Spacio-temporal determination of intrinsically-disordered polypeptides and interactions thereof

Abstract

Provided herein are methods for interrogating interactions between a binding molecule and a polypeptide of interest using hydrogen-deuterium exchange mass spectrometry, optionally in combination with in silico modelling of the binding interaction between the binding molecule and the polypeptide. The polypeptides of interest that are interrogated in a disordered domain include, without limitation, androgen receptor polypeptides, alpha-synuclein polypeptides, p27 polypeptides, c-myc polypeptides, and Tau polypeptides. Further described herein are enrichment of molecules that bind to a polypeptide described herein with high affinity, as well as methods of treating diseases or conditions by administering a binding molecule described herein to a subject in need thereof.

Description

[0001] Attorney Docket No.199589-705601 / PCT SPACIO-TEMPORAL DETERMINATION OF INTRINSICALLY-DISORDERED POLYPEPTIDES AND INTERACTIONS THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present disclosure claims priority to U.S. Patent Application No.63 / 618,117, filed on 5 January 2024, the contents of which is incorporated herein by reference in its entirety. SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in ST .26 xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on January 2025 is named 199589-705601_SL.xml and is ……. bytes in size. BACKGROUND

[0003] Determination of polypeptide structures holds the promise of unlocking druggability of proteins implicated in various diseases. While techniques such as X-ray crystallography can be used to elucidate a polypeptide structure, such techniques are hampered in poorly expressed or poorly folded proteins. Accordingly, there is a need to generate improved techniques that allow for accurate modeling of therapeutically relevant polypeptide structures, as well as interactions between such polypeptides and drugs for treatment of diseases. BRIEF SUMMARY

[0004] Provided herein are methods of interrogating a binding interaction between a disordered domain of an androgen receptor polypeptide and a binding molecule, the method comprising: (a) determining by mass spectrometry a hydrogen-deuterium exchange rate of the disordered domain of the androgen receptor polypeptide as a function of time in the absence of a binding molecule by: (i) labeling the androgen receptor polypeptide with a D2O buffer in a labeling reaction, (ii) quenching the labeling reaction after a reaction time period, (iii) generating peptide fragments of the androgen receptor polypeptide; and (iv) measuring incorporation of deuterium into the peptide fragments as a function of the reaction time period; (b) determining by mass spectrometry a hydrogen-deuterium exchange rate of the disordered domain of the androgen receptor polypeptide as a function of time in the presence of the binding molecule by contacting the androgen receptor polypeptide with the binding molecule and repeating (i)-(iv); (c) surveying a binding interaction Attorney Docket No.199589-705601 / PCT between the disordered domain of the androgen receptor polypeptide and the binding molecule by: (i) comparing the hydrogen-deuterium exchange rate in the peptide fragments in the presence and absence of the binding molecule, and (ii) identifying peptide fragments in which the hydrogen- deuterium exchange rate decreased in the presence of the binding molecule, relative to the hydrogen-deuterium exchange rate of the peptide fragments in the absence of the binding molecule, whereby the change in the hydrogen-deuterium exchange rate indicates (i) an increase in structural order in the disordered domain of the androgen receptor polypeptide resulting from binding of the binding molecule to the disordered domain of the androgen receptor polypeptide, or (ii) a protection of the amide backbone of the polypeptide by the binding molecule in absence of a structural change in the androgen receptor polypeptide thereby interrogating the binding interaction between the disordered domain of the androgen receptor polypeptide and the binding molecule. In some embodiments, the method is a method of altering order in the disordered region of the androgen receptor polypeptide, where the binding of the binding molecule to the disorder region results in a change in order in the disordered region of the androgen receptor polypeptide. Further provided herein are methods, wherein a plurality of binding molecules are independently contacted with the androgen receptor polypeptide. Further provided herein are methods, comprising selecting the binding molecules that produce significant changes in the hydrogen- deuterium exchange rate of peptide fragments of the androgen receptor polypeptide in the presence of the binding molecule relative to the absence of the binding molecule. Further provided herein are methods, wherein the peptide fragments comprise 20 to 30 amino acid residues. Further provided herein are methods, wherein the androgen receptor polypeptide is the human androgen receptor polypeptide deposited under Uniprot accession number P10275. Further provided herein are methods, wherein the androgen receptor polypeptide comprises one or more mutations or splice variants. Further provided herein are methods, wherein the binding molecule has a binding affinity to the disordered domain of the androgen receptor polypeptide of less than 10 µM (for example, a binding affinity of 1 µM, 100 nM, 10 nM, or less than 1 nM). Further provided herein are methods, wherein the disordered domain of the androgen receptor polypeptide comprises an N-terminal domain. Further provided herein are methods, wherein the binding molecule binds to an epitope in the disordered domain of the androgen receptor polypeptide capable of inhibiting an activity of the androgen receptor polypeptide. Further provided herein are methods, wherein the binding molecule binds to an epitope in the disordered domain of the androgen receptor polypeptide that has a polypeptide sequence of SEQ ID NO: 6 (MEVQLGLGRVYPRPPSKTYRGAFQNLFQSVREVIQN). Further provided herein are Attorney Docket No.199589-705601 / PCT methods, wherein the binding molecule is a peptide or a peptide mimic. Further provided herein are methods, wherein the binding molecule is a small molecule having a molecular weight of less than 40 kDa. Further provided herein are methods, wherein the reaction time period is from 0.05 seconds to 300 seconds. Further provided herein are methods, further comprising generating a model of the binding site of the disordered domain of the androgen receptor polypeptide to the binding molecule using at least one model trained to predict a polypeptide structure, wherein at least one model: (a) compares the hydrogen-deuterium exchange rate of the binding site determined by mass spectrometry to a calculated hydrogen-deuterium exchange rate of androgen receptor polypeptide docking conformers produced in modeling tools; and (b) identifies an androgen receptor polypeptide docking conformer that has a calculated hydrogen-deuterium exchange rate that is closest to the hydrogen-deuterium exchange rate determined by mass spectrometry, thereby generating the model of the binding site of the disordered domain of the androgen receptor polypeptide to the binding molecule. Further provided herein are methods, wherein the modeling tools comprises molecular dynamics (MD) simulations and / or machine- learning models. Further provided herein are methods, wherein the methods further comprise selecting the binding molecule as a treatment for a disease or condition. Further provided herein are methods, wherein the disease or condition is a cancer. Further provided herein are methods, wherein the cancer is androgen-dependent prostate cancer. Further provided herein are methods, wherein the methods further comprise administering a therapeutically effective amount of the binding molecule to a subject in need thereof. Provided herein are molecules that bind to the polypeptide sequence of SEQ ID NO: 6 (MEVQLGLGRVYPRPPSKTYRGAFQNLFQSVREVIQN) of an androgen receptor polypeptide, wherein the molecule binds to the polypeptide sequence with a binding affinity of less than 10 µM (for example, a binding affinity of 1 µM, 100 nM, 10 nM, or less than 1 nM). Further provided herein is a molecule, wherein the molecule is a peptide or peptide mimic. Further provided herein is a molecule, wherein the molecule is a small molecule having a molecular weight of less than 40 kDa. Further provided herein are pharmaceutical compositions that comprise the molecules disclosed herein and a pharmaceutically-acceptable excipient, diluent, or carrier. Provided herein are methods of treating androgen-dependent prostate cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition that comprises the molecules described herein and a pharmaceutically-acceptable excipient, diluent, or carrier. Further provided herein are methods, wherein the androgen-dependent prostate cancer is a metastatic castration-resistant cancer. Attorney Docket No.199589-705601 / PCT

[0005] Provided herein are pharmaceutical compositions for use in a method of treating an androgen-dependent prostate cancer in a subject, comprising a molecule that binds to the polypeptide sequence of SEQ ID NO: 6 of an androgen receptor polypeptide, wherein the molecule binds to the polypeptide sequence with a binding affinity of less than 10 μM, and a pharmaceutically-acceptable excipient, diluent, or carrier.

[0006] Provided herein are methods of treating a prostate cancer in a subject in need thereof, the method comprising administering to the subject: a therapeutically effective amount of an androgen receptor N-terminal domain binding molecule, wherein the androgen receptor N-terminal domain binding molecule provides for an increase in structural order of an androgen receptor upon contact, and wherein the increase in structural order is measured by comparing by mass spectrometry a hydrogen-deuterium exchange rate of the androgen receptor as a function of time in the presence and absence of the androgen receptor N-terminal domain binding molecule; and an additional therapeutic agent. Further provided herein are methods, wherein the additional therapeutic agent comprises an androgen receptor ligand-binding domain inhibitor or an inhibitor of CYP17. Further provided herein are methods, wherein the additional therapeutic agent comprises enzalutamide, apalutamide, darolutamide, bicalutamide, nilutamide, flutamide, ODM-204, TAS3681, galeterone, abiraterone, abiraterone acetate, or any combination thereof. Further provided herein are methods, wherein the additional therapeutic agent comprises enzalutamide. Further provided herein are methods, wherein the additional therapeutic agent comprises apalutamide. Further provided herein are methods, wherein the additional therapeutic agent comprises darolutamide. Further provided herein are methods, wherein the additional therapeutic agent comprises abiraterone. Further provided herein are methods, wherein the additional therapeutic agent comprises abiraterone acetate. Further provided herein are methods, wherein the additional therapeutic agent comprises bicalutamide.

[0007] Provided herein are methods of interrogating a binding interaction between a disordered domain of a c-myc polypeptide and a binding molecule, the method comprising: (a) determining by mass spectrometry a hydrogen-deuterium exchange rate of the disordered domain of the c-myc polypeptide as a function of time in the absence of a binding molecule by: (i) labeling the c-myc polypeptide with a D2O buffer in a labeling reaction, (ii) quenching the labeling reaction after a reaction time period, (iii) generating peptide fragments of the c-myc polypeptide, and (iv) measuring incorporation of deuterium into the peptide fragments as a function of the reaction time period; (b) determining by mass spectrometry a hydrogen-deuterium exchange rate of the disordered domain of the c-myc polypeptide as a function of time in the presence of the binding Attorney Docket No.199589-705601 / PCT molecule by contacting the c-myc polypeptide with the binding molecule and repeating (i)-(iv); (c) surveying a binding interaction between the disordered domain of the c-myc polypeptide and the binding molecule by: (i) comparing the hydrogen-deuterium exchange rate in the peptide fragments in the presence and absence of the binding molecule, and (ii) identifying peptide fragments in which the hydrogen-deuterium exchange rate decreased in the presence of the binding molecule, relative to the hydrogen-deuterium exchange rate of the peptide fragments in the absence of the binding molecule, whereby the change in the hydrogen-deuterium exchange rate indicates (i) an increase in structural order in the disordered domain of the c-myc polypeptide resulting from binding of the binding molecule to the disordered domain of the c-myc polypeptide, or (ii) a protection of the amide backbone of the polypeptide by the binding molecule, in absence of a structural change in the c-myc polypeptide thereby interrogating the binding interaction between the disordered domain of the c-myc polypeptide and the binding molecule. In some embodiments, the method is a method of altering order in the disordered region of the c-myc polypeptide, where the binding of the binding molecule to the disorder region results in a change in order in the disordered region of the c-myc polypeptide. Further provided herein are methods, wherein a plurality of binding molecules are independently contacted with the c-myc polypeptide, and wherein the method further comprises selecting the binding molecules that produce significant changes in the hydrogen-deuterium exchange rate of peptide fragments of the c-myc polypeptide in the presence of the binding molecule relative to the absence of the binding molecule. Further provided herein are methods, wherein the peptide fragments comprise 20 to 90 amino acids residues. Further provided herein are methods, wherein the c-myc polypeptide is the c-myc polypeptide deposited under Uniprot accession number P01106 or P01106-1. Further provided herein are methods, wherein the c-myc polypeptide comprises one or more mutations or slice variants. Further provided herein are methods, wherein the binding molecule has a binding affinity to the disordered domain of the c-myc polypeptide of less than 10 µM (for example, a binding affinity of 1 µM, 100 nM, 10 nM, or less than 1 nM). Further provided herein are methods, wherein the disordered domain of the c-myc polypeptide comprises an N-terminal domain. Further provided herein are methods, wherein the binding molecule binds to an epitope in the disordered domain of the c-myc polypeptide capable of inhibiting an activity of the c-myc polypeptide. Further provided herein are methods, wherein the binding molecule binds to an epitope in the disordered domain of the c-myc polypeptide that has a polypeptide sequence of SEQ ID NO: 1 (>sp|P01106|MYC_HUMAN Myc proto-oncogene protein OS=Homo sapiens OX=9606 GN=MYC PE=1 SV=2 Attorney Docket No.199589-705601 / PCT MDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPP APSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVT ELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPN PARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLS STESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGG HSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSP RSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILS VQAEEQKLISEEDLLRKRREQLKHKLEQLRNSCA) or SEQ ID NO: 2 (>sp|P01106- 1|MYC_HUMAN Isoform 1 of Myc proto-oncogene protein OS=Homo sapiens OX=9606 GN=MYC MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTP PLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDP DDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQ DLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVL HEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRC HVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTH NVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLL RKRREQLKHKLEQLRNSCA). Further provided herein are methods, wherein the binding molecule binds to an epitope comprising a polypeptide sequence of SEQ ID NO: 3 (IIIQDCMWSGFSAAAKLVSEKLASYQAAR).

[0008] Further provided herein are methods, wherein the binding molecule binds to an epitope comprising a polypeptide sequence of SEQ ID NO: 4 (KLDSVRVLRQISNNRKCTSPRSSDTEEN).

[0009] Further provided herein are methods, wherein the binding molecule binds to an epitope comprising a polypeptide sequence of SEQ ID NO: 5 (LERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLR KRREQLKHKLEQLRNSCA). Further provided herein are methods, wherein the binding molecule is a peptide or a peptide mimic. Further provided herein are methods, wherein the binding molecule is a small molecule having a molecular weight of less than 40 kDa. Further provided herein are methods, wherein the reaction time period is from 0.05 seconds to 300 seconds.

[0010] Further provided herein are methods, wherein the method further comprises generating a model of the binding site of the disordered domain of the c-myc polypeptide to the binding molecule using at least one model trained to predict a polypeptide structure, wherein the at least Attorney Docket No.199589-705601 / PCT one model: (a) compares the hydrogen-deuterium exchange rate for the binding site determined by mass spectrometry to a calculated hydrogen-deuterium exchange rate of c-myc polypeptide docking conformers produced in modeling tools; and (b) identifies a c-myc polypeptide docking conformer that has a calculated hydrogen-deuterium exchange rate that is closest to the hydrogen- deuterium exchange rate determined by mass spectrometry, thereby generating the model of the binding site of the disordered domain of the c-myc polypeptide to the binding molecule. Further provided herein are methods, wherein the modeling tools comprise molecular dynamics (MD) simulations and / or machine-learning models Further provided herein are methods, further comprising selecting the binding molecule as a treatment for a disease or condition. Further provided herein are methods, wherein the disease or condition is a cancer. Further provided herein are methods, wherein the cancer is a lymphoma. Further provided herein are methods, further comprising administering a therapeutically effective amount of the binding molecule to a subject in need thereof.

[0011] Provided herein are molecules that binds to the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 of a c-myc polypeptide, wherein the molecule binds to the polypeptide sequence with a binding affinity of less than 10 µM (for example, a binding affinity of 1 µM, 100 nM, 10 nM, or less than 1 nM). Further provided herein are molecules, wherein the molecule is a peptide or peptide mimic. Further provided herein are molecules, wherein the molecule is a small molecule having a molecular weight of less than 40 kDa.

[0012] Provided herein are pharmaceutical compositions that comprises the molecule disclosed herein and a pharmaceutically-acceptable excipient, diluent, or carrier.

[0013] Provided herein are methods of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition disclosed herein.

[0014] Provided herein are pharmaceutical compositions for use in a method of treating cancer in a subject, comprising a molecule that binds to the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, wherein the molecule binds to the polypeptide with a binding affinity of less than 10 μM, and a pharmaceutically-acceptable excipient, diluent, or carrier.

[0015] Provided herein are methods of interrogating a binding interaction between a disordered domain of an androgen receptor polypeptide and a binding molecule, the methods comprising: (a) determining by mass spectrometry a hydrogen-deuterium exchange rate of peptide fragments of the polypeptide as a function of time in the absence of a binding molecule by: (i) labeling the polypeptide with a D2O buffer in a labeling reaction, (ii) quenching the labeling reaction after a Attorney Docket No.199589-705601 / PCT reaction time period of from 0.05 seconds to 10 seconds, (iii) generating peptide fragments of the polypeptide and (iv) measuring incorporation of deuterium into the peptide fragments as a function of the reaction time period; (b) determining by mass spectrometry a hydrogen-deuterium exchange rate of the peptide fragments of the polypeptide as a function of time in the presence of the binding molecule by contacting the polypeptide with the binding molecule and repeating (i)-(iv); (d) surveying a binding interaction between the polypeptide and the binding molecule by: (i) comparing the hydrogen-deuterium exchange rate in the peptide fragments in the presence and absence of the binding molecule, and (ii) identifying peptide fragments in which the hydrogen- deuterium exchange rate changed in the presence of the binding molecule, relative to the hydrogen- deuterium exchange rate of the peptide fragments in the absence of the binding molecule, whereby the change in the hydrogen-deuterium exchange rate indicates an increase or decrease in structural order in a region of the polypeptide resulting from binding of the binding molecule to the region of the polypeptide, thereby interrogating the binding interaction between the polypeptide and the binding molecule. In some embodiments, the method is a method of altering order in the disordered region of the polypeptide, where the binding of the binding molecule to the disorder region results in a change in order in the disordered region of the polypeptide. Further provided herein are methods, wherein a plurality of binding molecules are independently contacted with polypeptide.

