WO2013086162A1 - Radioiodinateable angiotensin-converting enyzyme-2 (ace-2) modulating compounds, preparation thereof, and methods for use thereof - Google Patents

Abstract

The invention provides angiotensin-converting enzyme-2 (ACE-2) inhibitors and radioligands for measuring ACE-2 protein (quantifying amounts of protein), for determining tissue distribution of ACE-2 protein, and/or for measuring experimentally-induced changes in ACE-2 protein expression. The invention also provides pharmaceutical compositions containing the ACE-2 inhibitors and radioligands, processes and intermediates for preparing the ACE-2 inhibitors and radioligands, methods of using the ACE-2 inhibitors and radioligands for modulating the activity of angiotensin-converting enzyme-2 (ACE-2) protein, and methods of using the ACE-2 inhibitors and radioligands for treating ACE-2 associated conditions.

Description

RADIOIODINATEABLE ANGIOTENSIN-CONVERTING ENYZYME-2 (ACE-2) MODULATING COMPOUNDS, PREPARATION THEREOF, AND METHODS FOR USE

THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Patent Application No. 61/567,290, filed on December 6, 201 1, the content of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention generally relates to the characterization of proteins, particularly to the use of inhibitors and radioligands to quantify (measure amounts of protein) and localize (determine tissue location and/or anatomical distribution of protein quantified) proteins, and most particularly to the use of an angiotensin-converting enzyme-2 (ACE-2) inhibitor and radioligand to measure ACE-2 protein, to determine tissue distribution of ACE-2 protein, and to measure experimentally-induced changes in ACE-2 protein expression. BACKGROUND

Despite remarkable advances in our ability to treat high blood pressure and other cardiovascular diseases, cardiovascular disease still causes more deaths of Americans today than any other disease (831,272 lives in 2006; statistic obtained from the website of the American Heart Association). Estimates for 2006 are that 81.1 million people in the United States have one or more forms of cardiovascular disease (CVD) of which 73.6 million have hypertension; See the website of the American Heart Association. In addition, considerable morbidity arises from uncontrolled hypertension; stroke, angina, shortness of breath, and/or weakness. Clearly there is room for improvement in the prevention, diagnosis, management, and treatment of cardiovascular disease.

The continuing high rate of death and debility from hypertension is likely due to our limited knowledge of the mechanisms for regulation of the cardiovascular system. One example is our continuously evolving understanding of the renin-angiotensin system (RAS). The original concept of renin as a hormone (Tigerstedt et al. Scand Arch Physiol 8:223-271 1898), dormant for more than 70 years following the discovery that renin was the most critical enzyme for formation of angiotensin II (Braun-Menendez et al. J Physiol 98:283-298 1940; Page et al. J Exp Med 71 :495-519 1940), recently regained its role as a hormone with the discovery of a (pro)renin receptor (Nguyen et al. J Clin Invest 109: 1417-1427 2002).

Another major breakthrough in our understanding of the RAS was the recognition of the possible functional significance of angiotensin 1-7 (Ang 1-7), dating back to the late 1980's (Schiavone et al. Proc Natl Acad Sci USA 85:4095-4098 1988). While the

appreciation of its functional significance developed slowly, a seminal observation portending its importance was the discovery that the protein encoded by the mas oncogene, formerly an orphan G-protein receptor, was the receptor for Ang 1 -7 (Santos et al. Proc Natl Acad Sci USA 100:8258-8263 2003). Another important observation that has led to the inclusion of Ang 1-7 as a full-fledged member of the renin-angiotension system (RAS) is the discovery of angiotensin-converting enzyme-2 (ACE-2) (Donoghue et al. Circ Res 87:E1-E9 2000; Tipnis et al. J Biol Chem 275:33238-33243 2000), which is capable of cleaving angiotensin I (Ang I) to angiotensin 1-9, and most importantly, angiotensin II (Ang II) to Ang 1-7.

ACE-2 is a homologue of angiotensin-converting enzyme (ACE), sometimes now referred to as ACE-1. It has 49% similarity with the major variant of ACE (somatic ACE) in humans and 62% similarity with the ACE variant that is expressed in the testis. Both ACE and ACE-2 are zinc metalloproteases with nearly identical zinc-binding domains (Tipnis et al. J Biol Chem 275(43):33238-33243 2000).

ACE-2 is also the receptor for the severe acute respiratory syndrome (SARS) virus (Li et al. Nature 426:450-454 2003; Turner et al. Trends Pharmacol Sci 25:291-294 2004). Of note, binding of the SARS virus to ACE-2 initiates the internalization of ACE-2 (Wang et al. Cell Res 18:290-301 2008), a phenomenon often seen with ligand-bound receptor molecules. This gives rise to the question of whether ACE-2 may function as a receptor as well as an enzyme, akin to its close homolog ACE-1 (Kohlstedt et al. Circ Res 94:60-67 2004;

Guimaraes et al. Hypertension 57:965-972 2011).

