Methods for identifying compounds that inhibit angiogenin in complex with a ribosome

Abstract

Methods of identifying and validating compounds that inhibit angiogenin in complex with a ribosome.

Description

Methods for Identifying Compounds that Inhibit Angiogenin in Complex with a RibosomeCross-Reference to Related Applications

[0001] The present application claims priority from U.S. Provisional Application No. 63 / 520,982 filed August 22, 2023, and U.S. Provisional Application No. 63 / 509,902 filed June 23, 2023, the disclosure of each of which is hereby incorporated by reference herein in its entirety.Government Rights in Invention

[0002] This invention was made with government support under GM127094 awarded by the National Institutes of Health. The government has certain rights in the invention.Technical Field

[0003] The present invention relates to methods of identifying compounds that inhibit angiogenin in complex with a ribosome and, more particularly, to methods of identifying compounds that inhibit activated angiogenin and compounds that inhibit the activation of angiogenin.Background Art

[0004] Angiogenin, an RNase A-family protein, promotes angiogenesis and has been implicated in cancer, neurodegenerative diseases, and epigenetic inheritance. Upon activation during cellular stress, angiogenin cleaves tRNAs at the anticodon loop, producing tRNA “halves” (also known as tiRNAs or tsRNAs), resulting in translation repression. The catalytic activity of isolated angiogenin, however, is very low, and the mechanism of angiogenin’ s activation and tRNA specificity have remained unresolved.

[0005] Understanding the mechanism of angiogenin activation is essential in facilitating the development of therapeutics to treat cancer and neurodegenerative diseasesusing screens that can identify inhibitors of activated angiogenin, as well as inhibitors of angiogenin’ s activation.Summary of the Embodiments

[0006] In accordance with one embodiment, the invention provides a method for identifying whether a compound is an inhibitor of angiogenin in complex with a ribosome, the method comprising: (a) incubating, for a first period of time, a first mixture comprising the angiogenin, the compound, a first ribosome source, and a tRNA source, (b) incubating, for a second period of time, a second mixture formed by admixing, after the first period of time, the incubated first mixture and an mRNA encoding a protein, and (c) detecting whether the mRNA is translated in the second mixture so as to produce the protein, wherein, if the protein is detected, the compound is identified as said inhibitor of angiogenin in complex with the ribosome.

[0007] In some embodiments, prior to the incubating for the first period of time, the method comprises: (i) incubating, for an initial period of time, an initial mixture comprising the compound, the first ribosome source, and the tRNA source, and (ii) admixing, after the initial period of time, the incubated initial mixture and the angiogenin, thereby forming the first mixture.

[0008] The first ribosome source may be selected from the group consisting of rabbit reticulocyte lysate, a eukaryotic cell extract, an archaeal cell extract, a bacterial cell extract, and a reconstituted translation system.

[0009] The tRNA source may be selected from the group consisting of rabbit reticulocyte lysate, a eukaryotic cell extract, a bacterial cell extract, a reconstituted translation system, purified tRNA, synthetic tRNA, fluorophore-labeled tRNA, a human cell S-100 fraction, a non-human mammalian cell S-100 fraction, and a bacterial cell S-100 fraction.

[0010] In some embodiments, the first ribosome source and the tRNA source are the same.

[0011] In some embodiments, the protein is a reporter protein. The reporter protein may be an enzymatic protein. The enzymatic protein may be selected from the groupconsisting of a luciferase, a P-galactosidase, a P -glucuronidase, a horseradish peroxidase, a xylanase, and an alkaline phosphatase.

[0012] In some embodiments, the reporter protein is a fluorescent protein. The fluorescent protein may be selected from the group consisting of a green fluorescent protein, a red fluorescent protein, a yellow fluorescent protein, a cyan fluorescent protein, a blue fluorescent protein, and variants thereof.

[0013] In some embodiments, the first period of time may be 15 seconds-60 minutes. In other embodiments, the first period of time may be 1-30 minutes. In some embodiments, the first period of time may be 5-15 minutes.

[0014] In some embodiments, the initial period of time may be 15 seconds-60 minutes. In other embodiments, the initial period of time may be 1-30 minutes. In some embodiments, the initial period of time may be 5-15 minutes.

[0015] In accordance with one embodiment, the invention provides a method of validating whether an identified compound inhibits angiogenin in complex with a ribosome, the method comprising: (A) obtaining the identified compound, said identified compound being a compound identified as an inhibitor of angiogenin, (B) incubating, for a third period of time, a third mixture comprising the identified compound, the angiogenin, a second ribosome source, and a first tRNA substrate, and (C) comparing: (1) an amount of a first tRNA degradation product produced by cleavage of the first tRNA substrate caused by the angiogenin in the third mixture with (2) an amount of a control tRNA degradation product produced by cleavage of a control tRNA substrate by the angiogenin in a control mixture, the control mixture lacking the identified compound and comprising the angiogenin, the second ribosome source, and the control tRNA substrate, wherein, if the amount of the first tRNA degradation product is less than the amount of the control tRNA degradation product, the identified compound is validated as inhibiting angiogenin in complex with the ribosome.

[0016] In some embodiments, prior to the incubating for the third period of time, the method comprises: (i) admixing the obtained identified compound and the second ribosome source, thereby forming a mixture A; (ii) incubating the mixture A for a period of time A; (iii) admixing, after the period of time A, the mixture A and the angiogenin, thereby forming a mixture B; (iv) incubating the mixture B for a period of time B; (v) admixing, after theperiod of time B, the mixture B and the first tRNA substrate, thereby forming the third mixture.

[0017] In accordance with another embodiment, the invention provides a method for identifying whether a compound is an inhibitor of angiogenin in complex with a ribosome, the method comprising: (A) incubating, for a starting period of time, a starting mixture comprising the compound, the angiogenin, a second ribosome source, and a first tRNA substrate, and (B) comparing: (1) an amount of a first tRNA degradation product produced by cleavage of the first tRNA substrate caused by the angiogenin in the starting mixture with (2) an amount of a control tRNA degradation product produced by cleavage of a control tRNA substrate by the angiogenin in a control mixture, the control mixture lacking the compound and comprising the angiogenin, the second ribosome source, and the control tRNA substrate, wherein, if the amount of the first tRNA degradation product is less than the amount of the control tRNA degradation product, the compound is identified as said inhibitor of angiogenin in complex with the ribosome.

[0018] In some embodiments, prior to the incubating for the starting period of time, the method comprises: (i) admixing the compound and the second ribosome source, thereby forming a mixture A; (ii) incubating the mixture A for a period of time A; (iii) admixing, after the period of time A, the mixture A and the angiogenin, thereby forming a mixture B; (iv) incubating the mixture B for a period of time B; (v) admixing, after the period of time B, the mixture B and the first tRNA substrate, thereby forming the starting mixture.

[0019] In some embodiment, the second ribosome source comprises purified ribosomes. The purified ribosomes may be purified from a cell source selected from the group consisting of a eukaryotic cell, an archaeal cell, and a bacteria cell.