[0016] Also provided herein are methods of generating a model of a binding site of a polypeptide to a molecule, the methods comprising: (a) determining by mass spectrometry a hydrogen- deuterium exchange rate of the peptide fragments of the polypeptide as a function of time in the absence of the molecule by: (i) labeling the polypeptide with a D2O buffer in a labeling reaction, (ii) quenching the labeling reaction after a reaction time period, (iii) generating peptide fragments of the polypeptide, and (iv) measuring incorporation of deuterium into the peptide fragments as a function of the reaction time period; (b) determining by mass spectrometry a hydrogen-deuterium exchange rate of peptide fragments of the polypeptide as a function of time in the presence of the molecule by contacting the polypeptide with the molecule and repeating (i)-(iv); (c) surveying a binding interaction of the polypeptide to the molecule by: (i) comparing the hydrogen-deuterium exchange rate in the peptide fragments in the presence and absence of the molecule, and (ii) identifying peptide fragments in which the hydrogen-deuterium exchange rate decreased in the presence of the molecule, relative to the hydrogen-deuterium exchange rate of the peptide fragments in the absence of the molecule, whereby the decrease in the hydrogen-deuterium exchange rate indicates an increase in structural order in a region of the polypeptide resulting from binding of the molecule to the region of the polypeptide, thereby interrogating the binding Attorney Docket No.199589-705601 / PCT interaction between the polypeptide and the molecule; and (d) generating the model of the binding site of the polypeptide to the molecule using at least one model trained to predict a polypeptide structure, wherein the at least one model: (i) compares the hydrogen-deuterium exchange rate of the binding site of the polypeptide determined by mass spectrometry to a calculated hydrogen- deuterium exchange rate of polypeptide docking conformers produced in modeling tools, and (ii) identifies a polypeptide docking conformer that has a calculated hydrogen-deuterium exchange rate that is closest to the hydrogen-deuterium exchange rate determined by mass spectrometry, thereby generating the model of the binding site of the polypeptide to the molecule. Further provided herein are methods, wherein the modeling tools comprise molecular dynamics (MD) simulations and / or machine-learning models. Further provided herein are methods, wherein the polypeptide is an androgen receptor polypeptide. Further provided herein are methods, wherein the polypeptide is an alpha-synuclein polypeptide. Further provided herein are methods, wherein the polypeptide is a p27 polypeptide. Further provided herein are methods, wherein the polypeptide is a c-myc polypeptide. Further provided herein are methods, wherein the polypeptide is a Tau polypeptide. Further provided herein are methods, wherein the polypeptide is an Amyloid β (1-42) polypeptide. Further provided herein are methods, wherein the molecule is a peptide or peptide mimic. Further provided herein are methods, wherein the molecule is a small molecule having a molecular weight of less than 40 kDa.

[0017] Further provided herein are methods of altering order in regions of structural disorder of a polypeptide using mass spectrometry, the methods comprising: (a) determining by mass spectrometry a hydrogen-deuterium exchange rate of the polypeptide as a function of time by: (i) labeling the polypeptide with a D2O buffer in a labeling reaction, (ii) quenching the labeling reaction after a reaction time period of from 0.05 seconds to 10 seconds, (iii) generating peptide fragments of the polypeptide and (iv) measuring incorporation of deuterium into the peptide fragments as a function of the reaction time period; (b) identifying regions of structural disorder based on the hydrogen-deuterium exchange rate determined by the mass spectrometry by: (i) selecting peptide fragments that have the fastest rate of hydrogen-deuterium exchange during the reaction time period of from 0.05 seconds to 10 seconds; and (ii) aligning the selected peptide fragments to the polypeptide sequence, thereby identifying regions of structural disorder in the polypeptide. Further provided herein are methods, wherein the methods further comprise: validating the identified regions of structural disorder of the polypeptide determined by mass spectrometry by comparing the identified regions of structural disorder of the polypeptide to regions of structural disorder determined using modeling tools. Further provided herein are Attorney Docket No.199589-705601 / PCT methods, wherein the modeling tools comprise molecular dynamics (MD) simulations and / or machine-learning models. Further provided herein are methods, wherein MD simulation data, as detailed in this document, are collected. Further provided herein are methods, wherein the MD simulation data comprise an index score, which is computed using a structural prominence parameter and a disorder parameter. Further provided herein are methods, wherein the structural prominence parameter and the disorder parameter are determined through MD simulations of a homology model that incorporates combined structures of proteins similar to the polypeptide. Further provided herein are methods, wherein the MD simulation data comprise an index score that is calculated based on a structural prominence parameter and a disorder parameter, wherein the disorder parameter and the structural prominence parameter are derived from MD simulation of a homology model comprising aggregate structures of homologs of the polypeptide; and wherein the index score is proportional to the structural prominence parameter and is inversely proportional to the disorder parameter. Further provided herein are methods, wherein the identified regions of structural disorder of the polypeptide determined by mass spectrometry are validated when the regions have a low index score based on the MD simulation data. Further provided herein are methods, wherein the polypeptide is an androgen receptor polypeptide. Further provided herein are methods, wherein the polypeptide is alpha-synuclein polypeptide. Further provided herein are methods, wherein the polypeptide is p27 polypeptide. Further provided herein are methods, wherein the polypeptide is c-myc polypeptide. Further provided herein are methods, wherein the polypeptide is Tau polypeptide. Further provided herein are methods, wherein the polypeptide is Amyloid β (1-42) polypeptide.

[0018] The present disclosure further provides molecules that bind to the polypeptide sequence of SEQ ID NO: 7: >sp|P37840|SYUA_HUMAN Alpha-synuclein OS=Homo sapiens OX=9606 GN=SNCA PE=1 SV=1 MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATV AEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGI LEDMPVDPDNEAYEMPSEEGYQDYEPEA of an alpha-synuclein polypeptide. Also provided are methods of treating a synucleinopathy in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition that comprises the molecules provided herein and a pharmaceutically-acceptable excipient, diluent, or carrier. Further provided herein are methods, wherein the synucleinopathy is Parkinson’s disease. Attorney Docket No.199589-705601 / PCT

[0019] Provided herein are pharmaceutical compositions for use in a method of treating a synucleinopathy in a subject, comprising a molecule that binds to the polypeptide sequence of SEQ ID NO: 7 of an alpha-synuclein polypeptide, and a pharmaceutically-acceptable excipient, diluent, or carrier.

[0020] The present disclosure also provides molecules that bind to the polypeptide sequence of SEQ ID NO: 8: >sp|P10636|TAU_HUMAN OS=Homo sapiens OX=9606 GN=MAPT PE=1 SV=5 MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSE EPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDE AAGHVTQEPESGKVVQEGFLREPGPPGLSHQLMSGMPGAPLLPEGPREATRQPSGTGPE DTEGGRHAPELLKHQLLGDLHQEGPPLKGAGGKERPGSKEEVDEDRDVDESSPQDSPPS KASPAQDGRPPQTAAREATSIPGFPAEGAIPLPVDFLSKVSTEIPASEPDGPSVGRAKGQD APLEFTFHVEITPNVQKEQAHSEEHLGRAAFPGAPGEGPEARGPSLGEDTKEADLPEPSE KQPAAAPRGKPVSRVPQLKARMVSKSKDGTGSDDKKAKTSTRSSAKTLKNRPCLSPKH PTPGSSDPLIQPSSPAVCPEPPSSPKYVSSVTSRTGSSGAKEMKLKGADGKTKIATPRGAA PPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPT REPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINK KLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVE VKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSP VVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL of a Tau polypeptide. Also provided are methods of treating a tauopathy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition that comprises the molecules provided herein and a pharmaceutically-acceptable excipient, diluent, or carrier. Further provided herein are methods, wherein the tauopathy is Alzheimer’s disease.

[0021] Provided herein are pharmaceutical compositions for use in a method of treating a tauopathy in a subject, comprising a molecule that binds to the polypeptide sequence of SEQ ID NO: 8 of a Tau polypeptide, and a pharmaceutically-acceptable excipient, diluent, or carrier.

[0022] Also provided herein are molecules that bind to the polypeptide sequence of SEQ ID NO: 9: >sp|P46527|CDN1B_HUMAN OS=Homo sapiens OX=9606 GN=CDKN1B PE=1 SV=1 MSNVRVSNGSPSLERMDARQAEHPKPSACRNLFGPVDHEELTRDLEKHCRDMEEASQR KWNFDFQNHKPLEGKYEWQEVEKGSLPEFYYRPPRPPKGACKVPAQESQDVSGSRPAA Attorney Docket No.199589-705601 / PCT PLIGAPANSEDTHLVDPKTDPSDSQTGLAEQCAGIRKRPATDDSSTQNKRANRTEENVS DGSPNAGSVEQTPKKPGLRRRQT of a p27 polypeptide. Also provided herein are methods of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition that comprises the molecules provided herein and a pharmaceutically-acceptable excipient, diluent, or carrier. Further provided herein are methods, wherein the disease or condition is a hydatidiform mole or Beckwith-Wiedemann syndrome.

[0023] Provided herein are pharmaceutical compositions for use in a method of treating a disease or condition in a subject in need thereof, comprising a molecule that binds to the polypeptide sequence of SEQ ID NO: 9 of a p27 polypeptide, and a pharmaceutically-acceptable excipient, diluent, or carrier.

[0024] Also provided herein are methods of treating a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition that comprises the molecules provided herein and a pharmaceutically- acceptable excipient, diluent, or carrier. Further provided herein are methods, wherein the cancer is a lymphoma.

[0025] The present disclosure also provides molecules that bind to the polypeptide sequence of SEQ ID NO: 10: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA of an Amyloid β (1-42) polypeptide. Also provided herein are methods of treating Alzheimer’s disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition that comprises the molecules provided herein and a pharmaceutically-acceptable excipient, diluent, or carrier.

[0026] Provided herein are pharmaceutical compositions for use in a method of treating Alzheimer’s disease in a subject, comprising a molecule that binds to the polypeptide sequence of SEQ ID NO: 10 of an Amyloid β (1-42) polypeptide, and a pharmaceutically-acceptable excipient, diluent, or carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Novel features of exemplary embodiments are set forth with particularity in the appended claims. A better understanding of the features and advantages will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of Attorney Docket No.199589-705601 / PCT the disclosed systems and methods are utilized, and the accompanying drawings of which:

[0028] Figure 1 depicts an exemplary workflow for interrogating polypeptide binding information and increasing order in a disordered domain of a polypeptide using either a traditional hydrogen- deuterium exchange-mass spectrometry (HDX-MS) automation which determines hydrogen- deuterium exchange rates of polypeptides at reaction times of 15 seconds to 24 hours, or using an ultrafast HDX-MS automation consistent with embodiments described herein, where hydrogen- deuterium exchange rates of polypeptides can measured at reaction times of from 50 milliseconds to 5 to 10 minutes.

[0029] Figure 2 depicts an exemplary HDX-MS automation system consistent with embodiments described herein, wherein a rapid mixing quench-flow prototype for running millisecond to minute hydrogen-deuterium exchange experiments comprises a temperature controlled labelling chamber in which an equilibrium buffer and a labelling buffer are injected through labelling valves to a labelling mixer. The mixed buffers are then injected through 6 loops for millisecond to second labelling reactions and transferred to a temperature controlled quench chamber where a quench buffer is added to quench the labelling reactions. The mixed buffers are then transferred to an liquid chromatography (LC) separation chamber for desalting and online proteolysis before being analyzed by a mass spectrometer using electrospray mass spectrometric detection.

[0030] Figure 3 depicts an exemplary androgen receptor polypeptide that may be subjected to methods described herein, showing domains that include an intrinsically disordered region for binding of transcription factors, as the N-terminal domain (NTD) of the polypeptide and a structured domain comprising a DNA binding domain (DBD), a hinge (HR) region, and a hormone or ligand binding domain (LBD), where the protein folds according to an AlphaFold prediction. The amino acid residues corresponding to the various domains are also shown relative to their ADOPT z-scores ([Redl et al., “ADOPT: intrinsic protein disorder prediction through deep bidirectional transformers, NAR Genomics and Bioinformatics, Volume 5, Issue 2, 2023 and PCT publication WO 2022 / 243940A1).

[0031] Figure 4 depicts an overlay of 272 peptide fragments of the androgen receptor antagonist, with 91.5% coverage and 6.55 redundancy, as used to elicit sequence characterization information of the androgen polypeptide, obtained using methods disclosed herein.

[0032] Figures 5A–5F depict percent deuterium uptake using HDX-MS and deuterium uptake curves, obtained using the methods disclosed herein. FIG.5A shows the percent deuterium uptake as determined using a traditional HDX-MS method, where the percent deuterium uptake at 10 sec, 60 sec, and 600 seconds are all in the region of about 100%. FIG.5B shows the percent deuterium Attorney Docket No.199589-705601 / PCT uptake as determined using a fast HDX-MS method consistent with the embodiments described herein, where the percent deuterium uptake can be seen at different levels at 0.05 seconds, 0.15 seconds, 0.5 seconds, 1 second and 5 seconds before reaching 100% uptake at 300 seconds. FIG.5C shows a graph of the deuterium uptake (%) versus exchange time (seconds) of a peptide fragment comprising amino acid residues 27 to 55, using both traditional and fast HDX-MS methods. FIG.5D shows a graph of the deuterium uptake (%) versus exchange time (seconds) of a peptide fragment comprising amino acid residues 32-56, using both traditional and fast HDX- MS methods. FIG.5E shows a graph of the deuterium uptake (%) versus exchange time (seconds) of a peptide fragment comprising amino acid residues 409-436, using both traditional and fast HDX-MS methods. FIG. 5F shows a graph of the deuterium uptake (%) versus exchange time (seconds) of a peptide fragment comprising amino acid residues 539-550, using both traditional and fast HDX-MS methods.

[0033] Figures 6A–6B depicts information on more structured and less structured regions of the androgen receptor polypeptide, analyzed using the methods disclosed herein. FIG. 6A shows the percent deuterium uptake using the HDX-MS methods described herein, and which are used to identify more structured regions of the polypeptide, indicated by asterisks on the left of the figure and less structured regions indicated by asterisks on the right side of the figure. FIG.6B show the ADOPT z-scores of the amino acid residues 1-900, where more structured regions of the polypeptide are indicated by darker asterisks above the curve of the graph and less structured regions are depicted by lighter asterisks below the curve of the graph, corresponding to the information obtained from the HDX-MS methods described herein.

[0034] Figures 7A-7C depict binding information of masofaniten to androgen receptor polypeptides, obtained using the methods disclosed herein. FIG.7A shows the percent deuterium uptake using the HDX-MS methods described herein of the androgen receptor polypeptide with no masofaniten bound to the polypeptide. FIG.7B shows the percent deuterium uptake using the HDX-MS methods described herein of the androgen receptor polypeptide with masofaniten bound to the polypeptide. FIG.7C shows difference in the percent deuterium uptake using the HDX-MS methods described herein of the androgen receptor polypeptide with masofaniten bound to the polypeptide relative to the androgen receptor polypeptide with no masofaniten bound in order to interrogate the binding interaction using the methods described herein.

[0035] Figure 8 depicts exemplary structural information obtained using the methods disclosed herein using a fast HDX-MS method compared to a computational tool generating ADOPT scores to predict disorder in the polypeptide and the HDX-MS binding signal with androgen receptor Attorney Docket No.199589-705601 / PCT polypeptide bound to masofaniten indicates corresponding areas of disorder.

[0036] Figures 9A-9B depict model binding site structures, determined using the methods disclosed herein. FIG. 9A shows experimental deuterium uptake relative to time (ms) which are compared to calculated values of different snapshots of protein docking conformers to determine structural information based on how well the calculated values fit with the experimentally obtained curves. FIG.9B shows a molecular structure generated with the snapshot conformer having a good fit with the experimentally obtained data.

[0037] Figures 10A-10D depict high quality structural protein models, obtained using the methods disclosed herein. FIG.10A shows the relative deuterium (2H) uptake over the exposure time (ms) where the experimental data is incompatible with the calculated relative deuterium update. FIG. 10B depicts a protein model not compatible with measured data. FIG. 10C shows the relative deuterium (2H) uptake over the exposure time (ms) where the experimental data is compatible with the calculated relative deuterium update. FIG. 10D depicts a protein model compatible with data depicted in FIG.10C.

[0038] Figures 11A-11D depict exemplary experimental steps for determining protein active site binding information, obtained using the methods disclosed herein. FIG.11A shows the structural information using a traditional HDX-MS method on the left of the figure, versus an automated ensemble generation driven by fast HDX-MS consistent with the embodiments described herein. FIG. 11B shows a graph of the relative deuterium (2H) uptake versus exposure time (ms) which is used for automated selection of ensemble using generative and discriminative artificial intelligence (AI) forward models. FIG. 11C shows a graph of the deuterium (2H) uptake (Da) versus ligand concentrations (µM) for binding site validation through automated (ligand) titration. FIG. 11D shows an experimentally verified 3D target model generated from the foregoing steps shown in Figures 11A–11C, which may be used to suggest chemical group modifications and ligand information.