Now that ACE-2 has been recognized as a component of the RAS, it is viewed in a highly favorable light, in contrast to other components of the RAS. Indeed, the ACE-2/Ang 1-7/mas axis is viewed as a counterregulatory arm of the RAS. ACE-2 metabolically inactivates the pressor and other pathophysiological actions of Ang II. In addition, ACE-2 forms Ang 1-7, a vasodilatory peptide that opposes the pressor actions of Ang II (Santos et al. Proc Natl Acad Sci USA 100:8258-8263 2003; Speth et al. Proc West Pharmacol Soc 46: 11- 15 2003). Ang 1-7 also opposes the growth-promoting effects of Ang II (Tallant et al.

Hypertension 42:574-579 2003), the profibrotic effects (Iwata et al. Am J Physiol Heart Circ Physiol 289:H2356-H2363 2005), and its proinflammatory effects (Li et al. Hypertension Res 32:369-374 2009; Shenoy et al. Am JRespir Crit Care Med 182: 1065-1072 2010). See also recent reviews Castro-Chaves et al. Expert Opin Ther Targets 14:485-496 2010 and Shenoy et al. Curr Opin Pharmacol 11 : 150- 155 2011.

An important question surrounding the functionality of ACE-2 is: which of its effects are most important, degradation of Ang II or formation of Ang 1-7? (Ferrario, C. M. Curr Opin Nephrol Hypertension 20: 1-6 2011). In the brain, it appears that the formation of Ang 1-7 is of greater benefit based upon studies of mice overexpressing ACE-2 in neurons subjected to chronic Ang II infusion-induced hypertension (Feng et al. Circ Res 106:373-382 2010). When an antagonist of the Ang 1-7 receptor (A-779, He⁷ Ang 1-7) is administered to these mice, the blood pressure is reduced. A similar observation has been made in the lung wherein administration of an Ang 1-7 antagonist reverses the beneficial effects of Ang 1-7 administration (Shenoy et al. Am JRespir Crit Care Med 182: 1065-1072 2010).

The RAS is associated with other diseases that affect a variety of organs in the body. Of note, ACE-2 is widely, if not ubiquitously, distributed in the body (Bindom et al. Mol Cell Endocrinol 302: 193-202 2009), and may have beneficial actions in all of these tissues. To focus on a few of these tissues where there is considerable interest: ACE-2 is present in the pancreas. Located in both the endocrine and exocrine pancreas, it is co-localized with insulin in the islets of Langerhans (Fang et al. JInt Med Res 38:558-569 2010). In mice deficient in ACE-2 there is a significant increase in blood glucose levels (Bindom et al. Mol Cell

Endocrinol 302: 193-202 2009). Overexpression of ACE-2 improved glucose tolerance and preserved islet function in young db/db diabetic mice (Bindom et al. Diabetes 59:2540-2548 2010), mimicking the beneficial roles of ACE inhibitors and angiotensin receptor blockers in db/db mice (Tesch et al. Am J Physiol Renal Physiol 300:F301-F310 201 1).

ACE-2 in the heart may oppose the development of heart failure (Crackower et al.

Nature 417:822-828 2002). Formation of Ang 1-7 is upregulated in failing human heart, likely by ACE-2 (Zisman et al. Circulation 108: 1707-1712 2003). Deletion of ACE-2 in mice leads to increased susceptibility to stress-induced cardiac damage (Bodiga et al.

Cardiovasc Res 91 : 151-161 201 1). Overexpression of ACE-2 reverses Ang II induced cardiac hypertrophy and fibrosis (Huentelman et al. Exp Physiol 90:783-790 2005; Zhong et al. Circulation 122:717-728 2010). Of note, SARS virus was detected in the heart of 35% of people who died from SARS infection and was associated with decreased ACE-2 and myocardial damage (Oudit et al. Eur J Clin Invest 39:618-625 2009). ACE-2 in the lungs may counteract pulmonary hypertension. Overexpression of ACE-2 in the lungs of rats prevents monocrotaline-induced pulmonary hypertension and attenuates expression of proinflammatory cytokines (Yamazato et al. Hypertension 54:365-371 2009).

Overexpression of ACE-2 in the lungs of rats also prevents bleomycin-induced lung fibrosis (Shenoy et al. Am JRespir Crit Care Med 182: 1065-1072 2010). ACE-2 deficient mice have impaired vascular endothelial mediated relaxation, but when ACE-2 is virally transfected into vascular endothelial cells it counteracts the effects of Ang II by an Ang 1-7 dependent mechanism (Lovren et al. Am J Physiol Heart Circ Physiol 295:H1377-H1384 2008).

ACE-2 is found in the liver and appears to have antifibrotic actions. In a rat bile duct ligation model of liver injury and in human cirrhotic liver, ACE-2 expression is 23- and 97- fold increased respectively, suggesting that ACE-2 is exerting a compensatory response to the classical RAS of the liver (Paizis et al. Gut 54: 1790-1796 2005). ACE-2 is renoprotective in the kidney and its inhibition or deletion worsens kidney damage associated with animal models of diabetes (Bindom et al. Mol Cell Endocrinol 302: 193-202 2009) or renovascular hypertension (Burgelova et al. J Hypertension 27: 1988-2000 2009). Deletion of mas (Pinheiro et al. Kidney Int 75: 1 184-1 193 2009) or ACE-2 also leads to kidney damage suggesting that ACE-2 mediated formation of Ang 1 -7 is critical for maintenance of normal kidney function. Type-2 diabetics with nephropathy have a reduced expression of ACE-2 mRNA and immunoreactivity in the proximal tubule which may contribute to the renal injury, (Reich et al. Kidney Int 74: 1610-1616 2008). Exogenous ACE-2 reverses the renal pathology caused by chronic Ang II infusion (Zhong et al. Hypertension 57:314-322 2011) and diabetes (Oudit et al. Diabetes 59:529-538 2010) in mice.