[0020] In some embodiments, the first tRNA substrate may be a set of isoacceptor tRNA. In some embodiments, the control tRNA substrate may be a set of isoacceptor tRNA.

[0021] In some embodiments, the first tRNA substrate may be selected from the group consisting of a deacyl tRNA, an amino-acyl tRNA, and a fluorescent tRNA. In some embodiments, the control tRNA substrate may be selected from the group consisting of a deacyl tRNA, an amino-acyl tRNA, and a fluorescent tRNA.

[0022] The first tRNA substrate and the control tRNA substrate may be the same.

[0023] In some embodiments, the amount of the first tRNA degradation product is at most 80% the amount of the control tRNA degradation product. In other embodiments, the amount of the first tRNA degradation product is at most 50% the amount of the control tRNA degradation product. In some embodiments, the amount of the first tRNA degradation product is at most 10% the amount of the control tRNA degradation product.

[0024] The identified compound may be identified as an inhibitor of angiogenin by a method selected from the group consisting of a screen and in silico modeling. The screen may be a high-throughput screen. In some embodiments, the screen may be the method for identifying whether a compound is an inhibitor of angiogenin in complex with a ribosome.Brief Description of the Drawings

[0025] The foregoing features of embodiments will be more readily understood by reference to the following detailed description, taken with reference to the accompanying drawings, in which:

[0026] Fig. 1 shows a prior art screen for identifying compounds that inhibit angiogenin (top) and a method for validating that the identified compounds inhibit angiogenin (bottom), as disclosed in U.S. Publication No. US 2007 / 0032418.

[0027] Fig. 2A shows an exemplary screen for identifying compounds that inhibit angiogenin in complex with a ribosome, in accordance with an embodiment of the invention. Fig. 2B shows an exemplary ribosome-activated tRNA cleavage assay for validating compounds identified as inhibiting angiogenin in complex with a ribosome, in accordance with an embodiment of the invention. Additionally, the ribosome-activated tRNA cleavage assay of Fig. 2B may be utilized as an initial screen for identifying compounds that inhibit angiogenin in complex with a ribosome, not only as a validation assay.

[0028] Fig. 3A is a gel showing that Benzopurpurin B decreases tRNA cleavage by ribosome activated angiogenin at 20 pM and 200 pM, in accordance with an embodiment of the invention. Fig. 3B is a gel showing that neomycin decreases tRNA cleavage by ribosome activated angiogenin at >20 pM, in accordance with an embodiment of the invention. Fig. 3C is a plot showing that Benzopurpurin B partially rescues angiogenin inhibition of translation in RRL at 5-50 pM, in accordance with an embodiment of the invention. Fig. 3D is a plot showing that neomycin does not rescue angiogenin inhibition of translation in RRL due tothe inhibitory effect of neomycin on translation, in accordance with an embodiment of the invention. Fig. 3E is a gel showing that paromomycin has an effect on tRNA cleavage by ribosome activated angiogenin (at > 500 pM), in accordance with an embodiment of the invention.

[0029] Fig. 4A shows the structure of a ribosome and the binding site of angiogenin immediately next to a binding site for a ribosome-binding drug (neomycin). Figure 4B is a zoomed-in structure of angiogenin’ s binding site on the ribosome depicted in Figure 4 A, and shows the structural mechanism for disruption of angiogenin’ s binding site by neomycin which disrupts angiogenin’ s binding site on the ribosome, resulting in the inhibition of catalytic activity of angiogenin.Detailed Description of Specific Embodiments

[0030] Definitions. As used in this description and the accompanying claims, the following terms shall have the meanings indicated, unless the context otherwise requires:

[0031] A “set” includes at least one member.

[0032] The terms “a” and “an” and “the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0033] Ordinal numbers as used herein, including the claims, are not to be construed as necessarily denoting an order. Ordinal numbers may be used herein as a naming convention to distinguish various aspects of embodiments of the invention. For example, “a second ribosome source” should not be construed as implying that an embodiment necessarily comprises a first ribosome source unless explicitly disclosed as such for that embodiment.

[0034] Angiogenin was discovered as an angiogenesis factor required for vascularization and more recently was found to play roles in other biological processes. Angiogenin is upregulated in cancers due to the dependence of tumorigenesis onneovascularization. By contrast, missense mutations in angiogenin are correlated with the development of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson’s disease, and Alzheimer's disease, highlighting the angiogenin’s key role in neuroprotection.

[0035] “Angiogenin,” as used herein, means an angiogenin enzyme from a vertebrate organism including, but not limited to, a mammalian angiogenin, e.g., human angiogenin, mouse angiogenin, and zebrafish angiogenin. Angiogenin also includes angiogenin variants.

[0036] As used herein the term “variant” means the exhibition of qualities that deviate from what occurs in nature, e.g., a variant angiogenin is an angiogenin comprising one or more changes in amino acid residues as compared to a wild type angiogenin amino acid sequence, and which retains the same or substantially the same functional activity or activities of the wild type angiogenin (a reference sequence). The term “variant” encompasses homologous proteins having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% percent identity with a reference wild-type angiogenin sequence and having the same or substantially the same functional activity or activities as the reference sequence. The term also encompasses mutants, truncations, or domains of a reference sequence, and which display the same or substantially the same functional activity or activities as the reference sequence.

[0037] As used herein, a compound that “inhibits angiogenin in complex with a ribosome” includes (i) compounds that inhibit the RNase activity of activated angiogenin on tRNA, i.e., after angiogenin has been activated by a ribosome and / or (ii) compounds that inhibit activation of angiogenin by the ribosome, e.g., by preventing association of angiogenin with a ribosome.

[0038] Human angiogenin is a 14-kDa RNase-A family ribonuclease whose RNase activity is required for angiogenesis and is characterized by unusual properties. First, despite angiogenin’s structural and sequence homology with highly efficient RNase A-like nucleases, purified angiogenin is at least 10,000-fold less active (kcat / Kw) than RNase A. The large difference between the catalytic activities of angiogenin and RNase A is known to correlate with the conformations of the C-terminal tails in protein structures. The C-terminus of catalytically competent RNase A adopts a P-sheet conformation that forms a substrate binding surface containing the catalytic histidine corresponding to His l 14 in angiogenin(angiogenin numbering is for the 123-amino acid human protein, excluding the 24-amino acid signal peptide). By contrast, the C-terminal tail of free angiogenin (residues 116-123) folds into a short a-helix that partially occludes the substrate binding site, consistent with the weak enzymatic activity of purified angiogenin.

[0039] Second, angiogenin contrasts with RNase A in its high specificity toward cellular tRNAs. Angiogenin cleaves tRNAs at the anticodon stem loop, resulting in 30- to 40-nt-long tRNA fragments, leading to potent inhibition of translation and other downstream effects. The tRNA-cleaving function of angiogenin has also been implicated in transgenerational inheritance, stem cell maintenance, and astroglial support of motor neurons.