[0039] Figures 12A-12C depicts percent deuterium uptake information of alpha-synuclein polypeptides, obtained using the methods disclosed herein, compared to traditional HDX-MS. FIG.12A shows percent deuterium uptake of alpha synuclein polypeptides at 10 s, 1 m, and 10 m using a traditional HDX-MS method. FIG. 12B shows percent deuterium uptake of alpha synuclein polypeptides at 50, 80, 150, 200, 500, 1000, 5000, and 30000 ms using a fast HDX-MS method, consistent with embodiments, described herein. FIG. 12C shows percent deuterium uptake of alpha-synuclein and fasudil at 50, 80, 150, 200, 500, 1000, 5000, and 30000 ms using a fast HDX-MS method, consistent with embodiments, described herein. Attorney Docket No.199589-705601 / PCT

[0040] Figures 13A-13B depicts percent deuterium uptake information of p27 polypeptides, obtained using the methods disclosed herein, compared to traditional HDX-MS. FIG.13A shows percent deuterium uptake of p27 polypeptides at 10 s, 1 m and 10 m using a traditional HDX-MS method. FIG.13B shows percent deuterium uptake of p27 polypeptides at 50, 80, 150, 200, 500, 1000, 5000, and 30000 ms using a fast HDX-MS method, consistent with embodiments, described herein.

[0041] Figures 14A-14B depicts percent deuterium uptake information of Tau polypeptides, obtained using the methods disclosed herein, compared to traditional HDX-MS. FIG.14A shows percent deuterium uptake of Tau polypeptides at 10 s, 1 m, and 10 m using a traditional HDX-MS method. FIG.14B shows percent deuterium uptake of Tau polypeptides at 50, 80, 150, 200, 500, 1000, 5000, and 30000 ms using a fast HDX-MS method, consistent with embodiments, described herein.

[0042] Figures 15A-15D depicts percent deuterium uptake information of amyloid beta 42 polypeptides, obtained using the methods disclosed herein, compared to traditional HDX-MS. FIG. 15A shows percent deuterium uptake of amyloid beta polypeptides at 10 s, 1 m, and 10 m using a traditional HDX-MS method. FIG.15B shows percent deuterium uptake of amyloid beta polypeptides at 50, 80, 150, 200, 500, 1000, 5000, and 30000 ms using a fast HDX-MS method, consistent with embodiments, described herein. FIG. 15C shows percent deuterium uptake of amyloid beta polypeptides and 10074-G5 (a c-myc inhibitor) at 10 s, 1 m, and 10 m using a traditional HDX-MS method. FIG. 15D shows percent deuterium uptake of amyloid beta polypeptides and 10074-G5 at 50, 80, 150, 200, 500, 1000, 5000, and 30000 ms using a fast HDX- MS method, consistent with embodiments, described herein.

[0043] Figure 16 show a sequence coverage map for c-myc protein, where peptides that were identified and whose hydrogen-deuterium exchange was followed are indicated by bars.

[0044] Figure 17 shows percentage deuterium uptake profiles for c-myc obtained under traditional (left) and fast (right) HDX-MS conditions.

[0045] Figure 18 shows three sites identified in the binding of 10058-F4 to c-myc.

[0046] Figure 19 shows c-myc structure and binding sites identified by experiments described herein.

[0047] Figure 20 shows the effect of 10058-F4 to c-myc / MAX complex.

[0048] Figure 21 shows screening of binding compounds on intrinsically disordered proteins using HDX-MS described herein. DETAILED DESCRIPTION Attorney Docket No.199589-705601 / PCT Overview

[0049] Provided herein are methods of interrogating a binding interaction between a disordered domain of a polypeptide and a binding molecule. Further provided herein are methods for altering order in a disordered domain of a polypeptide. Further provided herein are processes utilizing hydrogen-deuterium exchange mass spectrometry (HDX-MS) to characterize a particular region of a polypeptide that may be useful to target with a binding molecule. Conventional methods that utilize HDX-MS to measure protein dynamics are typically limited by slow reaction times (generally in the order of 15 seconds to 24 hours). As a result, conventional methods are unable to accurately characterize intrinsically-disordered regions of polypeptides in which the hydrogen- deuterium exchange rate occurs on a faster time scale. In some embodiments, rapid HDX-MS methods accommodate reaction times as low as 0.05 seconds to 10 seconds and up to 300 seconds or more. Because the HDX-MS methods of the present disclosure can accommodate such quick reaction times, hydrogen-deuterium exchange rates can be accurately determined as a function of time for intrinsically-disordered regions of polypeptides. With the ability to accurately characterize intrinsically-disordered regions of polypeptides, the present disclosure provides the ability to identify and characterize binding interactions of binding molecules to intrinsically-disordered regions of polypeptides. For example, the present disclosure provides a method of detecting binding of binding molecules to the intrinsically-disordered N-terminal region of an androgen receptor polypeptide, among others. Indeed, designing therapeutics that target the androgen receptor has proven challenging by others. More specifically, others have attempted to design therapeutics that bind to the C-terminal ligand-binding domain (LBD) because the LBD is highly structured, while avoiding designing therapeutics against the N-terminal domain because it is intrinsically disordered. However, such therapeutics that target the LBD have proven to be ineffective as prolonged treatment due to mutations / splicing in the LBD. The present application provides a solution to this problem by providing a method to identify and characterize binding interactions between the intrinsically-disordered N-terminal domain of the androgen receptor and binding molecules utilizing the rapid HDX-MS methods described herein. Without wishing to be bound by theory, binding of the binding molecule to the intrinsically-disordered N-terminal domain results in structural changes of the N-terminal domain, which can be detected by a decrease in the hydrogen-deuterium exchange rate in the particular binding region. Because of the rapid reaction and quenching times described above, subtle structural changes in the N-terminal disordered region can be detected to successfully identify the binding site. Further, the present application provides integration with modelling tools such as molecular dynamics (MD) Attorney Docket No.199589-705601 / PCT simulations and machine-learning approaches / simulations to model the binding site. Hydrogen deuterium exchange rates of docking conformers of the polypeptide bound to the target in-silico can be calculated and fit to the empirical data provided by the rapid HDX-MS, thus allowing for selection of the appropriate docking conformer and modelling of the binding interaction of binding molecules to the polypeptide of interest (e.g., an androgen receptor). Further, the present application provides methods of treating diseases using binding molecules identified using methods described herein.

[0050] In some embodiments, the HDX-MS utilized in the methods described herein operate on a rapid time scale, allowing for detection and characterization of intrinsically-disordered regions of a polypeptide. For example, in some cases the HDX-MS methods comprise the following steps: (i) labeling a polypeptide of interest with a deuterium-containing buffer (such as a buffer containing D2O) in a labeling reaction, which results in exchange of exchangeable protons in the polypeptide chain with deuterium; (ii) quenching of the labeling reaction after a reaction time period, which stops the incorporation of deuterium into the polypeptide chain after the particular reaction time period, (iii) generating peptide fragments of the polypeptide of interest and (iv) measuring incorporation of deuterium into the peptide fragments as a function of the reaction time period. Without wishing to be bound by theory, portions of the polypeptide that are more structured have lower rates of deuterium incorporation into the polypeptide chain than portions of the polypeptide that are more disordered. Definitions

[0051] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described.

[0052] As used in the specification and claims, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a polypeptide” includes a plurality of polypeptides, including mixtures thereof.

[0053] The term “about” or “approximately” as used herein when referring to a measurable value such as an amount or concentration and the like and, unless stated otherwise, is meant to encompass variations of + / - 20%.

[0054] As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but do not exclude others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the intended use. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and Attorney Docket No.199589-705601 / PCT purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0055] The terms “subject,” “host,” “individual,” and “patient” are used interchangeably herein.

[0056] As used herein, the terms, “treat,” “treatment,” “treating,” and “amelioration,” refer to therapeutic and prophylactic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, prevent, slow down, or stop the progression or severity of a condition associated with a disease or disorder. Treating includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a disease or disorder. Treatment is “effective” if one or more symptoms or clinical markers are reduced or if the progression of a disease is reduced or halted.

[0057] As used herein, the terms, “polynucleotide,” and “nucleic acid molecule,” are used interchangeably, and refer to polymers of deoxyribonucleotides, ribonucleotides, modified nucleotides, and / or their analogs, of any length, and includes DNA and RNA.

[0058] The terms “protein,” “peptide,” and “polypeptide,” interchangeably and in their broadest sense, refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics.

[0059] As used herein the term “amino acid” refers to natural, unnatural, or synthetic amino acids.

[0060] As used herein, “homology” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology is determined by comparing a position in each sequence which is aligned for purposes of comparison using known computer programs such the Bestfit program. When a position in the compared sequence is the same base or amino acid, then the molecules are identical at that position.

[0061] The term “epitope” refers to a portion or structure on a polypeptide that a moiety (e.g., a polypeptide immunoglobulin, antibody, etc.) specifically binds to.

[0062] The term “paratope” refers to a structure of a moiety (e.g., a polypeptide immunoglobulin, antibody, etc.) that specifically binds to an epitope.

[0063] The term “supervised learning” refers to a deep learning training method in which a machine is provided data from human sources. The term “unsupervised learning” refers to a deep learning training method in which a machine is not provided data from human sources.

[0064] The term “semi-supervised learning” refers to a deep learning training method in which a machine is provided a small amount of data from human sources which is then compared to a Attorney Docket No.199589-705601 / PCT larger amount of data from other sources available to the machine.

[0065] The terms, “% identical,” “% identity,” “percent identity,” and grammatical equivalents thereof, as used herein, in the context of an amino acid sequence or nucleotide sequence, refer to the percent of residues that are identical between respective positions of two sequences when the two sequences are aligned for maximum sequence identity. The % identity is calculated by dividing the total number of the aligned residues by the number of the residues that are identical between the respective positions of the at least two sequences and multiplying by 100. Generally, computer programs can be employed for such calculations. Illustrative programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, Comput Appl Biosci. 1988 Mar;4(1):11-7), FASTA (Pearson and Lipman, Proc Natl Acad Sci U S A.1988 Apr;85(8):2444- 8; Pearson, Methods Enzymol. 1990;183:63-98) and gapped BLAST (Altschul et al., Nucleic Acids Res. 1997 Sep 1;25(17):3389-40), BLASTP, BLASTN, or GCG (Devereux et al., Nucleic Acids Res.1984 Jan 11;12(1 Pt 1):387-95).

[0066] The terms, “bind,” “binding,” “interact,” and “interacting,” as used herein, refer to a non- covalent interaction between macromolecules (e.g., between two polypeptides, between a polypeptide and a nucleic acid; between a polypeptide / guide nucleic acid complex and a target nucleic acid; and the like).

[0067] The term “nucleotide,” as used herein, can refer to a base-sugar-phosphate combination. The nucleotide can be composed of three subunit molecules: a nucleobase, a five-carbon sugar (ribose or deoxyribose), and a phosphate. The four nucleobases in DNA can include guanine, adenine, cytosine, and thymine; in RNA, uracil can be used in place of thymine. Where DNA sequences are included herein, the corresponding RNA sequences, wherein at least one, two, three, four, five, or all T are replaced with U, are contemplated. A nucleotide can comprise a synthetic nucleotide. A nucleotide can comprise a synthetic nucleotide analog. Nucleotides can be monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)). Methods of characterizing intrinsically-disordered regions using HDX-MS

[0068] Disclosed herein are methods of interrogating a binding interaction between a disordered domain of a polypeptide and a binding molecule. Also disclosed herein are methods of altering order in a disordered domain of a polypeptide. Also disclosed herein are methods of identifying regions of structural disorder of a polypeptide using mass spectrometry. In some embodiments, the methods comprise determining by mass spectrometry hydrogen-deuterium exchange in peptide fragments of the polypeptide as a function of time. In some embodiments, the hydrogen-deuterium Attorney Docket No.199589-705601 / PCT exchange is determined by labeling the polypeptide with a D2O buffer in a labeling reaction. In some embodiments, the method further comprises quenching the labeling reaction after a reaction time period of from 0.05 seconds to 300 seconds. In some embodiments, the reaction time period is from 0.05 seconds to 200 seconds, 0.05 seconds to 100 seconds, 0.05 seconds to 50 seconds, 0.05 seconds to 25 seconds, 0.05 seconds to 10 seconds, or any range or subrange therebetween. In some embodiments, the method further comprises the generation of polypeptide fragments of the polypeptide of interest. In some embodiments, the method further comprises measuring incorporation of deuterium into the peptide fragments as a function of the reaction time period. In some embodiments, the methods further comprise identifying regions of structural disorder based on the hydrogen-deuterium exchange determined by the mass spectrometry by selecting peptides that have the fastest rate of hydrogen-deuterium exchange during the reaction time period of from 0.05 seconds to 300 seconds. In some embodiments, the method further comprises aligning the selected peptides to the polypeptide sequence, thereby identifying regions of structural disorder in the polypeptide.

[0069] In some embodiments, the methods further comprise: validating the identified regions of structural disorder of the polypeptide determined by mass spectrometry by comparing the identified regions of structural disorder of the polypeptide to regions of structural disorder determined modeling tools such as molecular dynamics (MD) simulations and machine-learning models. MD simulation data are determined as described herein. In some embodiments, MD simulation data, as detailed in this document, is collected. Further provided herein are methods, wherein the MD simulation data comprise an index score, which is computed using a structural prominence parameter and a disorder parameter. Further provided herein are methods, wherein the structural prominence parameter and the disorder parameter are determined through MD simulations of a homology model that incorporates combined structures of proteins similar to the polypeptide. In some embodiments, the MD simulation data comprises an index score that is calculated based on a structural prominence parameter and a disorder parameter, wherein the disorder parameter and the structural prominence parameter are derived from MD simulation of a homology model comprising aggregate structures of homologs of the polypeptide; and wherein the index score is proportional to the structural prominence parameter and is inversely proportional to the disorder parameter.

[0070] In some embodiments, the identified regions of structural disorder of the polypeptide are determined by mass spectrometry as validated when the regions have a low index score based on the MD simulation data. In some embodiments, the polypeptide is an androgen receptor Attorney Docket No.199589-705601 / PCT polypeptide. In some embodiments, the polypeptide is an alpha-synuclein polypeptide. In some embodiments, the polypeptide is a p27 polypeptide. In some embodiments, the polypeptide is c- myc polypeptide. In some embodiments, the polypeptide is a Tau polypeptide. In some embodiments, the polypeptide is an Amyloid β (1-42) polypeptide. Methods of detecting binding interactions between binding molecules and polypeptides using HDX-MS

[0071] Disclosed herein are methods of interrogating a binding interaction between a disordered domain of a polypeptide and a binding molecule. Also disclosed herein are methods of altering order in a disordered region of a polypeptide. In some embodiments, the methods comprise determining by mass spectrometry a hydrogen-deuterium exchange rate of peptide fragments of the polypeptide as a function of time in the absence of the binding molecule. In some embodiments, the hydrogen-deuterium exchange rate is determined by labeling the polypeptide with a D2O buffer in a labeling reaction. In some embodiments, the labeling reaction is quenched after a reaction time period of from 0.05 seconds to 10 seconds. In some embodiments, the reaction time period is from 0.05 seconds to 9 seconds, 0.05 seconds to 8 seconds, 0.05 seconds to 7 seconds, 0.05 seconds to 6 seconds, 0.05 seconds to 5 seconds, 0.05 seconds to 6 seconds, 0.05 seconds to 7 seconds, 0.05 seconds to 8 seconds, 0.05 seconds to 9 seconds, 1 seconds to 10 seconds, 2 seconds to 10 seconds, 3 seconds to 10 seconds, 4 seconds to 10 seconds, 5 seconds to 10 seconds, 6 seconds to 10 seconds, 7 seconds to 10 seconds, 8 seconds to 10 seconds, 9 seconds to 10 seconds, or any range or subrange therebetween. In some embodiments, the method further comprises the generation of polypeptide fragments of the polypeptide of interest. In some embodiments, the method further comprises measuring incorporation of deuterium into the peptide fragments as a function of the reaction time period. In some embodiments, the method further comprises determining by mass spectrometry a hydrogen-deuterium exchange rate of peptide fragments of the polypeptide as a function of time in the presence of the molecule by contacting the polypeptide with the molecule and repeating the foregoing steps of labeling the peptide fragments with a D2O buffer in a labeling reaction, quenching the labeling reaction after a reaction time period of from 0.05 seconds to 10 seconds, generating polypeptide fragments and measuring incorporation of deuterium into the peptide fragments as a function of the reaction time period. In some embodiments, the methods further comprise detecting the binding interaction between the polypeptide and the binding molecule by comparing the hydrogen-deuterium exchange in the peptide fragments in the presence and absence of the molecule, and identifying peptide fragments in which the hydrogen-deuterium exchange rate decreased in the presence of the binding molecule, relative to the hydrogen- Attorney Docket No.199589-705601 / PCT deuterium exchange of the peptide fragments in the absence of the binding molecule. In some embodiments, the decrease in the hydrogen-deuterium exchange rate indicates an increase in structural order in a region of the polypeptide. Without wishing to be bound by theory, the increase in structural order may result from binding of the binding molecule to the region of the polypeptide, thereby allowing interrogation of the binding interaction between the polypeptide and the binding molecule. In some embodiments, a plurality of binding molecules are independently contacted with polypeptide.