There is abundant evidence that ACE-2 expression is altered in disease states and with therapeutic treatment. It is upregulated in failing human hearts (Zisman et al. Circulation 108: 1707-1712 2003), although it was not altered in heart of coronary artery-ligated Lewis rats showing signs of failure unless an AT-1 receptor blocker was co-administered (Ishiyama et al. Hypertension 43 :970-976 2004). ACE-2 is decreased in the aorta of spontaneously hypertensive rats (SHR) compared to Wistar-Kyoto rats (Zhong et al. Regul Pept 166:90-97 201 1). Other studies show that increased Ang II decreases ACE-2 mRNA (Ferrario, C. M. Curr Opin Nephrol Hypertension 20: 1-6 2011).

There are at least three other enzymes that can act on Ang II to form Ang 1-7: Prolyl carboxypeptidase (E.C. 3.4.12.4; identification number from "The Comprehensive Enzyme Information System" database; recently reclassified as E.C. 3.4.16.2), which was first called angiotensinase C (Yang et al. Nature 218: 1224-1226 1968), carboxypeptidase A (Pereira et al. Regul Pept 151 : 135-138 2008), and carboxypeptidase N (Axelband et al. Regul Pept 158:47-56 2009). In addition, neutral endopeptidase (EP 24.1 1, neprilysin, NEP) and prolyl endopeptidase (E.C. 3.4.21.26) can form Ang 1-7 from Ang I (Rice et al. Biochem J383 :45- 51 2004; Velez et al. Hypertension 53 :790-797 2009; Stephenson et al. Biochem J 241 :237- 247 1987; Welches et al. J Hypertension 9:631-638 1991). However, inhibition of neutral endopeptidase does not affect coronary vascular responses to Ang I in the guinea pig heart (Kozlovski et al. Pharmacol Rep 59:421-427 2007). Inhibition of carboxypeptidase A with potato carboxypeptidase inhibitor does not affect pressor responses to Ang I or Ang II (Pereira et al. Regul Pept 151 : 135-138 2008), and it appears that ACE-2 is the most important enzyme for the production of Ang 1-7, especially from Ang II (Zisman et al. Circulation 108: 1707-1712 2003; Garabelli et al. Exp Physiol 93 :613-621 2008; Shaltout et al. Am J Physiol Renal Physiol 292:F82-F91 2007). Of note, ACE-2 can play a small role in the formation of Ang 1-7 from Ang I via formation of Ang 1-9 which can then be acted upon by ACE to form Ang 1-7 (Karamyan et al. Regul Pept 143: 15-27 2007). However, the low rate of conversion of Ang I to Ang 1-9 by ACE-2 (Vickers J Biol Chem 277: 14838-14843 2002) makes this pathway of minor significance. Also of note, Ang II is not the only substrate of ACE-2. It is reported to hydrolyze a variety of substrates including des Arg⁹ bradykinin, apelin-13, beta casomorphin, neocasomorphin, dynorphin A, neurotensin, and ghrelin.

In view of its leading role in the production of Ang 1-7 from Ang II, it is important to determine the concentration of ACE-2 in various tissues. It is also critical to determine its localization within tissues with a diversity of functions, e.g. brain, and to determine conditions in which the enzyme is up- or down-regulated.

At present, measurement of ACE-2 regulation is based on measurement of ACE-2 mRNA expression, immunological assays (Western blotting, immunohistochemistry) or determination of the rate of specific substrate metabolism. All of these techniques provide useful information about ACE-2 but have limitations. Measurement of changes in mRNA expression does not always translate into changes in protein expression (Vickers J Biol Chem 277: 14838-14843 2002; Wang et al. J Am Soc Nephrol 8: 193-198 1997). The discovery of microRNAs (miRNA) provides a basis for this dichotomy. MicroRNAs and other small interfering RNAs can inhibit translation of mRNA into protein (Zamore et al. Sci 309: 1519- 1524 2005). A dichotomy between mRNA expression and protein expression has been shown for ACE-2 in transgenic mouse brains (Doobay et al. Am J Physiol Regul Integr Comp Physiol 292:R373-R381 2007), as well as for ACE-2, renin, and ACE-lin the placenta of mice exposed to a hypoxic stress (Goyal et al. Placenta 32: 134-139 201 1). Assays of protein immunoreactivity have four major limitations: specificity, quantification, reproducibility, and functionality. Antibodies used to assay a protein are frequently polyclonal and can recognize a variety of antigenic sites in a protein, and sometimes in other proteins which have similar antigenic sites. This lack of specificity can lead to false positive signals for the protein of interest. The heterogeneity of antigenic sites can be eliminated using monoclonal antibodies, but questions of specificity of the antigenic site to a single protein of interest remain. There is no established stoichiometric ratio of polyclonal antibody staining to antigen. Thus, only semiquantitative estimates of the amount of protein of interest can be made, often indicated as (-), (+), (++), and (+++) (Doobay et al. Am J Physiol Regul Integr Comp Physiol 292:R373-R381 2007). The reproducibility issue relates to the fact that supplies of polyclonal antibodies are finite and continued