[0040] How angiogenin achieves high specificity toward tRNA in cells has been the subject of intense investigation. The catalytic activity of angiogenin substantially increases in cells and cell extracts. Moreover, activation of angiogenin during cellular stress coincides with its colocalization with the translational machinery.

[0041] We have discovered that the RNase activity of angiogenin is potently activated by a mammalian 80S ribosome. Loveland et al. Structural mechanism of angiogenin activation by the ribosome. Nature. 2024 May 8. doi: 10.1038 / s41586-024- 07508-8. Epub ahead of print. PMID: 38718836 (hereinafter “Loveland et al.”), which is hereby incorporated by reference in its entirety. Cryogenic electron microscopy structures reveal the structural rearrangement of 80S ribosome-bound angiogenin into an RNase- A-like conformation, explaining the catalytic activation of angiogenin by the mammalian 80S ribosome. The C-terminal tail of angiogenin is rearranged by interactions with the 80S ribosome to activate the RNase catalytic center, making the enzyme several orders of magnitude more efficient in tRNA cleavage. Moreover, our findings suggest that angiogenin is activated by ribosomes with a vacant A site, whose abundance increases during cellular stresses.

[0042] In addition, we have discovered that ribosomal positioning of the angiogenin active site next to newly delivered tRNAs underlies the angiogenin’ s specificity for the cleavage of tRNA anticodon stem loops. We have thus uncovered that an 80S ribosome activates, and acts as a specificity factor for, angiogenin. See Loveland et al.

[0043] Our discovery of the mechanism of angiogenin’s activation and specificity allows for the screening and identification of compounds that modulate angiogenin’s activity in complex with the ribosome. For example, compounds that inhibit the activity of activated angiogenin, as well as compounds that inhibit activation of angiogenin at the ribosome, may be identified by methods described herein.

[0044] Prior efforts to discover compounds that inhibit angiogenin failed to sufficiently identify compounds that inhibit angiogenin in complex with an 80S ribosome. For example, Fig. 1 (top) shows a high-throughput screening method of the prior art (U.S. Publication No. US 2007 / 0032418) for identifying inhibitors of angiogenin. Briefly, the method involves incubating a compound library with purified angiogenin and a non- physiological fluorogenic RNase substrate, and detecting fluorescence as a readout of angiogenin activity (reduced fluorescence was an indication of inhibition of angiogenin activity). The ribosome was absent in these assays and therefore it was inhibitors of nonactivated angiogenin that were screened. A secondary assay (Fig. 1 (bottom)) is disclosed in the same prior art (U.S. Publication No. US 2007 / 0032418) for validating a hit compound’s ability to inhibit angiogenin-catalyzed mRNA degradation. This secondary assay involves incubating purified angiogenin with a protein-encoding mRNA (the translation of which can be detected at a later stage, for example an mRNA encoding luciferase) and a hit compound identified from the initial high-throughput screen of Fig. 1 (top). After a first period of incubation, rabbit reticulocyte lysate is admixed with the incubated samples and, after a second incubation period, translation of the mRNA is detected. For example, if the mRNA encodes luciferase, luciferin may be added to the reactions after the second incubation period and the presence of translated luciferase may be detected optically if the hit compound inhibits angiogenin’s RNase activity.

[0045] Thus, the screen of the prior art identified and validated inhibitors of the nonactivated form of angiogenin and, furthermore, these inhibitors reduced angiogenin’s RNase activity on mRNA and on non-physiological substrates.

[0046] Unlike the screens of prior art, based on our discovery of angiogenin’s mechanism of activation and tRNA substrate specificity, we have developed a high- throughput screen for identifying compounds that inhibit angiogenin in complex with a ribosome — including, but not limited to, compounds that inhibit the activity of activatedangiogenin on tRNA and compounds that inhibit activation of angiogenin by the ribosome. Moreover, we have developed a method for validating that compounds identified by our screen are indeed inhibitors of angiogenin activation, or inhibitors of angiogenin’ s RNase activity with respect to tRNA, while angiogenin is in complex with a ribosome. The method of validation may also be used as a screen for identifying compounds that inhibit angiogenin in complex with a ribosome.

[0047] Fig. 2A and 2B show a schematic overview of an exemplary screen (suitable for high-throughput screening) of a library of compounds for inhibitors of angiogenin in complex with a ribosome (Fig. 2A), and a ribosome-activated tRNA cleavage assay for validating compound hits obtained from the aforementioned screen (Fig. 2B), in accordance with an embodiment of the invention. Although a tRNA cleavage assay such as the exemplary assay shown in Fig. 2B may be used to validate compounds that have been identified as inhibitors of angiogenin in complex with a ribosome, it is also contemplated as part of the present disclosure that such a tRNA cleavage as may also be used as a screen to identify inhibitors of angiogenin in complex with a ribosome, not only to validate previously identified inhibitors of angiogenin in complex with a ribosome.

[0048] Figure 2A, step 1, shows the creation of a set of initial mixtures, each initial mixture comprising a first ribosome source (rabbit reticulocyte lysate (“RRL”), in this instance), a tRNA source (here, RRL), and a compound (from the library) to be screened.

[0049] The initial mixture is incubated for an initial period of time and is then admixed with angiogenin (Fig. 2A, step 2), thereby forming a first mixture, and incubated for a first period of time. Creating and incubating the initial mixture, prior to admixing with angiogenin, allows time for the compound to preferentially bind to the ribosome if the given compound and angiogenin compete for the same spot on the ribosome. In some embodiments, the initial period of time is 15 seconds-360 minutes, or longer. In some embodiments, the initial period of time is 15 seconds-180 minutes. In some embodiments, the initial period of time is 15 seconds-60 minutes. In other embodiments, the initial period of time is 1-30 minutes. In some embodiments, the initial period of time is 1-10 minutes. In still other embodiments, the initial period of time is 5-15 minutes. In some embodiments, the initial period of time is 15 seconds-5 minutes.

[0050] In other embodiments, the first mixture comprising a first ribosome source, a tRNA source, angiogenin, and a compound (from the library) to be screened may be admixed together in a single step, without creating an initial mixture.

[0051] In either embodiment, we have discovered that incubation of the first mixture for a first period of time is desirable so as to increase the fidelity of our screen in identifying compounds that inhibit angiogenin in complex with a ribosome, in accordance with embodiments of the invention. In some embodiments, the first period of time is 15 seconds- 360 minutes, or longer. In some embodiments, the first period of time is 15 seconds-180 minutes. In some embodiments, the first period of time is 15 seconds-60 minutes. In other embodiments, the first period of time is 1-30 minutes. In some embodiments, the first period of time is 1-10 minutes. In still other embodiments, the first period of time is 5-15 minutes. In some embodiments, the first period of time is 15 seconds-5 minutes.

[0052] Although RRL is used in the exemplary screen of Fig. 2A, the first ribosome source may be a human cell extract, a mammalian cell extract, a eukaryotic cell extract, an archaeal cell extract, a bacterial cell extract, or a reconstituted translation system.