[0072] The present disclosure also provides for methods of generating a model of a binding site of a polypeptide to a molecule. In some embodiments, the method comprises determining by mass spectrometry a hydrogen-deuterium exchange rate of peptide fragments of the polypeptide as a function of time in the absence of the molecule. In some embodiments, the hydrogen-deuterium exchange rates are determined by labeling the peptide fragments with D2O buffer in a labeling reaction. In some embodiments, the labeling reaction is quenched after a reaction time period. In some embodiments, the incorporation of deuterium into the peptide fragments is measured as a function of the reaction time period. In some embodiments, the methods further comprise determining by mass spectrometry a hydrogen-deuterium exchange rate of peptide fragments of the polypeptide as a function of time in the presence of the molecule by contacting the polypeptide with the molecule and repeating the steps of labeling the peptide fragments with a D2O buffer in a labeling reaction, quenching the labeling reaction after a reaction time period, and measuring incorporation of deuterium into the peptide fragments as a function of the reaction time period. In some embodiments, the methods further comprise identifying a binding site of the polypeptide to the molecule. In some embodiments, the binding site is identified by comparing the hydrogen- deuterium exchange in the peptide fragments in the presence and absence of the molecule, and identifying peptide fragments in which the hydrogen-deuterium exchange rate decreased in the presence of the molecule, relative to the hydrogen-deuterium exchange of the peptide fragments in the absence of the molecule. Without wishing to be bound by theory, the decrease in the hydrogen-deuterium exchange rate may indicate an increase in structural order in a region of the polypeptide resulting from binding of the molecule to the region of the polypeptide, thereby allowing for interrogation of the binding interaction between the polypeptide and the molecule. In some embodiments, the methods provide for generating the model of the binding site of the polypeptide to the molecule using at least one model trained to predict a polypeptide structure. In some embodiments, the at least one model compares the hydrogen-deuterium exchange rate of the binding site of the polypeptide determined by mass spectrometry to a calculated hydrogen- Attorney Docket No.199589-705601 / PCT deuterium exchange rate of polypeptide docking conformers produced in modeling tools such as molecular dynamics (MD) simulations and machine-learning models, and identifies a polypeptide docking conformer that has a calculated hydrogen-deuterium exchange rate that is closest to the hydrogen-deuterium exchange rate obtained by mass spectrometry, thereby generating the model of the binding site of the polypeptide to the molecule. Detecting binding interactions between a molecule and a disordered domain of a c-myc polypeptide

[0073] The Myc family consists of three related human genes: c-myc (MYC), l-myc (MYCL), and n-myc (MYCN). The gene c-myc was the first gene to be discovered in this family, due to homology with the viral gene v-myc. In cancer, c-myc is often constitutively (persistently) expressed. C-myc controls global gene expression and regulates cell proliferation, differentiation, the cell cycle, metabolism, and apoptosis. According to estimates, myc is dysregulated in about 70% of human cancers and strong evidence implicates aberrant myc expression in both tumor initiation and maintenance.

[0074] C-myc is an intrinsically disordered protein, meaning its extended, unstructured surface, particularly in the unbound basic helix-loop-helix leucine zipper (bHLHZip) domain, lacks pockets and hydrophobic regions typically targeted by conventional small molecule drugs. Additionally, small molecule modulators may not selectively bind to c-myc over the many bHLHZip motifs found in other transcriptional factors. Despite significant efforts by the pharmaceutical sector, only few compounds have been reported to interfere directly with the c- myc / Max / DNA complex in vivo, highlighting the challenge of developing potent and selective c- myc inhibitors.

[0075] Most current molecules targeting the c-myc protein are designed to target the bHLHZip domain, but the structural details of their binding are largely unknown due to the difficulty of performing biophysical experiments on intrinsically disordered proteins. Consequently, many drug discovery programs aimed at targeting c-myc cannot progress due to a lack of structural information and the challenge of rationalizing steep structure-activity relationships.

[0076] The present disclosure addresses the problem of identifying and characterizing small molecules that bind to full-length c-myc using hydrogen-deuterium exchange mass spectrometry (HDX-MS) measurements. In some embodiments, the small molecule binding is characterized in the presence of endogenous binding partners. In some embodiments, the small molecule binding is characterized in the absence of endogenous binding partners. In some embodiments, the endogenous binding partners is MAX. Attorney Docket No.199589-705601 / PCT

[0077] Conventional structural investigational techniques such as X-ray crystallography and NMR spectroscopy often fail to determine the structure of intrinsically disordered proteins. This is primarily because intrinsically disordered proteins are too flexible to crystallize or too large for effective NMR analysis. In addition, these conventional methods often have other limitations that hinder the study of intrinsically disordered proteins. They require large amounts of material (e.g., about 200 micrograms) and high protein purity. Intrinsically disordered proteins are often aggregation prone proteins and are challenging to purify.

[0078] Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful analytical technique used to study protein-small molecule interactions. It works by monitoring the exchange of hydrogen atoms with deuterium in the protein’s backbone amides when exposed to deuterated solvents, which provides insights into the protein’s conformational changes and binding sites. This method allows for detailed mapping of the protein’s dynamic regions and the identification of binding sites for small molecules, making it valuable for drug discovery and structural biology.

[0079] Provided herein are methods for the characterization of the confirmational states and the dynamics of the full length proto-oncogene c-myc protein, an intrinsically disordered protein, by both traditional and fast HDX-MS experiments, c-myc was studied both in isolated form and in complex with MAX partner and small molecules.

[0080] Provided herein are methods of interrogating a binding interaction between a disordered domain of a c-myc polypeptide and a binding molecule. Also provided herein are methods for altering order in a disordered region of c-myc polypeptides. Also provided herein are methods for identifying regions of structural disorder of c-myc polypeptides. Further provided herein are methods, wherein the c-myc polypeptide is the c-myc polypeptide deposited under Uniprot accession number P01106. Further provided herein are methods, wherein the c-myc polypeptide is the c-myc polypeptide deposited under Uniprot accession number P01106-1.

[0081] In some embodiments, the method comprises determining by mass spectrometry a hydrogen-deuterium exchange rate of peptide fragments of the disordered domain of the c-myc polypeptide as a function of time in the absence of the binding molecule. In some embodiments, the hydrogen-deuterium exchange rate detection comprises labeling the polypeptide with D2O buffer in a labeling reaction. In some embodiments, the labeling reaction is quenched after a reaction time period. In some embodiments, polypeptide fragments of the c-myc polypeptide are generated. In some embodiments, determination of the hydrogen-deuterium exchange rate comprises measuring incorporation of deuterium into the peptide fragments as a function of the reaction time period. In some embodiments, the method further comprises determining by mass Attorney Docket No.199589-705601 / PCT spectrometry a hydrogen-deuterium exchange rate of peptide fragments of the c-myc polypeptide as a function of time in the presence of the binding molecule by contacting the c-myc polypeptide with the binding molecule and repeating the steps of labeling the peptide fragments with D2O buffer in a labeling reaction, quenching the labeling reaction after a reaction time period, generating polypeptide fragments of the c-myc polypeptide and measuring incorporation of deuterium into the peptide fragments as a function of the reaction time period. In some embodiments, the method further comprises surveying the binding interaction between the disordered domain of the c-myc polypeptide and the binding molecule by comparing the hydrogen- deuterium exchange in the peptide fragments in the presence and absence of the binding molecule, and identifying peptide fragments in which the hydrogen-deuterium exchange rate decreased in the presence of the binding molecule, relative to the hydrogen-deuterium exchange of the peptide fragments in the absence of the binding molecule. Without wishing to be bound by theory, the change in the hydrogen-deuterium exchange rate may indicate a change in structural order in the disordered domain of the c-myc polypeptide resulting from binding of the binding molecule to the disordered domain of the c-myc polypeptide, thereby allowing for interrogation of the binding interaction between the disordered domain of the c-myc polypeptide and the binding molecule. C-myc Polypeptide Related Diseases

[0082] In some embodiments, the polypeptides contemplated in the methods of the present disclosure are c-myc polypeptides. In some embodiments, the c-myc polypeptide is the c-myc polypeptide deposited under Uniprot accession number P01106. In some embodiments, the c-myc polypeptide is the c-myc polypeptide deposited under Uniprot accession number P01106-1. In some embodiments, the molecules contemplated in the methods of the present disclosure are peptides or peptide mimics. In some embodiments, the molecules contemplated in the methods of the present disclosure are small molecules having a molecular weight of less than 40 kDa. The present disclosure also provides molecules that binds to the polypeptide sequence of SEQ ID NO: 1 of a c-myc polypeptide. The present disclosure also provides molecules that binds to the polypeptide sequence of SEQ ID NO: 2 of a c-myc polypeptide. The present disclosure also provides molecules that binds to the polypeptide sequence of SEQ ID NO: 3 of a c-myc polypeptide. The present disclosure also provides molecules that binds to the polypeptide sequence of SEQ ID NO: 4 of a c-myc polypeptide. The present disclosure also provides molecules that binds to the polypeptide sequence of SEQ ID NO: 5 of a c-myc polypeptide. In some embodiments, the molecule binds to the polypeptide sequence with a binding affinity of less than 10 µM (for example, a binding affinity of 1 µM, 100 nM, 10 nM, or less than 1 nM). In some Attorney Docket No.199589-705601 / PCT embodiments, the molecule is a peptide or peptide mimic. In some embodiments, the molecule is a small molecule having a molecular weight of less than 40 kDa.

[0083] Also provided herein are methods of treating a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition that comprises the molecules identified to bind to the c-myc polypeptide using the HDX-MS methods described herein.

[0084] The present disclosure further provides for methods of treating diseases or conditions associated with the c-myc polypeptide, or compositions comprising compounds for treating such diseases or conditions. In some embodiments, the modulation provides for inhibiting c-myc polypeptide activity. In some embodiments, the modulation provides for modulation of an alpha- synuclein neurodegenerative disease. In some embodiments, c-myc related diseases or conditions are chosen from: a lymphoma, immune disorder, cancer, myasthenia gravis, psoriasis, pemphigus vulgaris, atherosclerosis, long-term allogeneic survival among HSCT patients, primary inflammatory breast cancer, primary ovarian carcinoma, hematological malignancies, acute myeloid leukemia, chronic myelogenous leukemia, Hodgkin’s lymphoma, or diffuse large B-cell lymphoma. Detecting binding interactions between a molecule and the disordered domain of an androgen receptor polypeptide

[0085] The androgen receptor has a diverse range of biological actions, including important roles in the development and regulation of reproductive, musculoskeletal, cardiovascular, immune, neural, and hematopoietic systems. Provided herein are methods interrogating a binding interaction between a disordered domain of an androgen receptor polypeptide and a binding molecule. Also provided herein are methods for altering order in a disordered region of androgen receptor polypeptides. Also provided herein are methods for identifying regions of structural disorder of androgen receptor polypeptides. Further provided herein are methods, wherein the androgen receptor polypeptide is the androgen receptor polypeptide deposited under Uniprot accession number P10275.

[0086] In some embodiments, the method comprises determining by mass spectrometry a hydrogen-deuterium exchange rate of peptide fragments of the disordered domain of the androgen receptor polypeptide as a function of time in the absence of the binding molecule. In some embodiments, the hydrogen-deuterium exchange rate detection comprises labeling the polypeptide with D2O buffer in a labeling reaction. In some embodiments, the labeling reaction is quenched after a reaction time period. In some embodiments, polypeptide fragments of the androgen receptor Attorney Docket No.199589-705601 / PCT polypeptide are generated. In some embodiments, determination of the hydrogen-deuterium exchange rate comprises measuring incorporation of deuterium into the peptide fragments as a function of the reaction time period. In some embodiments, the method further comprises determining by mass spectrometry a hydrogen-deuterium exchange rate of peptide fragments of the androgen receptor polypeptide as a function of time in the presence of the binding molecule by contacting the androgen receptor polypeptide with the binding molecule and repeating the steps of labeling the peptide fragments with D2O buffer in a labeling reaction, quenching the labeling reaction after a reaction time period, generating polypeptide fragments of the androgen receptor polypeptide and measuring incorporation of deuterium into the peptide fragments as a function of the reaction time period. In some embodiments, the method further comprises surveying the binding interaction between the disordered domain of the androgen receptor polypeptide and the binding molecule by comparing the hydrogen-deuterium exchange in the peptide fragments in the presence and absence of the binding molecule, and identifying peptide fragments in which the hydrogen-deuterium exchange rate decreased in the presence of the binding molecule, relative to the hydrogen-deuterium exchange of the peptide fragments in the absence of the binding molecule. Without wishing to be bound by theory, the change in the hydrogen-deuterium exchange rate may indicate a change in structural order in the disordered domain of the androgen receptor polypeptide resulting from binding of the binding molecule to the disordered domain of the androgen receptor polypeptide, thereby allowing for interrogation of the binding interaction between the disordered domain of the androgen receptor polypeptide and the binding molecule. In some embodiments, the disordered domain of the androgen receptor polypeptide comprises an N-terminal domain. Androgen Receptor Polypeptide and Related Disease Conditions

[0087] Polypeptides contemplated in the methods of the present disclosure are androgen receptor polypeptides. In some embodiments, the androgen receptor polypeptide is the androgen receptor polypeptide deposited under Uniprot accession number P10275. Disclosed herein are molecules that bind to the polypeptide sequence of SEQ ID NO: 6 of an androgen receptor polypeptide. In some embodiments, the molecule binds to the polypeptide sequence with a binding affinity of less than 10 µM (for example, a binding affinity of 1 µM, 100 nM, 10 nM, or less than 1 nM). In some embodiments, the molecule is a peptide or peptide mimic. In some embodiments, the molecule is a small molecule having a molecular weight of less than 40 kDa.

[0088] Also disclosed herein is a method of treating androgen-dependent prostate cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a binding molecule identified to bind to the N-terminal domain of the androgen Attorney Docket No.199589-705601 / PCT receptor polypeptide using the HDX-MS methods described herein.

[0089] The present disclosure further provides for methods of treating diseases or conditions associated with the androgen receptor polypeptide, or compositions comprising compounds for treating such diseases or conditions.

[0090] In some embodiments, the diseases or conditions associated with the androgen receptor polypeptide comprise a cancer. In some embodiments, the cancer is selected from prostate cancer, breast cancer, ovarian cancer, bladder cancer, pancreatic cancer, hepatocellular cancer, endometrial cancer, or salivary gland carcinoma. In some embodiments, the cancer is prostate cancer. In some embodiments, the prostate cancer is primary or localized prostate cancer, locally advanced prostate cancer, recurrent prostate cancer, advanced prostate cancer, metastatic prostate cancer, non-metastatic castration-resistant prostate cancer, metastatic castration-resistant prostate cancer, or hormone-sensitive prostate cancer. In some embodiments, the prostate cancer is metastatic castration-resistant prostate cancer. In some embodiments, the prostate cancer expresses full-length androgen receptor or truncated androgen receptor splice variant.

[0091] In some embodiments, administration of a composition generated using methods described herein modulates activity of an androgen receptor in a subject. In some embodiments, the modulation provides for inhibiting androgen receptor activity. In some embodiments, the modulation provides for modulation of an androgen-dependent cancer. In some embodiments, the modulation provides for reduction of a tumor size in an androgen-dependent cancer. In some embodiments, the modulation results in a reduction of a tumor volume of an androgen-dependent cancer. In some embodiments, the modulation results in a reduction of an androgen-dependent cancer recurrence. In some embodiments, androgen receptor polypeptide related diseases or conditions are chosen from: an androgen insensitivity syndrome, spinal bulbar muscular atrophy, benign prostatic hyperplasia, and prostate cancer.