immunological assay requires new antibodies that may not provide equivalent targeting of the protein of interest. Also, it may not be possible to use a specific antibody in a species because it was raised in that species and secondary antibodies would not be able to distinguish the antiprotein antibody from all the other antibodies in the animal. The problem of functionality may be the most critical. The presence of the antigenic site does not provide any evidence for the functionality of the protein. The protein might not even be intact but a remnant can still be displaying the antigenic site to the antibody.

Assays of functional activity of enzymes are of great value but do not have high anatomical resolution because it is often necessary to take a whole organ or a substantial part of it to be able to have sufficient enzyme for assay of its activity. In addition, there can be some question of specificity of the enzymatic activity and the need for chromogenic substrate which may not accurately represent the naturally-occurring substrate of interest.

Alternatively, a radiolabeled substrate can be used to monitor the formation of radiolabeled product, however this requires sophisticated chromatographic procedures to isolate the product from the precursor (Zisman et al. Circulation 108: 1707-1712 2003; Joyner et al. Am J Physiol Regul Integr Comp Physiol 293 :R169-R177 2007). Thus, there is considerable value in developing additional means of directly assaying ACE-2 expression which can make up for the limitations of known techniques.

SUMMARY OF THE INVENTION

At present there is no radioligand that can be used for binding assays to quantify functional ACE-2 protein in tissue samples. Given the developing appreciation of the benefits ascribed to the activity of ACE-2, e.g. its ability to reverse the pathophysiological effects of angiotensin II, it is important to have a rapid and reliable means of assaying this enzyme.

The ACE-2/angiotensin 1-7 /mas axis antagonizes many pathophysiological effects of the classical renin-angiotensin system (RAS). Thus, as noted above, there is considerable interest in measuring ACE-2 and determining its tissue locations.

The invention provides a small molecule ACE-2 inhibitor that can be radioiodinated to assess the localization of ACE-2 by in vitro autoradiography. This ACE-2 inhibitor reduced metabolism of the synthetic ACE-2 substrate Mea-APK (Dnp) in the micromolar range.

The ACE-2 inhibitor was radioiodinated using the chloramine T procedure and the mono¹²⁵I-ACE-2 inhibitor was purified by reverse phase high performance liquid

chromatography (HPLC). The radioligand displayed saturable binding to rat kidney homogenate that was displaced by ethylenediaminetetraacidic acid (EDTA) (lOmM). The radioligand (50pM) was incubated with 20-micron thick coronal sections of mouse brain in the presence of captopril (1 μΜ) with or without the peptidic human ACE-2 inhibitor DX-600 (1.3 μΜ). An alternate set of sections was incubated with captopril (1 μΜ) and EDTA. There was a diffuse pattern of ¹²⁵I-labeled ACE-2 inhibitor binding throughout the mouse brain. Binding in the presence to DX-600 was only moderately decreased; however it was almost completely eliminated in the presence of EDTA. These results indicate the utility of a radioiodinated ACE-2 inhibitor for characterization and localization of ACE-2.

The research described below is innovative inasmuch as the technology of using a radiolabeled ACE-2 inhibitor to assay ACE-2 has to the inventor's knowledge never been considered. Moreover, the technologies used to validate this research are highly innovative. Based on the utility of the ¹²⁵I-351A for measuring ACE and the need for more reliable ways of measuring ACE-2, the inventive ACE-2 inhibitor and radioligand promises to be an extremely valuable new tool to use to study ACE-2. An additional benefit is that the inhibitor promises to be a useful small molecule inhibitor of ACE-2.

At the present time, only three ACE-2 inhibitors are available and all three have severe limitations: DX-600 is a peptide and prohibitively expensive for in vivo use, and it is also a poor inhibitor of rodent ACE-2 (Pedersen et al. Am J Physiol 301 :R1293-1299 2011) MLN-4760 is a patented compound and can only be used and under strict limitations from the company (Millennium Pharamaceticals, Inc., Cambridge, MA) that developed it, and phosphinic pseudopeptide inhibitors are not widely available nor are they well characterized. One embodiment of the invention provides an inhibitor and radioligand that can be used to measure functional angiotensin-converting enzyme-2 (ACE-2) protein and determine its tissue location and anatomical distribution. Radioreceptor and autoradiography technology could be applied with the radioligand in experiments directed at enhancing expression of ACE-2.