[0053] The tRNA source may be rabbit reticulocyte lysate, a human cell lysate, a mammalian cell extract, a eukaryotic cell extract, a bacterial cell extract, a reconstituted translation system, purified tRNA, synthetic tRNA, fluorophore-labeled tRNA, a human cell S-100 fraction, a non-human mammalian cell S-100 fraction, an archaeal cell S-100 fraction, or a bacterial cell S-100 fraction.

[0054] In some embodiments, the first ribosome source and the tRNA source may be the same.

[0055] After the first mixture is incubated for the first period of time, a second mixture is formed by admixing the first mixture, which had been incubated for the first period of time, with an mRNA encoding a protein (Fig. 2A, Step 3). For example, in the exemplary screen of Fig. 2A, mRNA encoding nanoluciferase (NANOLUC®, Promega Corp., Madison, WI) is admixed with the first mixture, along with NANO-GLO® nanoluciferase substrate (Promega Corp., Madison, WI) or NANO-GLO® endurazine™ Promega Corp., Madison, WI).

[0056] The protein encoded by the mRNA may be any reporter protein, as is readily understood by those of skill in the art. For example, the reporter protein may be anenzymatic protein, nanoluciferase being but one non-limiting example. Other enzymatic proteins include, but are not limited to, luciferase, a P-galactosidase, a P-glucuronidase, a horseradish peroxidase, a xylanase, and an alkaline phosphatase.

[0057] The reporter protein may be a fluorescent protein. Suitable fluorescent proteins include, but are not limited to, a green fluorescent protein, a red fluorescent protein, a yellow fluorescent protein, a cyan fluorescent protein, a blue fluorescent protein, and variants thereof.

[0058] The second mixture is incubated for a second period of time followed by detection of whether the mRNA is translated so as to produce the protein that it encodes. Step 4 of the exemplary screen of Fig. 2A shows detection of the production nanoluciferase. Nanoluciferase enzymatically converts NANO-GLO® reagents into a light emitting compound, allowing for optical detection of nanoluciferase production.

[0059] In some embodiments, the second period of time is 15 seconds-360 minutes, or longer. In some embodiments, the second period of time is 15 seconds-180 minutes. In some embodiments, the second period of time is 15 seconds-60 minutes. In other embodiments, the second period of time is 1-30 minutes. In some embodiments, the second period of time is 1-10 minutes. In still other embodiments, the second period of time is 5-15 minutes. In some embodiments, the second period of time is 15 seconds-5 minutes.

[0060] If the protein encoded by the mRNA is detected, then the compound is identified as an inhibitor of angiogenin in complex with the ribosome due to the fact that angiogenin’s RNase activity with respect to tRNA was inhibited, thus allowing for the translation of the mRNA to produce the protein.

[0061] We have further developed a tRNA cleavage assay to identify whether a compound, or to validate whether an identified compound, inhibits angiogenin in complex with a ribosome (Fig. 2B). In some embodiments, the identified compound is a compound that has been identified — by a screen for identifying whether a compound is an inhibitor of angiogenin (e.g., a high-throughput screen performed using the methods disclosed herein), by in silico modeling, or by other methods known to those of skill in the art — as an inhibitor of angiogenin. The compound identified as an inhibitor of angiogenin may be any compound that is identified as inhibiting angiogenin’s RNase activity. In other embodiments, the tRNA cleavage assay may be used to identify an inhibitor angiogenin incomplex with a ribosome, regardless of whether the inhibitor was previously identified as an inhibitor of angiogenin in complex with a ribosome, i.e., not for validation, but for identification.

[0062] A third mixture, comprising the identified compound (or a starting mixture comprising a compound to be assayed in order to identify whether the compound is an inhibitor angiogenin in complex with a ribosome), angiogenin, a second ribosome source, and a first tRNA substrate, is incubated for a third period of time (or a starting period of time when the assay is used to identify whether the compound is an inhibitor angiogenin in complex with a ribosome). After incubation, an amount of a first tRNA degradation product produced by cleavage of the first tRNA substrate caused by the angiogenin in the third mixture (or the starting mixture) is compared with an amount of a control tRNA degradation product produced by cleavage of a control tRNA substrate by angiogenin in a control mixture, the control mixture lacking the identified compound and comprising the angiogenin, the second ribosome source, and the control tRNA substrate. Amounts of tRNA degradation products may, for example, be determined by gel electrophoresis.

[0063] In some embodiments, the third period of time is 15 seconds-360 minutes, or longer. In some embodiments, the third period of time is 15 seconds-180 minutes. In some embodiments, the third period of time is 15 seconds-60 minutes. In other embodiments, the third period of time is 1-30 minutes. In some embodiments, the third period of time is 1-10 minutes. In still other embodiments, the third period of time is 5-15 minutes. In some embodiments, the third period of time is 15 seconds-5 minutes.

[0064] In some embodiments, the starting period of time is 15 seconds-360 minutes, or longer. In some embodiments, the starting period of time is 15 seconds-180 minutes. In some embodiments, the starting period of time is 15 seconds-60 minutes. In other embodiments, the starting period of time is 1-30 minutes. In some embodiments, the starting period of time is 1-10 minutes. In still other embodiments, the starting period of time is 5- 15 minutes. In some embodiments, the starting period of time is 15 seconds-5 minutes.

[0065] In some embodiments, the third mixture comprising the identified compound (or the starting mixture comprising the compound to be assayed in order to identify whether the compound is an inhibitor angiogenin in complex with a ribosome), angiogenin, a second ribosome source, and a first tRNA substrate may be admixed together in a single step andincubated for the third period of time (or the starting period of time). Here, the control mixture is formed in similar fashion, but lacking the identified compound.

[0066] In other embodiments, the identified compound (or a compound to be assayed in order to determine whether it is an inhibitor angiogenin in complex with a ribosome) is admixed with a second ribosome source, thereby forming mixture A (Fig. 2B, step 1). Mixture A is then incubated for a period of time A. After mixture A is incubated for the period of time A, mixture A is admixed with angiogenin, thereby forming mixture B (Fig. 2B, step 2). Mixture B is then incubated for a period of time B. After mixture B is incubated for the period of time B, mixture B is admixed with a first tRNA substrate, thereby forming the third mixture (Fig. 2B, step 3) (or the starting mixture). Here, the control mixture is formed in similar fashion, but, rather than the mixture A, a control mixture A is formed, having the second ribosome source but lacking the identified compound.

[0067] In some embodiments, the period of time A is 15 seconds-360 minutes, or longer. In some embodiments, the period of time A is 15 seconds-180 minutes. In some embodiments, the period of time A is 15 seconds-60 minutes. In other embodiments, the period of time A is 1-30 minutes. In some embodiments, the period of time A is 1-10 minutes. In still other embodiments, the period of time A is 5-15 minutes. In some embodiments, the period of time A is 15 seconds-5 minutes.

[0068] In some embodiments, the period of time B is 15 seconds-360 minutes, or longer. In some embodiments, the period of time B is 15 seconds-180 minutes. In some embodiments, the period of time B is 15 seconds-60 minutes. In other embodiments, the period of time B is 1-30 minutes. In some embodiments, the period of time B is 1-10 minutes. In still other embodiments, the period of time B is 5-15 minutes. In some embodiments, the period of time B is 15 seconds-5 minutes.