[0092] In some embodiments, the binding molecule identified to bind to the N-terminal domain of the androgen receptor polypeptide using the HDX-MS methods described herein is administered with one or more additional therapeutic agents. In some embodiments, the binding molecules identified using the disclosed HDX-MS method, are used to improve the efficacy of the one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents is a poly (ADP-ribose) polymerase (PARP) inhibitor comprising olaparib, niraparib, rucaparib, talazoparib; an androgen receptor ligand-binding domain inhibitor comprising enzalutamide, apalutamide, darolutamide, bicalutamide, nilutamide, flutamide, ODM-204, TAS3681; an inhibitor of CYP17 comprising galeterone, abiraterone, abiraterone acetate; a Attorney Docket No.199589-705601 / PCT microtubule inhibitor comprising docetaxel, paclitaxel, cabazitaxel (XRP-6258); a modulator of PD-1 or PD-L1 comprising pembrolizumab, durvalumab, nivolumab, atezolizumab; a gonadotropin releasing hormone agonist comprising cyproterone acetate, leuprolide; a 5-alpha reductase inhibitor comprising finasteride, dutasteride, turosteride, bexlosteride, izonsteride, FCE 28260, SKF105,111; a vascular endothelial growth factor inhibitor comprising bevacizumab (Avastin); a histone deacetylase inhibitor comprising OSU-HDAC42; an integrin alpha-v-beta-3 inhibitor comprising VITAXIN; a receptor tyrosine kinase comprising sunitumib; a phosphoinositide 3-kinase inhibitor comprising alpelisib, buparlisib, idealisib; an anaplastic lymphoma kinase (ALK) inhibitor comprising crizotinib, alectinib; an endothelin receptor A antagonist comprising ZD-4054; an anti-CTLA4 inhibitor comprising MDX-010 (ipilimumab); an heat shock protein 27 (HSP27) inhibitor comprising OGX 427; an androgen receptor degrader comprising ARV-330, ARV-110; a androgen receptor DNA-binding domain inhibitor comprising VPC-14449; a bromodomain and extra-terminal motif (BET) inhibitor comprising BI-894999, GSK25762, GS-5829; an N-terminal domain inhibitor comprising a sintokamide; an alpha-particle emitting radioactive therapeutic agent comprising radium 233 or a salt thereof; niclosamide; or related compounds thereof; a selective estrogen receptor modulator (SERM) comprising tamoxifen, raloxifene, toremifene, arzoxifene, bazedoxifene, pipindoxifene, lasofoxifene, enclomiphene; a selective estrogen receptor degrader (SERD) comprising fulvestrant, ZB716, OP- 1074, elacestrant, AZD9496, GDC0810, GDC0927, GW5638, GW7604; an aromatase inhibitor comprising anastrazole, exemestane, letrozole; selective progesterone receptor modulators (SPRM) comprising mifepristone, lonaprison, onapristone, asoprisnil, lonaprisnil, ulipristal, telapristone; a glucocorticoid receptor inhibitor comprising mifepristone, COR108297, COR125281, ORIC-101, PT150; HER2 receptor antagonist comprising trastuzumab, neratinib; or a mammalian target of rapamycin (mTOR) inhibitor comprising everolimus, temsirolimus, an AKT inhibitor comprising MK-2206; a Bcl-2 inhibitor comprising venetoclax; an aurora kinase inhibitor comprising alisertib; a Wnt-targeting antagonist comprising DKK-1-4 proteins (Dikhopf), secreted Frazzle related proteins (sFRP); a CYP11a inhibitor comprising ODM-208; a selective androgen receptor N-terminal domain inhibitor comprising LY2452473; or EZH2 inhibitor comprising CPI-1205. In another embodiment, the second therapeutically active agent is a nonsteroidal antiandrogen (NSAA). Alpha-synuclein Polypeptide Related Diseases

[0093] In some embodiments, the polypeptides contemplated in the methods of the present disclosure are alpha-synuclein polypeptides. In some embodiments, the alpha-synuclein Attorney Docket No.199589-705601 / PCT polypeptide is the alpha-synuclein polypeptide deposited under Uniprot accession number P37840. In some embodiments, the molecules contemplated in the methods of the present disclosure are peptides or peptide mimics. In some embodiments, the molecules contemplated in the methods of the present disclosure are small molecules having a molecular weight of less than 40 kDa. Disclosed herein are molecules that bind to the polypeptide sequence of SEQ ID NO: 7 of an alpha-synuclein polypeptide. In some embodiments, the molecule binds to the polypeptide sequence with a binding affinity of less than 10 µM(for example, a binding affinity of 1 µM, 100 nM, 10 nM, or less than 1 nM). In some embodiments, the molecule is a peptide or peptide mimic. In some embodiments, the molecule is a small molecule having a molecular weight of less than 40 kDa.

[0094] Also provided are methods of treating a synucleinopathy in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition that comprises a therapeutically effective amount of a binding molecule identified to bind to the alpha-synuclein polypeptide using the HDX-MS methods described herein.

[0095] The present disclosure further provides for methods of treating diseases or conditions associated with the alpha-synuclein polypeptide, or compositions comprising compounds for treating such diseases or conditions. In some embodiments, the modulation provides for inhibiting alpha-synuclein activity. In some embodiments, the modulation provides for modulation of an alpha-synuclein neurodegenerative disease. In some embodiments, the alpha-synuclein related diseases or conditions are chosen from: Parkinson’s disease, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), Huntington’s disease, Alzheimer’s disease, primary age-related tauopathy, progressive supranuclear palsy (PSP), frontotemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, argyrophilic grain dementia, amyotrophic lateral sclerosis / parkinsonism-dementia complex of Guam, corticobasal degeneration, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down’s syndrome, familial British dementia, familial Danish dementia, Gerstmann-Straussler-Scheinker disease, globular glial tauopathy, Guadeloupean parkinsonism with dementia, Guadelopean PSP, Hallevorden-Spatz disease, inclusion-body myositis, myotonic dystrophy, neurofibrillary tangle-predominant dementia, Niemann-Pick disease type C, pallido- ponto-nigral degeneration, Pick’s disease, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, subacute sclerosing panencephalitis, tangle only dementia, or other alpha-synuclein related neurodegenerative diseases. Attorney Docket No.199589-705601 / PCT Tau Polypeptide Related Diseases

[0096] In some embodiments, the polypeptides contemplated in the methods of the present disclosure are Tau polypeptides. In some embodiments, the Tau polypeptide is the Tau polypeptide deposited under Uniprot accession number P10636. In some embodiments, the molecules contemplated in the methods of the present disclosure are peptides or peptide mimics. In some embodiments, the molecules contemplated in the methods of the present disclosure are small molecules having a molecular weight of less than 40 kDa. The present disclosure also provides molecules that bind to the polypeptide sequence of SEQ ID NO: 8 of a Tau polypeptide. In some embodiments, the molecule binds to the polypeptide sequence with a binding affinity of less than 5 µM. In some embodiments, the molecule is a peptide or peptide mimic. In some embodiments, the molecule is a small molecule having a molecular weight of less than 40 kDa.

[0097] Also provided are methods of treating a tauopathy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition that comprises the binding molecule identified to bind to the Tau polypeptide using the HDX-MS methods described herein.

[0098] The present disclosure further provides for methods of treating diseases or conditions associated with the Tau polypeptide, or compositions comprising compounds for treating such diseases or conditions. In some embodiments, the modulation provides for inhibiting Tau polypeptide activity. In some embodiments, the modulation provides for degradation of Tau protein in a subject in need thereof. In some embodiments, Tau polypeptide related diseases or conditions are chosen from: Alzheimer’s disease, frontotemporal dementia with parkinsonism-17 (FTDP-17), Pick disease (PiD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), primary age-related tauopathy (PART) / neurofibrillary tangle-predominant senile dementia, chronic traumatic encephalopathy, dementia pugilistica, progressive supranuclear palsy, corticobasal degeneration, Lytico-Bodig disease, ganglioglioma, gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, Huntington’s disease, Alzheimer’s disease, or argyrophilic grain disease, or other Tau-related neurodegenerative diseases. P27 Polypeptide Related Diseases

[0099] In some embodiments, the polypeptides contemplated in the methods of the present disclosure p27 polypeptides. In some embodiments, the p27 polypeptide is the p27 polypeptide deposited under Uniprot accession number P46527. In some embodiments, the molecules Attorney Docket No.199589-705601 / PCT contemplated in the methods of the present disclosure are peptides or peptide mimics. In some embodiments, the molecules contemplated in the methods of the present disclosure are small molecules having a molecular weight of less than 40 kDa. The present disclosure also provides molecules that bind to the polypeptide sequence of SEQ ID NO: 9 of a p27 polypeptide. In some embodiments, the molecule binds to the polypeptide sequence with a binding affinity of less than 10 µM (for example, a binding affinity of 1 µM, 100 nM, 10 nM, or less than 1 nM). In some embodiments, the molecule is a peptide or peptide mimic. In some embodiments, the molecule is a small molecule having a molecular weight of less than 40 kDa.

[0100] Also provided herein are methods of treating a disease or condition associated with the p27 polypeptide in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition that comprises the binding molecules identified to bind to the p27 polypeptide using the HDX-MS methods described herein.

[0101] The present disclosure further provides for methods or treating diseases or conditions associated with the p27 polypeptide, or compositions comprising compounds for treating such diseases or conditions. In some embodiments, the modulation provides for inhibiting p27 polypeptide activity. In some embodiments, the modulation provides for inhibiting the activation of a cyclin E-Cdk2 complex. In some embodiments, the modulation provides for treating a hyperproliferative disorder in a subject in need thereof. In some embodiments, p27 diseases or conditions are chosen from: a hydatidiform mole or Beckwith-Wiedemann syndrome, IMAGe and Silver–Russell syndromes, or other genetic diseases. Amyloid Beta (1-42) Polypeptide Related Diseases

[0102] In some embodiments, the polypeptides contemplated in the methods of the present disclosure are Amyloid β (1-42) polypeptides. In some embodiments, the Amyloid β (1-42) polypeptide is the Amyloid β (1-42) polypeptide deposited under Uniprot accession number P05067. In some embodiments, the molecules contemplated in the methods of the present disclosure are peptides or peptide mimics. In some embodiments, the molecules contemplated in the methods of the present disclosure are small molecules having a molecular weight of less than 40 kDa. The present disclosure also provides molecules that binds to the polypeptide sequence of SEQ ID NO: 10 of an Amyloid β (1-42) polypeptide. In some embodiments, the molecule binds to the polypeptide sequence with a binding affinity of less than 10 µM (for example, a binding affinity of 1 µM, 100 nM, 10 nM, or less than 1 nM). In some embodiments, the molecule is a peptide or peptide mimic. In some embodiments, the molecule is a small molecule having a molecular weight of less than 40 kDa. Attorney Docket No.199589-705601 / PCT

[0103] Also provided herein are methods of treating Alzheimer’s disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition that comprises the molecules identified to bind to the amyloid beta polypeptide using the HDX-MS methods described herein.

[0104] The present disclosure further provides for methods for treating diseases or conditions associated with the amyloid beta polypeptide, or compositions comprising compounds for treating such diseases or conditions. In some embodiments, the modulation provides for inhibiting amyloid beta activity. In some embodiments, the modulation provides for modulation of amyloid beta aggregation. In some embodiments, amyloid beta polypeptide diseases or conditions are chosen from: Alzheimer’s disease, hereditary amyloidosis, or cerebral amyloid angiopathy. Binding molecules and methods of treating diseases

[0105] Also disclosed herein are binding molecules identified using the HDX-MS methods described herein to bind to polypeptides described herein. In some embodiments, the binding molecules bind to an intrinsically-disordered region of a polypeptide described herein, which may result in structural stabilization upon binding. In some embodiments, the binding molecules bind to the polypeptide with a binding affinity of from about 1 nM to about 100 µM. In some embodiments, the binding affinity is in a range of about 1 nM to about 75 µM, about 1 nM to about 50 µM, about 1 nM to about 25 µM, about 1 nM to about 10 µM, about 100 nM to about 100 µM, about 500 nM to about 100 µM, about 1 µM to about 100 µM, about 10 µM to about 100 µM, about 25 µM to about 100 µM, about 50 µM to about 100 µM, about 75 µM to about 100 µM, or any range or subrange therebetween.

[0106] Also disclosed herein are methods of treating diseases or conditions in a subject in need thereof, the method comprising administering a therapeutically effective amount of a binding molecule described herein, or a pharmaceutical composition containing a binding molecule described herein. In some embodiments, the pharmaceutical compositions disclosed herein are used for the treatment of a disease or condition in a subject or a subject in need thereof. In some embodiments the disease or condition is a health-related, a health condition associated with damaged cells, a population of tumorous cells, or a cancer.

[0107] In some embodiments, the pharmaceutical compositions described herein are used for the treatment of a cancer. In some embodiments, the cancer is a prostate cancer. In some embodiments, the cancer is an androgen-dependent prostate cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a blood cancer. In some embodiments, the solid cancer is a melanoma, lung, liver, head and neck, hepatocellular cancer, or pancreatic cancer. In some Attorney Docket No.199589-705601 / PCT embodiments, the solid cancer is a hepatocellular cancer, melanoma, or lung cancer. In some embodiments, a composition described herein is used for reduction of a tumor size. In some embodiments, a composition described herein is used for reduction of a tumor volume. In some embodiments, a composition described herein is used for reduction of a cancer recurrence. In some embodiments, a composition described herein is used for reduction of tumor metastasis.

[0108] In some embodiments is provided a method of treating a subject in need thereof with a therapeutic effective dose of a composition or a pharmaceutical composition described herein. In some embodiments the therapeutic effective dose is a dose sufficient to induce an inflammatory response, to promote tumor reduction, or both. In some embodiments, a therapeutically effective amount is an amount sufficient to reduce, ameliorate, or prevent at least one symptom of a disease or condition. In some embodiments, contacting or applying to a tissue of, or administering to a patient or a patient in need thereof, occurs daily, every other day, every third day, every fourth day, every fifth day, every sixth day, weekly, every two weeks, every three weeks, once a month, once every three months, once every six months, once a year, or as needed. In some embodiments, the contacting is once, twice, three, four, five, six, seven, eight, nine, or ten times in a 24-hour period. In some embodiments, the subject is chosen from: a mammal, a human, a primate, a non- human primate, a mouse, a rat, a hamster, a rabbit, a pig, a bovine, a deer, a sheep, a goat, a chicken, a cat, a dog, a ferret, a bird, a llama, a horse, or a non-human primate (e.g., marmoset, rhesus monkey). In some embodiments, the non-human animal is a domesticated mammal or an agricultural mammal etc. Pharmaceutical Compositions

[0109] A binding molecule identified using the HDX-MS methods described herein can be formulated in a pharmaceutical composition for use in treatment. The compositions and pharmaceutical compositions herein, and / or any therapeutic or further therapeutic herein can be formulated as neutral or salt forms, including as a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. In some embodiments, a salt or a pharmaceutically acceptable salt can comprise an HCl salt, an ascorbic acid salt, a mandelic acid salt, an aspartic acid salt, a carbonic acid salt, a citric acid salt, a formic acid salt, a glutamic acid salt, a lactic acid salt, a lauric acid salt, a maleic acid salt, a palmitic acid salt, a phosphoric acid salt, or any combination thereof. In some embodiments, a salt or a Attorney Docket No.199589-705601 / PCT pharmaceutically acceptable salt can include, but is not limited to, a metal salt such as sodium salt, potassium salt, cesium salt and the like; an alkaline earth metal salt such as calcium salt, magnesium salt and the like; an organic amine salt such as a triethylamine salt, a pyridine salt, a picoline salt, an ethanolamine salt, a triethanolamine salt, a dicyclohexylamine salt, an N,N′- dibenzylethylenediamine salt and the like; an inorganic acid salt such as hydrochloride, hydrobromide, phosphate, sulphate and the like; an organic acid salt such as citrate, lactate, tartrate, maleate, fumarate, mandelate, acetate, dichloroacetate, trifluoroacetate, oxalate, formate and the like; a sulfonate such as methanesulfonate, benzenesulfonate, p-toluenesulfonate and the like; and / or an amino acid salt such as arginate, asparginate, glutamate and the like.

[0110] The compositions and pharmaceutical compositions disclosed herein can comprise a preservative, e.g., a compound which can be added to essentially reduce bacterial and / or fungal action or presence in or on any composition or pharmaceutical composition herein. Examples of preservatives include but are not limited to octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkoniurn chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride.

[0111] A composition or pharmaceutical composition herein can be formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, topical, systemic, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), intratumoral, transdermal (e.g., topical), transmucosal, and rectal administration. In some embodiments, a composition or pharmaceutical composition can be formulated as a composition or pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, transdermal, or topical administration to a human being or subject or subject in need thereof. Compositions and pharmaceutical compositions for intravenous administration can be solutions in sterile isotonic aqueous buffer. Compositions and pharmaceutical compositions here can be sterile or aseptic.

[0112] Compositions and pharmaceutical compositions herein may also include a solubilizing agent and / or a local anesthetic such as lignocaine or a pharmaceutically acceptable salt of any of these, for example, to ease pain at the site of the administration, contacting, or injection. In some embodiments, the methods of the disclosure can comprise administration of a composition formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain agents such as Attorney Docket No.199589-705601 / PCT suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.

[0113] In some embodiments, a composition or a pharmaceutical composition comprises a surfactant. Surfactants can lower the surface tension of a liquid, the interfacial tension between two liquids, or that between a liquid and a solid. The surfactant can be a detergent, a wetting agent, an emulsifier, a foaming agent, a dispersant, or any combination thereof. In some embodiments, the surfactant can be a polysorbate-type emulsifier. In some embodiments, the polysorbate is a PEG (polyethylene glycol)-ylated sorbitan esterified with one or more fatty acids. In some embodiments, the surfactant is selected from: Polysorbate 20 (polyoxyethylene 20 sorbitan monolaurate), Polysorbate 60, Polysorbate 80, or any combination thereof.

[0114] In other embodiments, compositions and pharmaceutical compositions provided herein can be provided in an oral form, a transdermal form, an oil formulation, an edible food, or a food substrate, an aqueous dispersion, an emulsion, an oil-in-water emulsion, a water-in-oil emulsion, a solution, a suspension, an elixir, a gel, a syrup, an aerosol, a mist, a powder, a pill, a tablet, a lozenge, a gel, a lotion, a paste, a formulated stick, a balm, a cream, an ointment, or comprised in a bandage or a dressing.

