptor bound to a morphinan antagonist. Nature 485, 321–326 (2012). 130. Feng, G.-J. et al.
• the MyLO CRISPR-Cas9 toolkit a markerless yeast localization and overexpression CRISPR-Cas9 toolkit.

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