[0069] If the amount of the first tRNA degradation product is less than the amount of the control tRNA degradation product, the identified compound is validated as inhibiting angiogenin in complex with the ribosome and shows that the compound inhibits angiogenin’ s RNase activity with respect to tRNA when in complex with the ribosome.

[0070] In some embodiments, the amount of the first tRNA degradation product is at most 80% the amount of the control tRNA degradation product. In other embodiments, the amount of the first tRNA degradation product is at most 50% the amount of the controltRNA degradation product. In still other embodiments, the amount of the first tRNA degradation product is at most 25% the amount of the control tRNA degradation product. In additional embodiments, the amount of the first tRNA degradation product is at most 10% the amount of the control tRNA degradation product.

[0071] In some embodiments, the first tRNA substrate is a set of isoacceptor tRNA. The first tRNA substrate may be deacyl tRNA, amino-acyl tRNA, or fluorescent tRNA.

[0072] In some embodiments, the control tRNA substrate is a set of isoacceptor tRNA. The control tRNA substrate may be deacyl tRNA, amino-acyl tRNA, or fluorescent tRNA.

[0073] In some embodiments, the second ribosome source comprises purified ribosomes. The purified ribosomes may be purified from a mammalian cell, a eukaryotic cell, an archaeal cell, or a bacteria cell. The first tRNA substrate and the control tRNA substrate may be the same.

[0074] Example 1: First screen for compounds that inhibit angiogenin in complex with a ribosome

[0075] A two-step screen, comprising an initial screen and a validation assay, was designed to identify inhibitors of ribosome bound angiogenin (Figure 2A screen and Figure 2B validation) and was initially tested against a LOP AC® 1280 compound library (Sigma- Aldrich). First, aliquots of rabbit reticulocyte lysate were dosed with compounds from the library, then challenged with an inhibitory concentration of angiogenin and assayed for translation ability by supplementation with a Nanoluc reporter mRNA (Figure 2A).

[0076] Specifically, rabbit reticulocyte lysate (Green Hectares) was diluted to 33% (final concentration) and supplemented with hemin, buffer and salts, an ATP regeneration system, GTP, all 20 amino-acids and Nano-Gio® endurazine™ (Promega). The final translation mix (with 10 pM of each test compound, 66 nM angiogenin, and 2.5 ng / pl of the nanoluciferase (Nanoluc) mRNA) also contained: 60 pM hemin (Sigma-Aldrich), 75 mM potassium chloride, 0.5 mM MgCh, 6 mM betamercapto ethanol, 1 mM ATP (Promega), 2.1 mg / ml creatine phosphate (Roche), 0.9 mg / ml creatine kinase (Roche), 0.2 mM GTP (Promega), 1 mM each of the 20 amino acids, and 0.4% Nano-Gio® endurazine™ (Promega). 384 well white plates were first dispensed with 0.2 pl with each test compound of the LOP AC® 1280 compound library, one compound per well, in DMSO. 8 pl of ice-cold,supplemented RRL was added and the plates were kept on ice for a 1-30 minute incubation (on ice, to allow for plate dosing and compound interaction with the ribosome). Next, 1 pl of 666 nM angiogenin (RND) was added to inhibit translation. 1 pl of 666 nM angiogenin (RND) with 0.8 U / ml rRNAsin (Promega) was used as a positive control of angiogenin inhibition.

[0077] After a 1-30 minute incubation (on ice, to allow for plate dosing and angiogenin action on the ribosome and tRNA), 1 pl of 25 ng / pl nanoluciferase (Nanoluc) mRNA was added to each well and the plates were shifted to 37°C to begin translation and this incubation continued for a minimum of 15 minutes. Translation output was read on a plate reader as luminescence from nanoluciferase converting its substrate, NANO-GLO® END UR. AZINE™ (Promega).

[0078] In the presence of 0.2% DMSO, angiogenin alone fully inhibited translation (average counts 130) while angiogenin+RNasin allowed translation to proceed robustly (average counts 699,000) yielding a Z-score of 0.57 overall. Of the 960 compounds assayed in triplicate none restored translation to levels comparable to RNasin when dosed at 10 pM concentration. However, 29 compounds restored translation to 5 standard deviations above angiogenin alone (average counts 1000-4000).

[0079] Example 2: Hit validation by in vitro tRNA cleavage assay

[0080] Seven of the 29 hit compound from the LOP AC® screen were validated by an assay for tRNA cleavage by ribosome-activated angiogenin (Figure 2B). Ribosome complexes were assembled as described in Loveland et al., hereby incorporated by reference for its teaching of ribosome complex assembly, and first incubated with 50 pM (final concentration) of each compound, an equivalent volume of DMSO (0.5%) for the negative control, or 0.08 U / pl rRNAsin (Promega) for the positive control. 10 nM angiogenin was added to potentially interact with the ribosome followed by 250 nM Ala-tRNAAlain complex with the GTPase eEFl A and GTP (Loveland et al. manuscript in preparation). Cleavage of tRNA^ was resolved by 15% Urea-PAGE and SybrGold staining. While rRNasin robustly inhibited tRNA cleavage, none of the seven compounds tested altered tRNA cleavage, failing to confirm that these compounds inhibit angiogenin in complex with a ribosome.

[0081] Example 3: Second screen for compounds that inhibit angiogenin in complex with a ribosome

[0082] A second screen of 1320 FDA-approved compounds was run similarly to the screen of Example 1, except that at the final translation mix was at 20 pM for each FDA approved compound and the angiogenin final concentration was increased to 100 nM, This second screen yielded 7 hit compounds that reproducibly restored translation 2.5-fold standard deviations over background. A dilution series of these compounds found none that showed a dose-response, although two compounds were found to inhibit translation in a dose-dependent manner. These preliminary results suggest that larger libraries will need to be screened, higher compound concentrations may need to be used, and / or a higher significance cutoff may need to be used to identify true positives.

[0083] Example 4: Third screen for compounds that inhibit angiogenin in complex with a ribosome

[0084] A third screen of 429 kinase inhibitors was run similarly to the screen of Example 2, except that at the final translation mix was at 40 pM for each kinase inhibitor. The first set of plates had very high signal-to-noise (300-10,000) and yielded no hits. The second set of plates had much lower signal-to-noise due to translation occurring in negative control lanes and across sections of the plate. While 5 hit compounds that reproducibly restored translation, a dilution series found the best compound did not show a dose-response. These preliminary results further demonstrate the stringency of the assay and suggest that larger libraries will need to be screened, higher compound concentrations may need to be used, and / or a higher significance cutoff may need to be used to identify true positives.