[0115] Provided herein are also kits comprising compositions or pharmaceutical compositions disclosed herein. The kits can include packaging, instructions, and / or a container. The kits can comprise a further therapeutic, which can be comprised in composition or a pharmaceutical composition, or comprised in the kit separately from the composition or the pharmaceutical composition. In some embodiments, the kits can contain additional compositions used to generate various formulation precursors. Pharmacological carriers and diluents

[0116] In some embodiments, pharmacologically acceptable carriers and pharmaceutically acceptable carriers may be referred to interchangeably herein. Exemplary pharmaceutically acceptable carriers include but are not limited to buffered solutions. In some embodiments, a buffered solution can be a solution that resists changes in pH when acid or alkali is added to it. In some embodiments, a buffered solution is or comprises phosphate buffered saline (PBS). In some embodiments, a carrier or a pharmaceutically acceptable carrier can be or include a penetrant. In some cases, a carrier can be a substrate used in the process of drug delivery. In some cases, a carrier can contribute to a composition’s or pharmaceutically acceptable composition’s attributes such as stability, biopharmaceutical profile, appearance, and / or patient acceptability. In some cases, a carrier or pharmaceutically acceptable carrier can be or comprise an organic excipient. Attorney Docket No.199589-705601 / PCT Excipients include functional and / or non-functional ingredients in a composition or a pharmaceutical composition. In some cases, an excipient or a pharmaceutically acceptable excipient can comprise an oil, water, an aqueous solution, an acid, a salt, an alcohol, a carbohydrate, a sugar (i.e., a cyclodextrin), a buffer, a powder, a filler, a gum, a wax (e.g., carnauba, cetyl esters, microcrystalline, nonionic emulsifying, white, yellow), or any combination thereof.

[0117] In some cases, a carrier or a diluent or pharmaceutically acceptable carrier or diluent can be or comprise a solid such as a filling agent used in the production of a pill, for example lactose or another carbohydrate.

[0118] In some embodiments, the release and / or administration of a composition or pharmaceutical composition described herein is facilitated by a delivery system. In some embodiments, the delivery system requires at least one administration or contacting. In some embodiments, the delivery system can be administered or contacted with a subject or a subject in need thereof more than once, for example 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or more. In some embodiments, the delivery system requires multiple administrations.

[0119] In some embodiments, the delivery system is or can comprise a polymer-based system. In some embodiments, the polymer-based system is selected from at least one of: a poly(lactideglycolide), a copolyoxalate, a polycaprolactone, a polyesteramide, a polyorthoester, a polyhydroxybutyric acid, a polyanhydride, and any combination thereof. Delivery Systems

[0120] In some embodiments, compositions or pharmaceutical compositions described herein are administered or contacted for treatment of a skin or a tissue, for example of a subject or a subject in need thereof. In some embodiments, delivery is intravenous.

[0121] In some embodiments, the compositions and / or pharmaceutical compositions described herein are administered once, twice, three, four, five, six, seven, eight, nine, ten or multiple times. In some embodiments, compositions and / or pharmaceutical compositions described herein are administered directly to or contacted directly or indirectly to immune cells.

[0122] In some embodiments, compositions and / or pharmaceutical compositions described herein are formulated in extended-release formulations, to a tissue over a defined duration of time. In some embodiments the duration of time is at least about: 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 24 hours, at least about 22 days, at least about 3 days, at Attorney Docket No.199589-705601 / PCT least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days wherein a day is 24 hours, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, or for as long as necessary or desired.

[0123] The following subsections below illustrate compositions and methods of treatment using binding molecules that target certain polypeptides having intrinsically-disordered binding regions. Systems

[0124] Also disclosed herein are systems for performing methods described herein. For example, disclosed herein are HDX-MS systems configured to perform the rapid HDX-MS methods described herein. For example, the system comprises a rapid mixing HDX-specific stopped flow prototype for running millisecond to minute hydrogen-deuterium exchange experiments, connected to a LEAP HDX Extended MF System capable of automatically running HDX experiments from 15 seconds to 24 hours, and to an LC-MS system consisting of two I-Class UPLC Pumps, and a SELECT Series CyclicIMS mass spectrometer, equipped with an optional ECD cell (Electron Capture Dissociation).

[0125] A system can comprise a computer readable memory storing instructions for performing methods described herein. For example, the computer readable memory can comprise instructions for in silico determination of polypeptide structures as described herein. In some embodiments, the computer readable memory can comprise instructions for epitope determination as described herein.

[0126] A system can further comprise computer systems utilizing the computer readable memory. Computer systems can include a processor operatively coupled to the computer readable memory, and can be configured to execute the instructions to perform a method described herein. A computer system can further include user input and output means, such as a keyboard, monitor, and mouse.

[0127] A system as described herein can be configured to access a database. For example, a system can be configured to access local or online (e.g., cloud) databases such as protein structure database, protein sequence databases, homology databases, nucleic acid sequence databases, and the like.

[0128] Upon execution of a method described herein, a system can further comprise data obtained by executing a method described herein. For example, a system upon execution of a method described herein can comprise druggability index scores for determining novel epitopes. A system Attorney Docket No.199589-705601 / PCT can comprise structural information obtained from MD simulations described herein. Further, a system can comprise empirical structural data such as protein structures obtained from NMR or X-ray crystallography. A system can comprise an optimized polypeptide structure obtained using the in silico methods described herein.

[0129] Such systems can include storage means for storing or transferring data obtained by the methods described herein. In some instances, the systems can include means to transmit data obtained by the methods described herein into an external database (e.g., a local database or an online database). Molecular Dynamics Simulations

[0130] Methods described herein that utilize HDX-MS can also utilize molecular dynamics (MD) simulations as described herein. Molecular dynamics simulations can be performed in silico to model polypeptide structural conformations and biophysical features. Molecular dynamics simulations can predict structural dynamics, such that the secondary and tertiary structure of a polypeptide can vary within the timeline of the simulation along allowed conformations. Generally, allowed conformations are those that represent minima along various free energy minima. As such, molecular dynamics simulations can be used to visualize and sample biologically relevant conformations that static structural techniques (e.g., x-ray crystallography) may not sample. Exemplary molecular dynamics simulations for inclusion in methods described herein include, without limitation, Classical Dynamics, Replica Exchange Molecular Dynamics, Metadynamics and variations thereof, Langevin Dynamics and enhanced sampling methods in general.

[0131] Provided herein are methods wherein data generated from molecular dynamic simulations are relied upon for modeling and predicting polypeptide structures. As described herein, data generated from molecular dynamics simulations and molecular modeling in general is used to study conformations or states that might be energetically unfavourable in standard molecular dynamics simulations and molecular modeling in general and may occur on longer timescales. In some embodiments, specific enhancements are introduced or biases modelled by machine learning-based models predicting the protection of the amide backbone of the polypeptide in both the presence and absence of the binding molecule and refined with the data provided by the Ultra Fast HDX-MS methods described herein. In some embodiments, molecular dynamics simulations are finally reweighted to correct for biases introduced by enhanced sampling methods and extracting unbiased information from the simulation data calculating probability distributions that describe how the system would have behaved in the absence of the biasing potential. In some Attorney Docket No.199589-705601 / PCT embodiments, the integrative modeling approach described herein takes the amino acid sequence of the polypeptide, the SMILES sequence of a binding molecule and the single residue level protection factors derived from deuterium uptake curves provided by the Ultra Fast HDX-MS methods described herein as input. In some embodiments, the protection factor ^^^^^of the ^^-thresidue belonging to the polypeptide sequence ^^ of length ^^ where ^^ ൌ 1^^. , ^^ is defined as^^^^^^^^ / ^^^^^^^^ where ^^^^^^^ constant, describes backbone amides in D-exchange-incompetentclosed state, with amide hydrogens hydrogen-bonded in secondary structures, and the ^^^^^^constant, describes backbone amides in a D-exchange-competent open state, with compromised hydrogen bonds.

[0132] In some embodiments, the output of the integrative modeling approach is a molecular ensemble of the polypeptide-molecule complex describing the interaction between the polypeptide and the binding molecule.

[0133] In some embodiments, data generated from molecular dynamics simulations is used as an input for machine learning to iterate among allowed and rare structural conformations to generate a more robust and fulsome predicted polypeptide structure. Such data can include residue-specific biophysical properties relevant to a single residue within the molecular dynamics simulation, as well as pairwise properties that relate to a set of biophysical properties relating to interactions between at least two residues within the molecular dynamics simulation. Examples of residue specific biophysical properties generated using molecular dynamics simulations include grand average of hydropathy (GRAVY) scores, a residue identity or label, coulombic energies, Van Der Waals energies, solvent accessible surface area (SASA), side chain order parameter (S2) and the like. Examples of pairwise biophysical properties generated using molecular dynamics simulations include distance between given residues, Coulombic energies, Van Der Waals energies, a fraction of native contacts (Q) and the like.

[0134] Such properties generated from molecular dynamics can be generated from a given conformation as a function of time. Accordingly, a data set of biophysical properties as a function of time from a set of polypeptide structures can be generated from the molecular dynamics simulations and used as input for machine learning algorithms. This data is arranged into a graph format prior to embedding. Each protein sequence of length is mapped into undirected graph functions. FIG.3 illustrates mapping of the individual graph functions as a function of time. Such graph functions can include: ● continuous-time dynamic graph where represents the set of nodes, represents temporal edges between vertices in and is a Attorney Docket No.199589-705601 / PCT function that maps each edge to a corresponding timestamp. Each edge is assigned to a unique time where represents a couple of residues. This approach takes into account a set of time frames in the molecular dynamics where, each time frame has time . ● discrete-time dynamic graph as a sequence of graphs from timestamps to , where each timestamp represents a time frame in the molecular dynamics simulation. Each graph at time is represented by where and are the nodes and edges active between the timespan of the graph ● static graph where represents nodes and represents the set of edges.

[0135] Each node graph, continuous-time dynamic graph, and discrete time dynamic graph represents a residue while each edge represents the related pairwise residue-residue interaction, obtained from the compression of the information (e.g., arithmetic average) related to each time frame in the molecular dynamics simulation into a single time frame. In each graph function, , i.e. the number of nodes is equal to the sequence length which can be different for protein. Data generated from the dynamic graph representation is then encoded to be used as input for machine learning algorithms described herein. In some embodiments, a function that maps each vertex in either the continuous-time dynamic graph discrete-time dynamic graph into a -dimensional vector is generated, where is the embedding dimension.

[0136] A function can be a conditional log-probability of certain sets of temporal random walks. These are random walks that preserve the time order or the temporal edges, i.e., along a path of such a walk, the timestamp of the consecutive edges are non-decreasing. Additionally, such a function can be represented as a dynamic Skip-gram model trained on evolving random walks where, pre-trained Skip-gram model is used as initial weight for the next Skip-gram model . Indeed, other such algorithms can be employed with the methods described herein.

[0137] After generation of graph representations as described herein, data is embedded for input into machine learning algorithms as described herein. In some instances, manifold learning techniques, (e.g., t-distributed stochastic neighbor embedding (t-SNE)) can be used. Embedding as described herein can include dynamic residue embedding and static protein embedding.

[0138] In dynamic residue embedding, each protein is mapped / embedded into a dense rank tensor , where is the residue index and is the embedding index, is the dynamic embedding dimension. Hence each residue is embedded into a Attorney Docket No.199589-705601 / PCT dense vector for and a separate dynamic residue embedding is trained for each continuous-time or continuous-time dynamic graph, representing an element of , the set Stacking is derived from each protein sequence in to generate where is the protein index. is the dynamic residue embedding vector where .

[0139] In static protein is mapped / embedded into a dense rank tensor . where is the static embedding dimension, where the data output from dynamic graph is used as an input. Hence each protein is embedded into a dense vector and a protein embedding is trained taking into account every static an element of . Hausdorff distance , as well as other types of distances such as Frobenius norm that involve can be used as graph proximity metric . In some cases, residue embedding tensors and can be two non-empty subsets of the metric space where embedding vectors and is the euclidean distance. Stacking coming from each protein sequence in is then generated to calculate . Machine Learning

[0140] Provided herein are methods wherein tensor representations and generated from the dynamic and static embedding, respectively, are used as input for machine learning to iteratively generate low energy predicted polypeptide structures. Such machine learning framework can be used to shorten an effective simulation time, execute prediction tasks, and perform design related tasks, such that a more robust and fulsome polypeptide structure can be generated from the limited data obtained from the molecular dynamics simulations. The tensor representations and generated from the dynamic and static embedding, respectively, allow for accurate prediction of structure beyond the current computational capabilities of molecular dynamics simulations.

[0141] In some embodiments, polypeptide structures can be generated using unstructured computation, artificial intelligence or deep learning. In some cases, unstructured computation can be employed such that calculations can be performed iteratively. Further, polypeptide structure calculation can rely on artificial intelligence or deep learning. For example, a method described herein such as random forest can employ deep learning to generate Gini impurity scores that can be used to parse out probes with improved predictive value. Attorney Docket No.199589-705601 / PCT

[0142] In some embodiments, methods of structural prediction as described herein can employ machine learning and computational intelligence techniques, such as deep neural networks, and combinations of supervised, semi-supervised and unsupervised learning techniques. In some embodiments, methods of structural prediction as described herein employ a supervised algorithm (by way of non-limiting example, linear region, random forest classification, decision tree learning, ensemble learning, bootstrap aggregating, and the like). In some embodiments, methods of structural prediction as described herein employ a non-supervised algorithm (by way of non- limiting example, clustering or association).

[0143] In some embodiments, the methods of structural prediction as described herein may be configured to utilize one or more exemplary AI / machine learning techniques chosen from, but not limited to, decision trees, boosting, support-vector machines, neural networks, nearest neighbor algorithms Naive Bayes, bagging, random forests, and the like. In some embodiments and, optionally, in combination of any embodiment described above or below, an exemplary neutral network technique may be one of, without limitation, feedforward neural network, radial basis function network, recurrent neural network, convolutional network (e.g., U-net) or other suitable network. In some embodiments and, optionally, in combination of any embodiment described above or below, an exemplary implementation of Neural Network may be executed as follows: a. define Neural Network architecture / model, b. transfer the input data to the exemplary neural network model, c. train the exemplary model incrementally, d. determine the accuracy for a specific number of timesteps, e. apply the exemplary trained model to process the newly-received input data, f. optionally and in parallel, continue to train the exemplary trained model with a predetermined periodicity.

[0144] In some embodiments and, optionally, in combination of any embodiment described above or below, the exemplary trained neural network model may specify a neural network by at least a neural network topology, a series of activation functions, and connection weights. For example, the topology of a neural network may include a configuration of nodes of the neural network and connections between such nodes. In some embodiments and, optionally, in combination of any embodiment described above or below, the exemplary trained neural network model may also be specified to include other parameters, including but not limited to, bias values / functions and / or aggregation functions. For example, an activation function of a node may be a step function, sine function, continuous or piecewise linear function, sigmoid function, hyperbolic tangent function, Attorney Docket No.199589-705601 / PCT or other type of mathematical function that represents a threshold at which the node is activated. In some embodiments and, optionally, in combination of any embodiment described above or below, the exemplary aggregation function may be a mathematical function that combines (e.g., sum, product, etc.) input signals to the node. In some embodiments and, optionally, in combination of any embodiment described above or below, an output of the exemplary aggregation function may be used as input to the exemplary activation function. In some embodiments and, optionally, in combination of any embodiment described above or below, the bias may be a constant value or function that may be used by the aggregation function and / or the activation function to make the node more or less likely to be activated.

[0145] In some embodiments, the machine learning model for structural prediction processes the biophysical properties encoded in the embeddings described above by applying the parameters of the machine learning model to produce a model output. In some embodiments, the model output may be decoded to generate one or more numerical output values and / or vectors indicative of polypeptide structure.

[0146] In some embodiments, the parameters of the machine learning model may be trained based on known polypeptide structures. For example, the biophysical properties may be paired with a target structure and / or measurement to form a training pair, such as historical biophysical properties and an observed structure representing a data point in the relationship between the historical biophysical properties and structure. In some embodiments, the biophysical properties may be provided to the machine learning model (e.g., encoded in the embeddings) to produce data representative of polypeptide structure. In some embodiments, an optimization problem associated with the machine learning model may then compare the polypeptide structure with the known output of a training pair including the historical biophysical properties to determine an error of the polypeptide structure. In some embodiments, the optimization problem may employ a loss function, (such as, e.g., Hinge Loss, Multi-class SVM Loss, Cross Entropy Loss, Negative Log Likelihood, or other suitable classification loss function) to determine the error of the polypeptide structure based on the known structure.

[0147] In some embodiments, the known output may be obtained after the machine learning model produces the prediction, such as in online learning scenarios. In such a scenario, the machine learning model may receive the biophysical properties and generate the model output vector to produce the data representative of polypeptide structure. Subsequently, a user may provide feedback (by, e.g., modifying, adjusting, removing, and / or verifying the predicted structure) via a suitable feedback mechanism, such as a user interface device (e.g., keyboard, mouse, touch screen, Attorney Docket No.199589-705601 / PCT user interface, or other interface mechanism of a user device or any suitable combination thereof). The feedback may be paired with the biophysical properties to form the training pair and the optimization problem may determine an error of the polypeptide structure using the feedback.

[0148] In some embodiments, based on the error, the optimization problem may update the parameters of the machine learning model using a suitable training algorithm (such as, e.g., backpropagation) for a prediction machine learning model. In some embodiments, backpropagation may include any suitable minimization algorithm such as a gradient method of the loss function with respect to the weights of the prediction machine learning model. Examples of suitable gradient methods include for example, stochastic gradient descent, batch gradient descent, mini-batch gradient descent, or other suitable gradient descent technique. As a result, the optimization problem may update the parameters of the machine learning model based on the error of predicted structure in order to train the machine learning model to model the correlation between biophysical properties and polypeptide structure in order to produce more accurate prediction of structure based on biophysical properties. EXAMPLES

[0149] For a better understanding of the present disclosure and of its many advantages, the following examples are given by way of illustration and without limiting the scope of this disclosure.