An angiotensin-converting enzyme-2 (ACE-2) inhibitor of the invention can be synthesized by modifying R² side chains in analogs of MLN-4760 (FIG. 1; Formula I) with replacement of the isopropyl (leucine-like) moiety with a phenol (tyrosine-like) moiety; for example the following compound (FIG. 2; Formula II) wherein R² represents the phenol m iety:

This phenolic moiety is the portion (of the inhibitor) that can be radiolabeled. One example of such a radiolabel is iodine-125. An alkyl linker/spacer chain of 1 to 4 methyl groups can be used to extend the phenolic moiety away from other parts of the

molecule/compound if there is steric hindrance to the binding of the ACE-2 inhibitor by the phenolic moiety and its radiolabel. The structures of the alkyl linker/spacer chains are shown in FIGS. 10A-D.

The term "angiotensin-converting enzyme-2 (ACE-2) inhibitor" refers to any substance which reduces, or is capable of reducing, the activity of ACE-2, in vitro and/or in vivo.

Another embodiment of the invention provides a method for studying a sample containing or suspected of containing angiotensin-converting enzyme-2 (ACE-2) protein. The method includes steps for contacting the sample with an inhibitor of ACE-2 and quantifying binding of the inhibitor to ACE-2 protein present in the sample.

Another method provided by the invention is a method for quantifying angiotensin- converting enzyme-2 (ACE-2) protein in a biological sample containing or suspected of containing ACE-2 protein. The method includes steps for obtaining at least one biological sample, contacting the sample with an inhibitor of ACE-2, quantifying binding of the ACE-2 inhibitor to ACE-2 protein present in the sample, and quantifying the amount of the ACE-2 protein detected in the biological sample. The method can also include a step for determining anatomical location and distribution of the ACE-2 protein quantified.

In the methods, the "sample" can be any substance containing or suspected of containing ACE-2. A "biological sample" refers to a sample including material derived from a living organism or organisms. One non-limiting example of a biological sample is a kidney tissue sample.

A radiolabled compound having the formula:

can be used in the methods. R² represents the radiolabeled phenol moiety.

The radiolabeling can be carried out using any known methods and known radiolabels/tags. One non-limiting example is radiolabeling with iodine- 125 using a chloramine T procedure.

The quantifying steps in the methods can be carried out using any methods known for quantifying a substance of interest. One non-limiting example is the use of autoradiography with quantitative densitometric analysis to quantify an amount of ACE-2 protein present in a sample as detected by inhibitor binding.

Another embodiment of the invention provides a pharmaceutical composition comprising a pharmaceutically-acceptable carrier and an effective amount of an angiotensin- converting enzyme-2 (ACE-2) inhibitor.

The term "pharmaceutically-acceptable carrier" refers to any material that can or is capable of carrying the ACE-2 inhibitor to the intended area of its (the inhibitor's) function. The pharmaceutically-acceptable carrier must be compatible with the ACE-2 inhibitor and not of any danger to an intended recipient of the pharmaceutical composition. One non- limiting example of a material that can be used as a pharmaceutically-acceptable carrier is polyethylene glycol.

The term "effective amount" refers to the amount of ACE-2 inhibitor necessary to achieve its (the inhibitor's) intended function.

An ACE-2 inhibitor that can be used in a pharmaceutical composition is a compound comprising the formula:

Another aspect of the invention provides a method for modulating activity of an angiotensin-converting enzyme-2 (ACE-2) protein comprising contacting said protein with angiotensin-converting enzyme-2 (ACE-2) inhibitor.

As used herein, the term "modulating" refers to any alteration of the activity of angiotensin-converting enzyme-2 (ACE-2) protein or to any alteration in the expression of the ACE-2 protein.

An ACE-2 inhibitor that can be used to modulate the activity of ACE-2 protein is a compound comprising the formula:

Yet another aspect of the invention involves a method of treating an angiotensin- converting enzyme-2 (ACE-2) associated condition in a subject in need thereof comprising administering a therapeutically-effective amount of an ACE-2 inhibitor to the subject such that the ACE-2 associated condition is treated.

The term "angiotensin-converting enzyme-2 (ACE-2) associated condition" refers to any condition associated with ACE-2, associated with substrates of ACE-2, and/or associated with any metabolic pathway of ACE-2. A non-limiting example of an "angiotensin- converting enzyme-2 (ACE-2) associated condition" is severe acute respiratory syndrome (SARS).

The term "therapeutically-effective amount" refers to the amount of ACE-2 inhibitor required to achieve the desired function, i.e. treatment of the ACE-2 associated condition.

An ACE-2 inhibitor that can be used to treat an ACE-2 associated condition is a compound comprising the formula:

The term "subject" as used herein refers to any human or animal that can or may benefit from the ACE-2 inhibitors of the invention and/or from the methods of using the ACE-2 inhibitors.

Other objectives and advantages of this invention will become apparent from the following description taken in conjunction with the accompanying drawings, wherein are set forth, by way of illustration and example, certain embodiments of this invention. The drawings constitute a part of this specification and include exemplary embodiments of the present invention and illustrate various objects and features thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete understanding of the present invention may be obtained by references to the accompanying drawings when considered in conjunction with the subsequent detailed description. The embodiments illustrated in the drawings are intended only to exemplify the invention and should not be construed as limiting the invention to the illustrated embodiments.