[0085] Example 5: Positive controls and new mechanisms of angiogenin inhibition

[0086] There are several known inhibitors of angiogenin’ s catalytic activity including a small-molecule inhibitor Benzopurpurin B (Jenkins et al. Biochemistry 42, 6674-6687 (2003).). Furthermore, neomycin has been shown to affect angiogenin uptake pathways, but has not been reported to be an inhibitor of angiogenin’s catalytic activity. (Hu. Proceedings of the National Academy of Sciences 95, 9791-9795 (1998), Skorupa et al. Journal of Neuroscience 32, 5024-5038, (2012).). These inhibitors were tested in the screen as shown in Figure 2A, validated in the tRNA cleavage assay as shown in Figure 2B, and were found to be active. Benzopurpurin B was previously identified as one of the most potent antagonists of isolated angiogenin with a Ki of 5 pM and is thought to occlude the substrate binding sitebased on in silica docking (Jenkins et al. Biochemistry 42, 6674-6687 (2003)). Neomycin was previously found to inhibit angiogenesis in response to angiogenin, but was suggested to act through its action on phospholipase C (Hu. Proceedings of the National Academy of Sciences 95, 9791-9795 (1998).) yet neomycin is an aminoglycoside antibiotic that affects both prokaryotic and eukaryotic ribosomes (Perez-Fernandez et al. Nature Communications 5, 3112 (2014)). We find that Benzopurpurin B and neomycin are differentially active in the assays of ribosome-activated angiogenin activity as shown in Fig. 2A and 2B. Both compounds inhibited tRNA cleavage by ribosome-activated angiogenin in vitro (Fig. 3A and 3B), but only Benzopurpurin B restored translation in RRL in the presence of angiogenin (Fig. 3C and 3D).

[0087] Neomycin alone (in the absence of angiogenin) was found to inhibit translation at 50 pM (data not shown), the same concentration at which it inhibits tRNA cleavage by ribosome-activated angiogenin (Fig. 3B). Of the other aminoglycosides tested, paromomycin, like neomycin, inhibited tRNA cleavage in vitro (Fig. 3E). These data are consistent with our and others’ structural data showing that neomycin and paromomycin bind to the decoding center of the ribosome in a manner that triggers a conformational change of ribosomal residues that impact angiogenin binding to the A site (Fig. 4A and 4B). These data suggest that the same binding site on the ribosome inhibits translation and angiogenin's cleavage of tRNA. Since translation is inhibited by neomycin alone, no signal is detected for neomycin in our translation assay for angiogenin activity despite neomycin's ability to inhibit tRNA cleavage by angiogenin.

[0088] Thus, these two inhibitors of angiogenin in complex with a ribosome demonstrate two different inhibition mechanisms for ribosome-activated angiogenin: 1) Benzopurpurin B likely directly binds angiogenin’s active site, while 2) neomycin perturbs angiogenin’s association with the ribosome by binding the ribosome, thereby inhibiting activation of angiogenin and, thus, tRNA cleavage by angiogenin. The assays disclosed herein will enable identification of novel small molecules with both of these modes of action, and with high specificity.

[0089] The publications (including patent and patent application publications), web sites, company names, books, manuals, treatise, and scientific literature referred to herein establish the knowledge that is available to those with skill in the art and are herebyincorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter.

[0090] Various embodiments of the present invention may be characterized by the potential claims listed in the paragraphs following this paragraph (and before the actual claims provided at the end of this application). These potential claims form a part of the written description of this application. Accordingly, subject matter of the following potential claims may be presented as actual claims in later proceedings involving this application or any application claiming priority based on this application. Inclusion of such potential claims should not be construed to mean that the actual claims do not cover the subject matter of the potential claims. Thus, a decision to not present these potential claims in later proceedings should not be construed as a donation of the subject matter to the public.