[0150] An HDX-MS experiment involves several steps: (i) an exchange reaction comprising initiating and halting a deuterium exchange process at specific time points; (ii) an LC-MS analysis comprising injecting a quenched sample into a liquid chromatography-mass spectrometry system for peptide separation and mass analysis; and (iii) data analysis comprising processing the mass spectrometry data to determine the relative amount of deuterium incorporation in each peptide Example 1: Interrogating androgen receptor binding sites and disordered region behavior using ultrafast HDX-MS

[0151] A method for detecting a binding interaction between an N-terminal domain of an androgen receptor polypeptide and a binding molecule can be performed using a traditional HDX-MS automation or ultrafast HDX-MS (FIG. 1). The ultrafast HDX-MS method of the present application applies mass spectrometry using an applied photophysics device capable of detecting hydrogen-deuterium exchange rate of peptide fragments from 50 msec to 5 min (FIG.2).

[0152] The fast HDX-MS method of the present application allows for characterization of the intrinsically disordered domain of the N-terminal domain (NTD) of the androgen receptor peptide Attorney Docket No.199589-705601 / PCT (FIG.3), providing the first characterization of the full length NTD, using 141 peptides as shown in FIG.4.

[0153] As shown in Figures 5A-5D, the novel fast HDX-MS method of the present application (FIG.5B) is capable of generating deuterium uptake curves in exchange times below 10 seconds (Figures 5C-5F), compared to traditional HDX-MS methods (FIG. 5A). The ultrafast HDX-MS method provides information on both more structured regions as well as less structured regions of the NTD, based on the percent of deuterium uptake (Figures 6A-6B).

[0154] Fast HDX-MS experiments were carried out using the compound, Masofaniten (Oral EPI- 7386), a known androgen receptor inhibitor (Figures 7A-7C). While masofaniten is known to bind to the NTD, there is limited structural information on the binding site. Using the methods of the present application, performed in triplicate with three time points (10 sec, 1 min, and 10 min), 97% D2O, and a P:L ratio = 1:100 (mol:mol) at 5 °C, the main binding region of masofaniten was observed at 1:100 mol:mol ratio around residues 10-36, as shown in FIG.7C.

[0155] The experimentally obtained dynamics of the androgen receptor polypeptide NTD were shown to be in qualitative agreement with those obtained by a computational tool, ADOPT (FIG. 8).

[0156] A model of the binding site for masofaniten was generated using molecular dynamics, by comparing the hydrogen-deuterium exchange rates obtained from the fast HDX-MS to a calculated hydrogen-deuterium exchange rate of the androgen receptor polypeptide docking conformer produced using molecular dynamics simulation methods as described herein (Figures 9A-9B). As shown in Figures 10A-10D, the technology of the present application yields high-quality structural protein models, thereby facilitating medicinal chemistry and biologics discovery, similar to X-ray and CryoEM for folded proteins.

[0157] The processes described above allow for identification of previously unascertainable binding sites using, as depicted in Figures 11A-11D, where novel ultrafast HDX-MS allows for automated ensemble generation, followed by automated selection using generative artificial intelligence forward models and binding site validation through automated ligand titration, to provide novel chemistry suggestions based on experimentally validated 3D target models. Example 2: Interrogating alpha-synuclein binding sites and disordered region behavior using ultrafast HDX-MS

[0158] The fast HDX-MS process described above allows for interrogation of binding interactions of an alpha-synuclein polypeptide, as depicted in Figures 12A-12C. FIG. 12A shows percent deuterium uptake of alpha synuclein polypeptides at 10 s, 1 m, and 10 m using a traditional HDX- Attorney Docket No.199589-705601 / PCT MS method, while FIG.12B shows percent deuterium uptake of alpha synuclein polypeptides at 50, 80, 150, 200, 500, 1000, 5000, and 30000 ms using a fast HDX-MS method. FIG.12C shows percent deuterium uptake of alpha-synuclein and fasudil at 50, 80, 150, 200, 500, 1000, 5000, and 30000 ms using a fast HDX-MS method. Example 3: Interrogating p27 binding sites and disordered region behavior using ultrafast HDX-MS

[0159] The fast HDX-MS process described above allows for interrogation of binding interactions of a p27 polypeptide, as depicted in Figures 13A-13B. FIG.13A shows percent deuterium uptake of p27 polypeptides at 10 s, 1 m and 10 m using a traditional HDX-MS method, while FIG.13B shows percent deuterium uptake of p27 polypeptides at 50, 80, 150, 200, 500, 1000, 5000, and 30 000 ms using a fast HDX-MS method. Example 4: Interrogating Tau binding sites and disordered region behavior using ultrafast HDX-MS

[0160] The fast HDX-MS process described above allows for interrogation of binding interactions of a Tau polypeptide, as depicted in Figures 14A-14B. FIG.14A shows percent deuterium uptake of Tau polypeptides at 10 s, 1 m, and 10 m using a traditional HDX-MS method, while FIG.14B shows percent deuterium uptake of Tau polypeptides at 50, 80, 150, 200, 500, 1000, 5000, and 30 000 ms using a fast HDX-MS method. Example 5: Interrogating amyloid beta 42 binding sites and disordered region behavior using ultrafast HDX-MS

[0161] The fast HDX-MS process described above allows for interrogation of binding interactions of an amyloid beta 42 polypeptide, as depicted in Figures 15A–15D. FIG. 15A shows percent deuterium uptake of amyloid beta polypeptides at 10 s, 1 m, and 10 m using a traditional HDX- MS method, while. FIG.15B shows percent deuterium uptake of amyloid beta polypeptides at 50, 80, 150, 200, 500, 1000, 5000, and 30000 ms using a fast HDX-MS method. FIG. 15C shows percent deuterium uptake of amyloid beta polypeptides and 10074-GS (a c-myc inhibitor) at 10 s, 1 m, and 10 m using a traditional HDX-MS method, compared to FIG.15D which shows percent deuterium uptake of amyloid beta polypeptides and 10074-GS at 50, 80, 150, 200, 500, 1000, 5000, and 30000 ms using a fast HDX-MS method. Example 6: Interrogating c-myc binding sites and disordered region behavior using ultrafast HDX-MS Attorney Docket No.199589-705601 / PCT

[0162] A protein sample of c-myc (1 mg / ml, 2 microliters) in an equilibration buffer (20 mM sodium phosphate buffer, pH 7.4, 150 mM NaCl, 1 mM TCEP, H2O) is diluted 18-fold with 36 ml of a labeling buffer (20 mM sodium phosphate buffer, pH 7.4, 150 mM NaCl, 1 mM TCEP, in D2O) to initiate the HDX reaction. At specific labeling time points the reaction is stopped by addition of 38 microliters (1:2 dilution) of ice / cold quenching buffer (2 M guanidine chloride, pH 2.0, 0.8% formic acid in H2O). The quenched sample is then injected into a 3-valve cooling chamber system for HDX-MS analysis. Sample labeling, quenching and injection can be performed either manually or automatically using either a liquid handling robot or the Fast HDX prototype.

[0163] Due to the high flexibility of c-myc, the labeling reaction was performed at 5 degrees C for relatively short time points: traditional HDX from 10 seconds to 10 minutes and fast HDX from 50 milliseconds to 5 minutes. Compounds were incubated at a concentration of 500 micromolar (with 5% DMSO final) while MAS was added to the c-myc solution to achieve an equimolar concentration. Once in the cooling chamber (3-valve system), the sample undergoes the following steps;

[0164] It is delivered to a digestion column, where the protein is digested into peptides. The digested sample is sent to a trap that retains the peptides while washing away hydrophilic components. The trapped peptides are directed to an analytical column for chromatographic separation. The separated peptides are then sent to the mass spectrometer (MS) for mass analysis.

[0165] The HDX system’s cooling chamber was maintained at 0.0 ±0.1 degree C throughout the measurements. Peptides were trapped and desalted on a VanGuard Pre-column trap (2.0 mm × 5 mm acquity UPLC BEH C18, 1.7 micrometer) for 3 minutes at a flow rate of 100 microliters / minute. Elution of peptides from the trap was achieved using a 5-35% acetonitrile gradient with 0.1% formic acid over 6 minutes at a flow rate of 100 microliters / minute, followed by separation on an acquity UPLC HSS T3 column (1.8 micrometers, 1.0 mm × 50 mm).

[0166] Mass spectra were acquired using a cyclic IMS mass spectrometer. Spectra were collected between 50 and 2000 m / z with a capillary voltage of 2.8 kV, a sample cone voltage of 40 V, and a transfer CE ramping from 30 to 50 V. One-pass cIM was performed. The error in determining the average deuterium incorporation for each peptide was at or below ±0.5 Da, based on deuterated peptide standards.

[0167] Peptides were identified from replicate HDMSE analyses of undeuterated control samples using PLGS 3.0.3. The identified peptides were then filtered in DynamX 3.0 with a minimum products per amino acid cut-off of 0.25 and at least two consecutive product ions. Peptides meeting Attorney Docket No.199589-705601 / PCT these criteria were further processed by DynamX 3.0. The software determined the relative amount of deuterium in each peptide by subtracting the centroid mass of the undeuterated peptide from that of the deuterated peptide at each time point. Deuterium levels were not corrected for back exchange and are reported as relative values.

[0168] By studying full-length c-myc, a more complete picture of intra-chain interactions and stabilizations is obtained, allowing detailed characterization of its conformational states and dynamics.

[0169] In additional, when full length c-myc is incubated in the presence of small molecule binders and binding partners, new binding sites were observed, potentially exploitable for drugging purposes.

[0170] Applicants have showed the conformational state and flexibility of c-myc and the effects of binding of compounds, such as 10058-F4, when incubated in the presence of c-myc alone or in complex with its obligate partner MAX.

[0171] As shown in FIG.16, sequence coverage for c-myc was 91.2%. FIG.17 shows percentage deuterium uptake profiles for c-myc obtained under traditional and fast HDX-MS conditions, where in the traditional experiment, labeling time points were at 10 seconds, 60 seconds, and 600 seconds, whereas in the fast experiment, time points were at 50 milliseconds, 80 milliseconds, 150 milliseconds, 200 milliseconds, 500 milliseconds, 1000 milliseconds, and 300000 milliseconds. The data were normalized to the last labeling time point (10 minutes for traditional and 5 minutes for fast), as saturation of uptake was reached at these time points. Peptides shown in FIG.17 can vary from those shown in FIG.16.

[0172] FIG.18 shows three sites identified in the binding of 10058-F4 to c-myc, as shown below: SEQ ID NO: 3: 128-IIIQDCMWSGFSAAAKLVSEKLASYQAAR-156 SEQ ID NO: 4: 326-KLDSVRVLRQISNNRKCTSPRSSDTEEN-353 SEQ ID NO: 5: 362- LERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKL ISEEDLLRKRREQLKHKLEQLRNSCA-439

[0173] To Applicant’s knowledge, Site A and Site B are newly identified binding sites, whereas Site C was known previously identified.

[0174] FIG. 19 shows c-myc structure and binding sites identified. A secondary structure of c- myc, as predicted by Alphafold 2 and disorder propensity predicted by ADOPT, where consensus myc box I (MBI), myc box II (MBII), myc box III (MBIII), and helix-loop-helix (HLH) consensus domains are indicated, as well as binding sites A, B and C of 10058-F4 as identified by the present Attorney Docket No.199589-705601 / PCT HDX-MS experiments described herein. FIG. 20 shows the effect of 10058-F4 to c-myc / MAX complex and FIG.21 shows screening of binding compounds, such as 10058-F4, EGCG, 10074- G5, MYCi361, MYCi975, EN4, and KJ-Pyr9, on intrinsically disordered proteins using HDX-MS described herein.

[0175] Without wishing to be bound by theory, Applicants believe the fast HDX-MS experiments described herein has several advantages over traditional structural investigation techniques. For instance, HDX-MS experiments of the present disclosure show no theoretical limitations on the size of the protein to be studied, e.g., it allows for study of the full length c-myc protein; it is compatible for studying intrinsically disordered proteins, and it allows for the detection of transient conformational states in flexible proteins such as c-myc.

[0176] While exemplary embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art. It should be understood that various alternatives to the embodiments described herein may be employed. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

Attorney Docket No.199589-705601 / PCT CLAIMS What is claimed is:

1. A method of interrogating a binding interaction between a disordered domain of a c-myc polypeptide and a binding molecule, the method comprising: (a) determining by mass spectrometry a hydrogen-deuterium exchange rate of the disordered domain of the c-myc polypeptide as a function of time in the absence of a binding molecule by: (i) labeling the c-myc polypeptide with a D2O buffer in a labeling reaction, (ii) quenching the labeling reaction after a reaction time period, (iii) generating peptide fragments of the c-myc polypeptide, and (iv) measuring incorporation of deuterium into the peptide fragments as a function of the reaction time period; wherein the reaction time period is from 0.5 milliseconds to 300 milliseconds (b) determining by mass spectrometry a hydrogen-deuterium exchange rate of the disordered domain of the c-myc polypeptide as a function of time in the presence of the binding molecule by contacting the c-myc polypeptide with the binding molecule and repeating (i)-(iv); (c) surveying a binding interaction between the disordered domain of the c-myc polypeptide and the binding molecule by: (i) comparing the hydrogen-deuterium exchange rate in the peptide fragments in the presence and absence of the binding molecule, and (ii) identifying peptide fragments in which the hydrogen-deuterium exchange rate decreased in the presence of the binding molecule, relative to the hydrogen-deuterium exchange rate of the peptide fragments in the absence of the binding molecule, whereby a difference in the hydrogen-deuterium exchange rate of the peptide fragments in presence and absence of the binding molecule indicates (i) an increase in structural order in the disordered domain of the c-myc polypeptide resulting from binding of the binding molecule to the disordered domain of the c-myc polypeptide; or (ii) a protection of the amide backbone of the polypeptide by the binding molecule, in absence of a structural change in the c-myc polypeptide, thereby interrogating the binding interaction between the disordered domain of the c-myc polypeptide and the binding molecule.Attorney Docket No.199589-705601 / PCT 2. The method of claim 1, wherein a plurality of binding molecules are independently contacted with the c-myc polypeptide, and wherein the method further comprises selecting the binding molecules that produce significant changes in the hydrogen-deuterium exchange rate of peptide fragments of the c-myc polypeptide in the presence of the binding molecule relative to the absence of the binding molecule.

3. The method of claim 1 or 2, wherein the peptide fragments comprise 20 to 90 amino acids residues.

4. The method of any one of claims 1-3, wherein the c-myc polypeptide is the c-myc polypeptide deposited under Uniprot accession number P01106 or P01106-1.

5. The method of any one of claims 1-4, wherein the c-myc polypeptide comprises one or more mutations or slice variants.

6. The method of any one of claims 1-5, wherein the binding molecule has a binding affinity to the disordered domain of the c-myc polypeptide of less than 10 µM.

7. The method of any one of claims 1-6, wherein the disordered domain of the c-myc polypeptide comprises an N-terminal domain.

8. The method of any one of claims 1-7, wherein the binding molecule binds to an epitope in the disordered domain of the c-myc polypeptide capable of inhibiting an activity of the c-myc polypeptide.

9. The method of any one of claims 1-8, wherein the binding molecule binds to an epitope in the disordered domain of the c-myc polypeptide that has a polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

10. The method of any one or claims 1-8, wherein the binding molecule binds to an epitope comprising a polypeptide sequence of SEQ ID NO:

3.

11. The method of any one or claims 1-8, wherein the binding molecule binds to an epitope comprising a polypeptide sequence of SEQ ID NO:

4.

12. The method of any one or claims 1-8, wherein the binding molecule binds to an epitope comprising a polypeptide sequence of SEQ ID NO: 5.Attorney Docket No.199589-705601 / PCT 13. The method of any one of claims 1-8, wherein the binding molecule is a peptide or a peptide mimic.

14. The method of any one of claims 1-8, wherein the binding molecule is a small molecule having a molecular weight of less than 40 kDa.

15. The method of any one of claims 1-14, further comprising generating a model of a binding site of the disordered domain of the c-myc polypeptide to the binding molecule using at least one model trained to predict a polypeptide structure, wherein the at least one model: (a) compares the hydrogen-deuterium exchange rate for the binding site determined by mass spectrometry to a calculated hydrogen-deuterium exchange rate of c-myc polypeptide docking conformers produced in modeling tools; and (b) identifies a c-myc polypeptide docking conformer that has a calculated hydrogen- deuterium exchange rate that is closest to the hydrogen-deuterium exchange rate determined by mass spectrometry, thereby generating the model of the binding site of the disordered domain of the c-myc polypeptide to the binding molecule.

16. The method of claim 15, wherein the modeling tools comprise molecular dynamics (MD) simulations and / or machine-learning models.

17. The method of any one of claims 1-16, further comprising selecting the binding molecule as a treatment for a disease or condition.

18. The method of claim 17, wherein the disease or condition is a cancer.

19. The method of claim 18, wherein the cancer is a lymphoma.

20. The method of any one of claims 1-19, further comprising administering a therapeutically effective amount of the binding molecule to a subject in need thereof.

21. A pharmaceutical composition comprising a molecule that binds to the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, wherein the molecule binds to the polypeptide sequence with a binding affinity of less than 10 μM, and a pharmaceutically-acceptable excipient, diluent, or carrier.Attorney Docket No.199589-705601 / PCT 22. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 21.