FIG. 1 shows the structural formula for MLN-4760 (Millennium Pharmaceuticals, Inc.; U.S. Patent 6,632,830 Bl). For the purpose of clarity, this compound is referred to as "Formula I" in the instant specification.

FIG. 2 shows the structural framework of an ACE-2 inhibitor (Dales et al. J Am Chem Soc 124: 1 1852-1 1853 2002). For the purpose of clarity, this compound is referred to as "Formula Π" in the instant specification.

FIG. 3 shows the structural formula for the radioiodinatable MLN-4760 analog of the invention. For the purpose of clarity, this compound is referred to as "Formula III" in the instant specification.

FIG. 4 shows a chromatogram (HPLC) of the purification of ¹²⁵I-351A from un- iodinated 351A.

FIG. 5 shows the steps for synthesis of radioiodinatable MLN-4760 analogs; JFS 101 (R conformation) and JFS102 (S conformation).

FIG. 6 shows a chromatogram (HPLC) of the purification of ¹²⁵I-JFS101 from un- iodinated JFS 101.

FIG. 7 shows a chromatogram (HPLC) of the purification of ¹²⁵I-JFS102 from un- iodinated JFS 102. FIGS. 8A-E show results of Matrix-Assisted Laser Desorption/Ionization (MALDI) mass spectrometry (MS) imaging in the murine kidney: FIG. 8A shows a murine kidney section; FIG. 8B shows a murine kidney section incubated with Ang II; FIG. C shows the murine kidney section incubated with Ang II resulting in detection of Ang (1-7) and Ang III (FIG. 8D); and FIG. 8E shows distinct regional distribution patterns as demonstrated by the overlay of MS signals for both peptide products (Ang (1-7) and Ang III). This data is from N. Grobe et al. of Wright State University.

FIGS. 9A-D show receptor autoradiography with ¹²⁵I-SarI-Ile8-Ang II: FIG. 9A shows non-specific binding; FIG. 9B shows angiotension I receptor (AT-1 receptor) binding; FIG. 9C shows angiotensin II receptor (AT -2 receptor) binding in the presence of losartan; and FIG. 9D shows a thionin stain of a section adjacent to the sections shown in FIGS. 9A-C.

FIGS. 10A-D show the structural formulas for alkyl chain linkers/spacers: FIG. 10A shows a linker/spacer chain with one methyl group; FIG. 10B shows a linker/spacer chain with two methyl groups; FIG. IOC shows a linker/spacer chain with three methyl groups; and FIG. 10D shows a linker/spacer chain with four methyl groups.

DETAILED DESCRIPTION OF THE INVENTION

For the purpose of promoting an understanding of the principles of the invention, reference will now be made to embodiments illustrated herein and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modification in the described compositions and methods and any further application of the principles of the invention as described herein, are contemplated as would normally occur to one skilled in the art to which the invention relates.

The experiments described herein were designed and carried out (or will be carried out) according to a pre-determined experimental plan as described below.

EXPERIMENTAL PLAN

The hypothesis (to be tested by carrying out the following experiments) is that a derivative of MLN-4760 which has a phenolic side chain (Formula III) would retain the high affinity and specificity of MLN-4760 for ACE-2 with and without an iodine- 125 on this side chain; that this derivative can be used to measure functional ACE-2 protein; that this derivative can be used to determine ACE-2 protein distribution in tissues using in vitro autoradiography with quantitative densitometric analysis, and that this derivative can be used to measure experimentally-induced changes in ACE-2 expression. In addition, it is also hypothesized that the compound developed through carrying out the described experiments can be used to study the physiological importance of ACE-2. First Specific Aim to Test the Hypothesis

To synthesize 1-3 analogs of MLN-4760 (FIG. 1 ; Formula I) with a modified R2 side chain in which the isopropyl (leucine-like) moiety is replaced with a phenol (tyrosine-like) moiety (FIG. 3; Formula III). An alkyl linker/spacer chain of 1 to 4 methyl groups (FIGS. 10A-D) will be used to extend the phenolic moiety away from the other parts of the molecule if there is steric hindrance to the binding of the ACE-2 inhibitor by the phenolic ring and its iodine- 125 tag. The analogs will be assayed for their ability to inhibit ACE-2 activity using both an in situ mass spectrometry method using mouse kidney slices as well as in vitro in membrane preparations derived from mouse kidneys and brains. Approach

Specific Aim 1: To synthesize analogs of MLN-4760 and test them for inhibition of ACE-2 activity.

Synthesis of MLN-4760 analogs: The first inhibitor analog, shown in FIG. 3 (Formula III), will be synthesized and an effort will be made to resolve all 4 stereoisomers from a small sample batch. However, if the 4 stereoisomers cannot be resolved initially, the racemic mixture of the product will be tested to determine its IC5₀ with the knowledge that the affinity of the most active stereoisomer could be underestimated by as much as 4 times. The synthesis will be scaled up to provide sufficient material to allow for resolution of multi-milligram quantities of each stereoisomer. The starting material used a histidine with the S

conformation, so there are two enantiomers.