[0091] Without limitation, potential subject matter that may be claimed (prefaced with the letter “P” so as to avoid confusion with the actual claims presented below) includes:Pl. A method for identifying whether a compound is an inhibitor of angiogenin in complex with a ribosome, the method comprising:(a) incubating, for a first period of time, a first mixture comprising the angiogenin, the compound, a first ribosome source, and a tRNA source,(b) incubating, for a second period of time, a second mixture formed by admixing, after the first period of time, the incubated first mixture and an mRNA encoding a protein, and(c) detecting whether the mRNA is translated in the second mixture so as to produce the protein, wherein, if the protein is detected, the compound is identified as said inhibitor of angiogenin in complex with the ribosome.P2. The method of potential claim Pl, wherein prior to the incubating for the first period of time, the method comprises:(i) incubating, for an initial period of time, an initial mixture comprising the compound, the first ribosome source, and the tRNA source, and(ii) admixing, after the initial period of time, the incubated initial mixture and the angiogenin, thereby forming the first mixture.P3. The method according to any one of the preceding potential claims, wherein the first ribosome source is selected from the group consisting of rabbit reticulocyte lysate, a eukaryotic cell extract, an archaeal cell extract, a bacterial cell extract, and a reconstituted translation system.P4. The method according to any one of the preceding potential claims, wherein the tRNA source is selected from the group consisting of rabbit reticulocyte lysate, a eukaryotic cell extract, a bacterial cell extract, a reconstituted translation system, purified tRNA, synthetic tRNA, fluorophore-labeled tRNA, a human cell S-100 fraction, a non-human mammalian cell S-100 fraction, and a bacterial cell S-100 fraction.P5. The method according to any one of the preceding potential claims, wherein the first ribosome source and the tRNA source are the same.P6. The method according to any one of the preceding potential claims, wherein the protein is a reporter protein.P7. The method of potential claim P6 wherein the reporter protein is an enzymatic protein.P8. The method of potential claim P7, wherein the enzymatic protein is selected from the group consisting of a luciferase, a P-galactosidase, a P-glucuronidase, a horseradish peroxidase, a xylanase, and an alkaline phosphatase.P9. The method of potential claim P6 wherein the reporter protein is a fluorescent protein.PIO. The method of potential claim P9, wherein the fluorescent protein is selected from the group consisting of a green fluorescent protein, a red fluorescent protein, a yellow fluorescent protein, a cyan fluorescent protein, a blue fluorescent protein, and variants thereof.Pl 1 . The method according to any one of the preceding potential claims, wherein the first period of time is 15 seconds-60 minutes.P12. The method according to any one of the preceding potential claims, wherein the first period of time is 1-30 minutes.Pl 3. The method according to any one of the preceding potential claims, wherein the first period of time is 5-15 minutes.P14. The method according to any one of potential claims P2-P13, wherein the initial period of time is 15 seconds-60 minutes.Pl 5. The method according to any one of potential claims P2-P14, wherein the initial period of time is 1-30 minutes.Pl 6. The method according to any one of potential claims P2-P15, wherein the initial period of time is 5-15 minutes.Pl 7. A method of validating whether an identified compound inhibits angiogenin in complex with a ribosome, the method comprising:(A) obtaining the identified compound, said identified compound being a compound identified as an inhibitor of angiogenin,(B) incubating, for a third period of time, a third mixture comprising the identified compound, the angiogenin, a second ribosome source, and a first tRNA substrate, and(C) comparing: (1) an amount of a first tRNA degradation product produced by cleavage of the first tRNA substrate caused by the angiogenin in the third mixture with (2) an amount of a control tRNA degradation product produced by cleavage of a control tRNA substrate by the angiogenin in a control mixture, the control mixture lacking the identified compound and comprising the angiogenin, the second ribosome source, and the control tRNA substrate, wherein, if the amount of the first tRNA degradation product is less than the amount of the control tRNA degradation product, the identified compound is validated as inhibiting angiogenin in complex with the ribosome.P18. The method according to potential claim P17, wherein the second ribosome source comprises purified ribosomes.Pl 9. The method according to potential claim Pl 8, wherein the purified ribosomes are purified from a cell source selected from the group consisting of a eukaryotic cell, an archaeal cell, and a bacteria cell.P20. The method according to any one of potential claims P17-P19, wherein the first tRNA substrate is a set of isoacceptor tRNA.P21. The method according to any one of potential claims P17-P20, wherein the control tRNA substrate is a set of isoacceptor tRNA.P22. The method according to any one of potential claims P17-P21, wherein the first tRNA substrate is selected from the group consisting of a deacyl tRNA, an amino-acyl tRNA, and a fluorescent tRNA.P23. The method according to any one of potential claims P17-P22, wherein the control tRNA substrate is selected from the group consisting of a deacyl tRNA, an aminoacyl tRNA, and a fluorescent tRNA.'llP24. The method according to any one of potential claims P17-P23, wherein the first tRNA substrate and the control tRNA substrate are the same.P25. The method according to any one of potential claims P17-P24, wherein the amount of the first tRNA degradation product is at most 80% the amount of the control tRNA degradation product.P26. The method according to any one of potential claims P17-P24, wherein the amount of the first tRNA degradation product is at most 50% the amount of the control tRNA degradation product.P27. The method according to any one of potential claims P17-P24, wherein the amount of the first tRNA degradation product is at most 10% the amount of the control tRNA degradation product.P28. The method according to any one of potential claims P17-P27, wherein the identified compound is identified as an inhibitor of angiogenin by a method selected from the group consisting of a screen and in silica modeling.P29. The method of potential claim P28, wherein the screen is a high-throughput screen.P30. The method according to any one of potential claims P17-P27, wherein the identified compound is identified as an inhibitor of angiogenin by the method according to any one of potential claims 1-16.P31. The method according to any one of potential claims P17-P30, wherein, prior to the incubating for the third period of time, the method comprises:(i) admixing the obtained identified compound and the second ribosome source, thereby forming a mixture A;(ii) incubating the mixture A for a period of time A;(iii) admixing, after the period of time A, the mixture A and the angiogenin, thereby forming a mixture B;(iv) incubating the mixture B for a period of time B;(v) admixing, after the period of time B, the mixture B and the first tRNA substrate, thereby forming the third mixture.P32. A method for identifying whether a compound is an inhibitor of angiogenin in complex with a ribosome, the method comprising:(A) incubating, for a starting period of time, a starting mixture comprising the compound, the angiogenin, a second ribosome source, and a first tRNA substrate, and(B) comparing: (1) an amount of a first tRNA degradation product produced by cleavage of the first tRNA substrate caused by the angiogenin in the starting mixture with (2) an amount of a control tRNA degradation product produced by cleavage of a control tRNA substrate by the angiogenin in a control mixture, the control mixture lacking the compound and comprising the angiogenin, the second ribosome source, and the control tRNA substrate, wherein, if the amount of the first tRNA degradation product is less than the amount of the control tRNA degradation product, the compound is identified as said inhibitor of angiogenin in complex with the ribosome.P33. The method according to potential claim P32, wherein the second ribosome source comprises purified ribosomes.P34. The method according to potential claim P33, wherein the purified ribosomes are purified from a cell source selected from the group consisting of a eukaryotic cell, an archaeal cell, and a bacteria cell.P35. The method according to any one of potential claims P32-P34, wherein the first tRNA substrate is a set of isoacceptor tRNA.P36. The method according to any one of potential claims P32-P35, wherein the control tRNA substrate is a set of isoacceptor tRNA.P37. The method according to any one of potential claims P32-P36, wherein the first tRNA substrate is selected from the group consisting of a deacyl tRNA, an amino-acyl tRNA, and a fluorescent tRNA.P38. The method according to any one of potential claims P32-P37, wherein the control tRNA substrate is selected from the group consisting of a deacyl tRNA, an aminoacyl tRNA, and a fluorescent tRNA.P39. The method according to any one of potential claims P32-P38, wherein the first tRNA substrate and the control tRNA substrate are the same.P40. The method according to any one of potential claims P32-P39, wherein the amount of the first tRNA degradation product is at most 80% the amount of the control tRNA degradation product.P41. The method according to any one of potential claims P32-P39, wherein the amount of the first tRNA degradation product is at most 50% the amount of the control tRNA degradation product.P42. The method according to any one of potential claims P32-P39, wherein the amount of the first tRNA degradation product is at most 10% the amount of the control tRNA degradation product.P43. The method according to any one of potential claims P32-P42, wherein, prior to the incubating for the starting period of time, the method comprises:(i) admixing the compound and the second ribosome source, thereby forming a mixture A;(ii) incubating the mixture A for a period of time A;(iii) admixing, after the period of time A, the mixture A and the angiogenin, thereby forming a mixture B;(iv) incubating the mixture B for a period of time B;(v) admixing, after the period of time B, the mixture B and the first tRNA substrate, thereby forming the starting mixture.

[0092] The embodiments of the invention described above are intended to be merely exemplary; numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention as defined in any appended claims.

Claims

What is claimed is:

1. A method for identifying whether a compound is an inhibitor of angiogenin in complex with a ribosome, the method comprising:(a) incubating, for a first period of time, a first mixture comprising the angiogenin, the compound, a first ribosome source, and a tRNA source,(b) incubating, for a second period of time, a second mixture formed by admixing, after the first period of time, the incubated first mixture and an mRNA encoding a protein, and(c) detecting whether the mRNA is translated in the second mixture so as to produce the protein, wherein, if the protein is detected, the compound is identified as said inhibitor of angiogenin in complex with the ribosome.

2. The method of claim 1, wherein prior to the incubating for the first period of time, the method comprises:(i) incubating, for an initial period of time, an initial mixture comprising the compound, the first ribosome source, and the tRNA source, and(ii) admixing, after the initial period of time, the incubated initial mixture and the angiogenin, thereby forming the first mixture.

3. The method according to any one of the preceding claims, wherein the first ribosome source is selected from the group consisting of rabbit reticulocyte lysate, a eukaryotic cell extract, an archaeal cell extract, a bacterial cell extract, and a reconstituted translation system.

4. The method according to any one of the preceding claims, wherein the tRNA source is selected from the group consisting of rabbit reticulocyte lysate, a eukaryotic cell extract, a bacterial cell extract, a reconstituted translation system, purified tRNA, synthetic tRNA, fluorophore-labeled tRNA, a human cell S-100 fraction, a non-human mammalian cell S-100 fraction, and a bacterial cell S-100 fraction.