23. A pharmaceutical composition for use in a method of treating cancer in a subject, comprising a molecule that binds to the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, wherein the molecule binds to the polypeptide sequence with a binding affinity of less than 10 μM, and a pharmaceutically-acceptable excipient, diluent, or carrier.

24. The pharmaceutical composition for use of claim 23, wherein the molecule is a peptide or peptide mimic.

25. The pharmaceutical composition for use of claim 23, wherein the molecule is a small molecule having a molecular weight of less than 40 kDa.

26. A method of interrogating a binding interaction between a disordered domain of an androgen receptor polypeptide and a binding molecule, the method comprising: (a) determining by mass spectrometry a hydrogen-deuterium exchange rate of the disordered domain of the androgen receptor polypeptide as a function of time in the absence of a binding molecule by: (i) labeling the androgen receptor polypeptide with a D2O buffer in a labeling reaction, (ii) quenching the labeling reaction after a reaction time period, (iii) generating peptide fragments of the androgen receptor polypeptide, and (iv) measuring incorporation of deuterium into the peptide fragments as a function of the reaction time period, wherein the reaction time period is from 0.5 milliseconds to 300 milliseconds; (b) determining by mass spectrometry a hydrogen-deuterium exchange rate of the disordered domain of the androgen receptor polypeptide as a function of time in the presence of the binding molecule by contacting the androgen receptor polypeptide with the binding molecule and repeating (i)-(iv); (c) surveying a binding interaction between the disordered domain of the androgen receptor polypeptide and the binding molecule by: (i) comparing the hydrogen-deuterium exchange rate in the peptide fragments in the presence and absence of the binding molecule, andAttorney Docket No.199589-705601 / PCT (ii) identifying peptide fragments in which the hydrogen-deuterium exchange rate decreased in the presence of the binding molecule, relative to the hydrogen-deuterium exchange rate of the peptide fragments in the absence of the binding molecule, whereby a difference in the hydrogen-deuterium exchange rate of the peptide fragments in the presence and absence of the binding molecule indicates (i) an increase in structural order in the disordered domain of the androgen receptor polypeptide resulting from binding of the binding molecule to the disordered domain of the androgen receptor polypeptide; or (ii) a protection of the amide backbone of the polypeptide by the binding molecule, in absence of a structural change in the androgen receptor polypeptide; thereby interrogating the binding interaction between the disordered domain of the androgen receptor polypeptide and the binding molecule.

27. The method of claim 26, wherein a plurality of binding molecules are independently contacted with the androgen receptor polypeptide, and wherein the method further comprises selecting the binding molecules that produce significant changes in the hydrogen-deuterium exchange rate of peptide fragments of the androgen receptor polypeptide in the presence of the binding molecule relative to the absence of the binding molecule.

28. The method of claim 26 or 27, wherein the peptide fragments comprise 20 to 30 amino acids residues.

29. The method of any one of claims 26-28, wherein the androgen receptor polypeptide is the androgen receptor polypeptide deposited under Uniprot accession number P10275.

30. The method of any one of claims 26-28, wherein the androgen receptor polypeptide comprises one or more mutations or slice variants.

31. The method of any one of claims 26-30, wherein the binding molecule has a binding affinity to the disordered domain of the androgen receptor polypeptide of less than 10 µM.

32. The method of any one of claims 26-31, wherein the disordered domain of the androgen receptor polypeptide comprises an N-terminal domain.

33. The method of any one of claims 26-32, wherein the binding molecule binds to an epitope in the disordered domain of the androgen receptor polypeptide capable of inhibiting an activity of the androgen receptor polypeptide.Attorney Docket No.199589-705601 / PCT 34. The method of claim any one of claims 26-33, wherein the binding molecule binds to an epitope in the disordered domain of the androgen receptor polypeptide that has a polypeptide sequence of SEQ ID NO:

6.

35. The method of any one of claims 26-34, wherein the binding molecule is a peptide or a peptide mimic.

36. The method of any one of claims 26-34, wherein the binding molecule is a small molecule having a molecular weight of less than 40 kDa.

37. The method of any one of claims 26-36, further comprising generating a model of a binding site of the disordered domain of the androgen receptor polypeptide to the binding molecule using at least one model trained to predict a polypeptide structure, wherein the at least one model: (a) compares the hydrogen-deuterium exchange rate for the binding site determined by mass spectrometry to a calculated hydrogen-deuterium exchange rate of androgen receptor polypeptide docking conformers produced in modeling tools; and (b) identifies an androgen receptor polypeptide docking conformer that has a calculated hydrogen-deuterium exchange rate that is closest to the hydrogen-deuterium exchange rate determined by mass spectrometry, thereby generating the model of the binding site of the disordered domain of the androgen receptor polypeptide to the binding molecule.

38. The method of claim 37, wherein the modeling tools comprise molecular dynamics (MD) simulations and / or machine-learning models.

39. The method of any one of claims 26-38, further comprising selecting the binding molecule as a treatment for a disease or condition.

40. The method of claim 39, wherein the disease or condition is a cancer.

41. The method of claim 40, wherein the cancer is androgen-dependent prostate cancer.

42. The method of any one of claims 26-41, further comprising administering a therapeutically effective amount of the binding molecule to a subject in need thereof.

43. A pharmaceutical composition that comprises a molecule that binds to the polypeptide sequence of SEQ ID NO: 6 of an androgen receptor polypeptide, wherein the molecule binds toAttorney Docket No.199589-705601 / PCT the polypeptide sequence with a binding affinity of less than 10 μM, and a pharmaceutically- acceptable excipient, diluent, or carrier.

44. A method of treating an androgen-dependent prostate cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 43.

45. The method of claim 44, wherein the androgen-dependent prostate cancer is a metastatic castration-resistant cancer.

46. A pharmaceutical composition for use in a method of treating an androgen-dependent prostate cancer in a subject, comprising a molecule that binds to the polypeptide sequence of SEQ ID NO: 6 of an androgen receptor polypeptide, wherein the molecule binds to the polypeptide sequence with a binding affinity of less than 10 µM, and a pharmaceutically-acceptable excipient, diluent, or carrier.

47. The pharmaceutical composition for use of claim 46, wherein the molecule is a peptide or peptide mimic.

48. The pharmaceutical composition for use of claim 46, wherein the molecule is a small molecule having a molecular weight of less than 40 kDa.

49. A method of treating a prostate cancer in a subject in need thereof, the method comprising administering to the subject: a therapeutically effective amount of an androgen receptor N-terminal domain binding molecule, wherein the androgen receptor N-terminal domain binding molecule provides for an increase in structural order of an androgen receptor upon contact, and wherein the increase in structural order is measured by comparing by mass spectrometry a hydrogen-deuterium exchange rate of the androgen receptor as a function of time in the presence and absence of the androgen receptor N-terminal domain binding molecule; and an additional therapeutic agent.

50. The method of claim 49, wherein the additional therapeutic agent comprise an androgen receptor ligand-binding domain inhibitor or an inhibitor of CYP17.Attorney Docket No.199589-705601 / PCT 51. The method of claim 49 or 50, wherein the additional therapeutic agent comprises enzalutamide, apalutamide, darolutamide, bicalutamide, nilutamide, flutamide, ODM-204, TAS3681, galeterone, abiraterone, abiraterone acetate, or any combination thereof.

52. The method of claim 51, wherein the additional therapeutic agent comprises enzalutamide.

53. The method of claim 51, wherein the additional therapeutic agent comprises apalutamide.

54. The method of claim 51, wherein the additional therapeutic agent comprises darolutamide.

55. The method of claim 51, wherein the additional therapeutic agent comprises abiraterone.

56. The method of claim 51, wherein the additional therapeutic agent comprises abiraterone acetate.

57. The method of claim 51, wherein the additional therapeutic agent comprises bicalutamide.

58. A method of interrogating a binding interaction between a disordered domain of a polypeptide and a binding molecule, the method comprising: (a) determining by mass spectrometry a hydrogen-deuterium exchange rate of peptide fragments of the polypeptide as a function of time in the absence of a binding molecule by: (i) labeling the peptide fragments with a D2O buffer in a labeling reaction, (ii) quenching the labeling reaction after a reaction time period of from 0.05 seconds to 10 seconds, (iii) generating peptide fragments of the polypeptide, and (iv) measuring incorporation of deuterium into the peptide fragments as a function of the reaction time period, wherein the reaction time period is from 0.5 milliseconds to 300 milliseconds; (b) determining by mass spectrometry a hydrogen-deuterium exchange rate of the peptide fragments of the polypeptide as a function of time in the presence of the binding molecule by contacting the polypeptide with the binding molecule and repeating (i)-(iv); (c) surveying a binding interaction between the polypeptide and the binding molecule by: (i) comparing the hydrogen-deuterium exchange rate in the peptide fragments in the presence and absence of the binding molecule, andAttorney Docket No.199589-705601 / PCT (ii) identifying peptide fragments in which the hydrogen-deuterium exchange rate changed in the presence of the binding molecule, relative to the hydrogen-deuterium exchange rate of the peptide fragments in the absence of the binding molecule, whereby a difference in the hydrogen-deuterium exchange rate of the peptide fragments in the presence and absence of the binding molecules indicates an increase or a decrease in structural order in a region of the polypeptide resulting from binding of the binding molecule to the region of the polypeptide, thereby interrogating the binding interaction between the polypeptide and the binding molecule.

59. The method of claim 58, wherein a plurality of binding molecules are independently contacted with the polypeptide.

60. A method of generating a model of a binding site of a polypeptide to a molecule, the method comprising: (a) determining by mass spectrometry a hydrogen-deuterium exchange rate of the peptide fragments of the polypeptide as a function of time in the absence of the molecule by: (i) labeling the polypeptide with a D2O buffer in a labeling reaction, (ii) quenching the labeling reaction after a reaction time period, (iii) generating peptide fragments of the polypeptide, and (iv) measuring incorporation of deuterium into the peptide fragments as a function of the reaction time period, wherein the reaction time period is from 0.5 milliseconds to 300 milliseconds; (b) determining by mass spectrometry a hydrogen-deuterium exchange rate of peptide fragments of the polypeptide as a function of time in the presence of the molecule by contacting the polypeptide with the molecule and repeating (i)-(iv); (c) surveying a binding interaction of the polypeptide to the molecule by: (i) comparing the hydrogen-deuterium exchange rate in the peptide fragments in the presence and absence of the molecule, and (ii) identifying peptide fragments in which the hydrogen-deuterium exchange rate decreased in the presence of the molecule, relative to the hydrogen-deuterium exchange rate of the peptide fragments in the absence of the molecule, whereby the decrease in the hydrogen-deuterium exchange rate indicates an increase in structural order in a region of the polypeptide resulting from binding of the molecule to the regionAttorney Docket No.199589-705601 / PCT of the polypeptide, thereby interrogating the binding interaction between the polypeptide and the molecule; and (d) generating the model of the binding site of the polypeptide to the molecule using at least one model trained to predict a polypeptide structure, wherein the at least one model: (i) compares the hydrogen-deuterium exchange rate of the binding site of the polypeptide determined by mass spectrometry to a calculated hydrogen-deuterium exchange rate of polypeptide docking conformers produced in modeling tools, and (ii) identifies a polypeptide docking conformer that has a calculated hydrogen- deuterium exchange rate that is closest to the hydrogen-deuterium exchange rate determined by mass spectrometry, thereby generating the model of the binding site of the polypeptide to the molecule.

61. The method of claim 60, wherein the modeling tools comprise molecular dynamics (MD) simulations and / or machine-learning models.

62. The method of claim 60 or 61, wherein the polypeptide is an androgen receptor polypeptide.

63. The method of claim 60 or 61, wherein the polypeptide is an alpha-synuclein polypeptide.

64. The method of claim 60 or 61, wherein the polypeptide is a p27 polypeptide.

65. The method of claim 60 or 61, wherein the polypeptide is a c-myc polypeptide.

66. The method of claim 60 or 61, wherein the polypeptide is a Tau polypeptide 67. The method of claim 60 or 61, wherein the polypeptide is an Amyloid β (1-42) polypeptide.

68. The method of any one of claims 60-67, wherein the molecule is a peptide or peptide mimic.

69. The method of any one of claims 60-67, wherein the molecule is a small molecule having a molecular weight of less than 40 kDa.

70. A method of altering order in regions of structural disorder of a polypeptide using mass spectrometry, the method comprising:Attorney Docket No.199589-705601 / PCT (a) determining by mass spectrometry a hydrogen-deuterium exchange rate of the polypeptide as a function of time by: (i) labeling the peptide fragments with a D2O buffer in a labeling reaction, (ii) quenching the labeling reaction after a reaction time period of from 0.05 seconds to 10 seconds, (iii) generating peptide fragments of the polypeptide, and (iv) measuring incorporation of deuterium into the peptide fragments as a function of the reaction time period, wherein the reaction time period is from 0.5 milliseconds to 300 milliseconds; (b) identifying regions of structural disorder based on the hydrogen-deuterium exchange rate determined by the mass spectrometry by: (i) selecting peptide fragments that have the fastest rate of hydrogen-deuterium exchange during the reaction time period of from 0.05 seconds to 10 seconds; and (ii) aligning the selected peptide fragments to the polypeptide sequence, thereby identifying regions of structural disorder in the polypeptide.

71. The method of claim 70, further comprising: validating the identified regions of structural disorder of the polypeptide determined by mass spectrometry by comparing the identified regions of structural disorder of the polypeptide to regions of structural disorder determined using modeling tools.

72. The method of claim 71, wherein the modeling tools comprise molecular dynamics (MD) simulations and / or machine-learning models.

73. The method of claim 72, wherein MD simulation data are collected.

74. The method of claim 73, wherein, the MD simulation data comprises an index score, which is computed using a structural prominence parameter and a disorder parameter.

75. The method of claim 74, wherein the structural prominence parameter and the disorder parameter are determined through MD simulations of a homology model that incorporates combined structures of proteins similar to the polypeptide.

76. The method of any one of claims 73-75, wherein the MD simulation data comprises an index score that is calculated based on a structural prominence parameter and a disorder parameter, wherein the disorder parameter and the structural prominence parameter are derived from MDAttorney Docket No.199589-705601 / PCT simulation of a homology model comprising aggregate structures of homologs of the polypeptide; and wherein the index score is proportional to the structural prominence parameter and is inversely proportional to the disorder parameter.

77. The method of any one of claims 73-76, wherein the identified regions of structural disorder of the polypeptide determined by mass spectrometry are validated when the regions have a low index score based on the MD simulation data.

78. The method of any one of claims 70-77, wherein the polypeptide is an androgen receptor polypeptide.

79. The method of any one of claims 70-77, wherein the polypeptide is an alpha-synuclein polypeptide.

80. The method of any one of claims 70-77, wherein the polypeptide is a p27 polypeptide.

81. The method of any one of claims 70-77, wherein the polypeptide is a c-myc polypeptide.

82. The method of any one of claims 70-77, wherein the polypeptide is a Tau polypeptide.

83. The method of any one of claims 70-77, wherein the polypeptide is an Amyloid β (1-42) polypeptide.

84. A method of treating a synucleinopathy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition that comprises a molecule that binds to the polypeptide sequence of SEQ ID NO: 7 of an alpha- synuclein polypeptide, and a pharmaceutically-acceptable excipient, diluent, or carrier.

85. The method of claim 84, wherein the synucleinopathy is Parkinson’s disease.

86. A pharmaceutical composition for use in a method of treating a synucleinopathy in a subject, comprising a molecule that binds to the polypeptide sequence of SEQ ID NO: 7 of an alpha-synuclein polypeptide, and a pharmaceutically-acceptable excipient, diluent, or carrier.

87. A method of treating a tauopathy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition that comprises a molecule that binds to the polypeptide sequence of SEQ ID NO: 8 of a Tau polypeptide, and a pharmaceutically-acceptable excipient, diluent, or carrier.Attorney Docket No.199589-705601 / PCT 88. The method of claim 87, wherein the tauopathy is Alzheimer’s disease.

89. A pharmaceutical composition for use in a method of treating a tauopathy in a subject, comprising a molecule that binds to the polypeptide sequence of SEQ ID NO: 8 of a Tau polypeptide, and a pharmaceutically-acceptable excipient, diluent, or carrier.

90. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition that comprises a molecule that binds to the polypeptide sequence of SEQ ID NO: 9 of a p27 polypeptide, and a pharmaceutically-acceptable excipient, diluent, or carrier.

91. The method of claim 90, wherein the disease or condition is a hydatidiform mole or Beckwith-Wiedemann syndrome.

92. A pharmaceutical composition for use in a method of treating a disease or condition in a subject in need thereof, comprising a molecule that binds to the polypeptide sequence of SEQ ID NO: 9 of a p27 polypeptide, and a pharmaceutically-acceptable excipient, diluent, or carrier.

93. A method of treating Alzheimer’s disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition that comprises molecule that binds to the polypeptide sequence of SEQ ID NO: 10 of an Amyloid β (1-42) polypeptide, and a pharmaceutically-acceptable excipient, diluent, or carrier.

94. A pharmaceutical composition for use in a method of treating Alzheimer’s disease in a subject, comprising a molecule that binds to the polypeptide sequence of SEQ ID NO: 10 of an Amyloid β (1-42) polypeptide, and a pharmaceutically-acceptable excipient, diluent, or carrier.

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