Fluorometric assay of ACE-2: The catalytic activity of ACE-2 will be determined in kidney homogenates using an established assay (Vickers et al. J Biol Chem 277: 14838-14843 2002; Elased et al. Exp Physiol 93 :665-675 2008). Briefly, tissues are homogenized in ice- cold 50 mM Tris HC1 buffer, pH 7.4 (50mg/ml) with 150 mM NaCl, 5 mM ZnCL₂, 2 mM PMSF and 1 μΜ captopril (to inhibit ACE activity). The homogenate is centrifuged at 9,000 x g for 10 minutes and the supernatant is decanted. 10 μΐ of this supernatant is pipette into the wells of a 96-well microtiter plate. Varying concentrations of the radioiodinatable MLN- 4760 analog dissolved in the Tris HC1 buffer: 1, 10, 100, 1000 nM, 10, 100 μΜ (final assay concentration) will be added to the wells in triplicate. Other wells will receive 10 μΐ of the Tris HC1 buffer to define 100% activity or 10 μΜ DX 600 (Phoenix Pharmaceuticals) to define 100% inhibition of ACE-2 activity. In a darkened room, 80 μΐ of a 50 μΜ

concentration of the fluorogenic substrate 7-Mca-APK-[DNP] (P-163, Biomol International) is added to each well to give a final concentration of 40 μΜ to initiate the hydrolysis reaction. The microtiter plate is placed in a plate reader to monitor fluorescence emission at 405 nm in response to excitation at 335 nm (time zero). Subsequent readings are taken at 1, 2, 3, 4 and 20 hours to determine an optimal assessment of the amount of inhibition of substrate metabolism. The % inhibition of ACE 2 activity will be calculated as (1 - emission at time t at inhibitor concentration X - emission at time t with DX-600) minus emission at time zero) divided by (emission at time t no inhibitor - emission at time t with DX-600) - emission at time 0)) * 100. The IC5₀ will be determined from the % inhibition at each concentration of the radioiodinatable MLN-4760 analog using a one-site inhibition model equation IC50 = the concentration of the radioiodinatable MLN-4760 analogs at which 1/(1 + IC5₀) = ½ (Prism, Graphpad Software).

To assess the specificity of the inhibition of ACE-2 by the radioiodinatable MLN-

4760 analogs and their ability to inhibit ACE and carboxypeptidase A (CPA), published assay procedures for ACE (Elased et al. Hypertension 46:953-959 2005), and CPA (Dales et al. J Am Chem Soc 124: 1 1852-1 1853 2002) will be used.

Matrix assisted laser desorption/ionization (MALDI) imaging technique for ACE-2 activity: Fresh frozen kidneys will be cut at 12 μιη thickness on a cryostat at -20° C. Kidney sections will be mounted on indium-tin-oxide coated glass slides, dried in a desiccator and incubated with peptide substrate at 37°C. MALDI matrix consisting of 10 mg/ml a-cyano-4- hydroxy-cinnamic acid (CHCA) in 60% methanol, 10% acetone and 0.3% trifluoroacetic acid will be spray coated onto the tissue using a thin layer chromatography nebulizer with nitrogen gas at 10 psi. The CHCA solution will be repetitively sprayed across the sections from a distance of 20 cm allowing 30 seconds of drying time between three passes until a uniform matrix coating is achieved. The images will be acquired using a Bruker Autoflex III MALDI TOF/TOF (matrix assisted laser desorption/ionization time of flight/time of flight) instrument. The mass spectrometer will be operated with positive polarity in reflectron mode and spectra will be acquired in the range of m/z 500-3000. A total of 200 spectra will be acquired at each spot position at a laser frequency of 100 Hz. The spectral analysis will be performed with proprietary Bruker Flex Analysis and Imaging software. Unknown ion peaks will be fragmented using the Bruker Lift method and identified upon comparison to standard compounds.

Second Specific Aim to Test the Hypothesis

To radioiodinate the ACE-2 inhibitor analogs with iodine- 125 using the chloramine T procedure and purify the mono-radioiodinated analogs from the unradioiodinated analogs and the di-radioiodinated analogs by reverse-phase HPLC. These radioligands will be used to develop a radioligand binding assay for the mono¹²⁵I-MLN-4760 analogs in rat and mouse brain and kidney homogenates and to develop a method for autoradiographic localization of ACE-2 in the brain and kidney as well.

Approach

Specific Aim 2: To radioiodinate and purify the mono-radioiodinated ACE-2 inhibitor analogs, and use them to develop a radioligand binding assay for ACE-2 in rat and mouse brain and kidney homogenates and develop a method for autoradiographic localization of ACE-2 in the brain and kidney.

The inhibitor analogs will be radioiodinated using the chloramine T procedure (Hunter et al. Nature 194:495-496 1962) which is routinely used to prepare radioligands for research (Speth et al. Radiolabeling of angiotensin peptides. In: Wang DH, ed. Angiotensin Protocols. Totowa NJ: Humana Press; 275-295 2001). Radiolabeling is done at American Radiolabeled Chemicals in St. Louis. A mobile phase made up of triethylamine phosphate, pH 3:acetonitrile in a ratio suitable to isocratically elute non-radioiodinated compound at ~ 4 minutes, monoradioiodinated compound at ~ 8 minutes and di-radio-iodinated compound at ~ 12 minutes. The monoradioiodinated MLN-4760 analogs will be diluted to a concentration of <200 nM with water and stored frozen in the presence of 2 mg/ml bovine albumin.