5. The method according to any one of the preceding claims, wherein the first ribosome source and the tRNA source are the same.

6. The method according to any one of the preceding claims, wherein the protein is a reporter protein.

7. The method of claim 6 wherein the reporter protein is an enzymatic protein.

8. The method of claim 7, wherein the enzymatic protein is selected from the group consisting of a luciferase, a P-galactosidase, a P -glucuronidase, a horseradish peroxidase, a xylanase, and an alkaline phosphatase.

9. The method of claim 6 wherein the reporter protein is a fluorescent protein.

10. The method of claim 9, wherein the fluorescent protein is selected from the group consisting of a green fluorescent protein, a red fluorescent protein, a yellow fluorescent protein, a cyan fluorescent protein, a blue fluorescent protein, and variants thereof.

11. The method according to any one of the preceding claims, wherein the first period of time is 15 seconds-60 minutes.

12. The method according to any one of the preceding claims, wherein the first period of time is 1-30 minutes.

13. The method according to any one of the preceding claims, wherein the first period of time is 5-15 minutes.

14. The method according to any one of claims 2-13, wherein the initial period of time is 15 seconds-60 minutes.

15. The method according to any one of claims 2-14, wherein the initial period of time is 1-30 minutes.

16. The method according to any one of claims 2-15, wherein the initial period of time is 5-15 minutes.

17. A method of validating whether an identified compound inhibits angiogenin in complex with a ribosome, the method comprising:(A) obtaining the identified compound, said identified compound being a compound identified as an inhibitor of angiogenin,(B) incubating, for a third period of time, a third mixture comprising the identified compound, the angiogenin, a second ribosome source, and a first tRNA substrate, and(C) comparing: (1) an amount of a first tRNA degradation product produced by cleavage of the first tRNA substrate caused by the angiogenin in the third mixture with (2) an amount of a control tRNA degradation product produced by cleavage of a control tRNA substrate by the angiogenin in a control mixture, the control mixture lacking the identified compound and comprising the angiogenin, the second ribosome source, and the control tRNA substrate, wherein, if the amount of the first tRNA degradation product is less than the amount of the control tRNA degradation product, the identified compound is validated as inhibiting angiogenin in complex with the ribosome.

18. The method according to claim 17, wherein the second ribosome source comprises purified ribosomes.

19. The method according to claim 18, wherein the purified ribosomes are purified from a cell source selected from the group consisting of a eukaryotic cell, an archaeal cell, and a bacteria cell.

20. The method according to any one of claims 17-19, wherein the first tRNA substrate is a set of isoacceptor tRNA.

21. The method according to any one of claims 17-20, wherein the control tRNA substrate is a set of isoacceptor tRNA.

22. The method according to any one of claims 17-21 , wherein the first tRNA substrate is selected from the group consisting of a deacyl tRNA, an amino-acyl tRNA, and a fluorescent tRNA.

23. The method according to any one of claims 17-22, wherein the control tRNA substrate is selected from the group consisting of a deacyl tRNA, an amino-acyl tRNA, and a fluorescent tRNA.

24. The method according to any one of claims 17-23, wherein the first tRNA substrate and the control tRNA substrate are the same.

25. The method according to any one of claims 17-24, wherein the amount of the first tRNA degradation product is at most 80% the amount of the control tRNA degradation product.

26. The method according to any one of claims 17-24, wherein the amount of the first tRNA degradation product is at most 50% the amount of the control tRNA degradation product.

27. The method according to any one of claims 17-24, wherein the amount of the first tRNA degradation product is at most 10% the amount of the control tRNA degradation product.

28. The method according to any one of claims 17-27, wherein the identified compound is identified as an inhibitor of angiogenin by a method selected from the group consisting of a screen and in silico modeling.

29. The method of claim 28, wherein the screen is a high-throughput screen.

30. The method according to any one of claims 17-27, wherein the identified compound is identified as an inhibitor of angiogenin by the method according to any one of claims 1-16.

31. The method according to any one of claims 17-30, wherein, prior to the incubating for the third period of time, the method comprises:(i) admixing the obtained identified compound and the second ribosome source, thereby forming a mixture A;(ii) incubating the mixture A for a period of time A;(iii) admixing, after the period of time A, the mixture A and the angiogenin, thereby forming a mixture B;(iv) incubating the mixture B for a period of time B;(v) admixing, after the period of time B, the mixture B and the first tRNA substrate, thereby forming the third mixture.

32. A method for identifying whether a compound is an inhibitor of angiogenin in complex with a ribosome, the method comprising:(A) incubating, for a starting period of time, a starting mixture comprising the compound, the angiogenin, a second ribosome source, and a first tRNA substrate, and(B) comparing: (1) an amount of a first tRNA degradation product produced by cleavage of the first tRNA substrate caused by the angiogenin in the starting mixture with (2) an amount of a control tRNA degradation product produced by cleavage of a control tRNA substrate by the angiogenin in a control mixture, the control mixture lacking the compoundand comprising the angiogenin, the second ribosome source, and the control tRNA substrate, wherein, if the amount of the first tRNA degradation product is less than the amount of the control tRNA degradation product, the compound is identified as said inhibitor of angiogenin in complex with the ribosome.

33. The method according to claim 32, wherein the second ribosome source comprises purified ribosomes.

34. The method according to claim 33, wherein the purified ribosomes are purified from a cell source selected from the group consisting of a eukaryotic cell, an archaeal cell, and a bacteria cell.

35. The method according to any one of claims 32-34, wherein the first tRNA substrate is a set of isoacceptor tRNA.

36. The method according to any one of claims 32-35, wherein the control tRNA substrate is a set of isoacceptor tRNA.

37. The method according to any one of claims 32-36, wherein the first tRNA substrate is selected from the group consisting of a deacyl tRNA, an amino-acyl tRNA, and a fluorescent tRNA.

38. The method according to any one of claims 32-37, wherein the control tRNA substrate is selected from the group consisting of a deacyl tRNA, an amino-acyl tRNA, and a fluorescent tRNA.

39. The method according to any one of claims 32-38, wherein the first tRNA substrate and the control tRNA substrate are the same.

40. The method according to any one of claims 32-39, wherein the amount of the first tRNA degradation product is at most 80% the amount of the control tRNA degradation product.

41. The method according to any one of claims 32-39, wherein the amount of the first tRNA degradation product is at most 50% the amount of the control tRNA degradation product.

42. The method according to any one of claims 32-39, wherein the amount of the first tRNA degradation product is at most 10% the amount of the control tRNA degradation product.

43. The method according to any one of claims 32-42, wherein, prior to the incubating for the starting period of time, the method comprises:(i) admixing the compound and the second ribosome source, thereby forming a mixture A;(ii) incubating the mixture A for a period of time A;(iii) admixing, after the period of time A, the mixture A and the angiogenin, thereby forming a mixture B;(iv) incubating the mixture B for a period of time B;(v) admixing, after the period of time B, the mixture B and the first tRNA substrate, thereby forming the starting mixture.

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