Anticipated results: Resolve the 2 R² diastereomers, radioiodinate each one individually, and resolve each monoradioiodinated isomer from its un-radioiodinated and di- radioiodinated isomers. Radioligand binding assays

Saturation Isotherms: The monoradioiodinated MLN-4760 analogs will be used for radioligand binding assays using rat brain and kidney homogenates similar to previous studies of ¹²⁵I-351A binding (Walters et al. Brain Res 507:23-27 1990; Speth et al. Biol Reprod 38:695-702 1988; Speth et al. Proc West Pharmacol Soc 31 : 185-188 1988). The use of these tissues is based upon reports of high levels of ACE-2 expression (Doobay et al. Am J Physiol Regul Integr Comp Physiol 292:R373-R381 2007; Elased et al. Exp Physiol 93:665- 675 2008) and functional significance of ACE-2 in these tissues (23 Feng et al. Circ Res 106:373-382 2010; Diz et al. Exp Physiol 93 :694-700 2008). Briefly, whole rat kidneys or rat forebrains will be homogenized with a mechanical homogenizer in 25 volumes of a hypotonic (20 mM) sodium phosphate buffer, pH 7.2 to lyse the cells. The homogenate is centrifuged for 20 minutes at 48,000 x g at 4° C. The supernatant is decanted and the pellet resuspended in 19 volumes of a 50 mM Tris HC1 buffer (pH 7.4 at 22 C) containing 100 mM NaCl, 5 mM ZnCl₂, 2 mM PMSF.

40 μΐ of ¹²⁵I-MLN-4760 analog is added to 12 x 75 mm culture tubes at 6 different concentrations (final assay volume) ranging from 5 nM to 0.25 nM. 10 μΐ of DX600 (to achieve a final concentration of 10 μΜ to saturate ACE-2 binding sites) or Tris HC1 buffer will be added to alternate tubes to define nonspecific and total binding, respectively. Specific binding to ACE-2 will be defined as total minus nonspecific binding. 50 μΐ of tissue homogenate is added to initiate the binding assay. After one-hour incubation at 22 °C, the samples are filtered over GF/B fiberglass filters using a Brandel M-24R cell harvester to collect the particulate. The filter disks are counted in a gamma counter. The counts for each tube are put into an Excel spreadsheet to calculate specific binding. The data for specific binding as well as the concentration of free ¹²⁵I-MLN-4760 analog at equilibrium is entered into Prism (Graphpad Software) for determination of the dissociation constant for specific binding (K_D) and the concentration of binding sites (B_max) using a one site saturation isotherm model.

Total and non-specific binding values can also be entered into Prism to show the ratio of total to nonspecific binding and to assess the linearity of the non-specific binding. If the best fit line calculated for ¾ and B_max shows a deviation from the data points for specific binding, it will be determined if it is possible that a two-site binding model B = B_max_i * S/(K_D-i = S) + B_max_₂ * S/(K_D-₂ = S) might give a better fit to the data, the binding assay will be repeated using 12 concentrations of ¹²⁵I-MLN-4760 analog ranging from 10 nM to 0.1 nM and re-evaluate specific binding comparing a one- versus two-site model using Prism to determine which fit is best.

If the initial radioligand binding assays are not successful in showing an abundance of high affinity binding sites for the ¹²⁵I-MLN-4760 analog, the binding assay conditions will be modified, e.g., adjust pH of the assay buffer, decrease the amounts of PMSF and ZnCl2 (to zero if needed) add small (1-5 mM) amounts of additional elements, e.g., KC1, MgCi₂, CaCi₂, switch to a HEPES, MOPS, or sodium phosphate buffer, extend the incubation time, and change assay temperature. Once the binding has been optimized, the time course of association and the dissociation rate for the binding will be determined to assess a kinetic ¾ from the association and dissociation rate constants (¾ = K_i/K₊i) using well characterized procedures (Yamamura et al. Neurotransmitter Receptor Binding. New York: Raven Press 1977; Speth et al. Brain Res 131 :350-355 1977; Speth et al. Life Sci 22:859-866 1978).

Anticipated results: That these assays will show a similar high affinity binding ¾ ~ 1 nM with a high density of ACE-2 binding sites in rat and mouse brain and kidney with low non-specific binding. They will also reveal which of the 4 stereoisomers has the highest affinity for ACE-2.

Competition binding assays: To assess the specificity of the binding of the ¹²⁵I-MLN- 4760 analogs to ACE-2, competition binding assays will use the ACE-2 inhibitor DX-600, the non-radiolabeled MLN-4760 analogs, captopril (ACE inhibitor), z-pro-prolinal (PRCP inhibitor), potato carboxypeptidase A inhibitor, and glutamate phosphonate (an

aminopeptidase A inhibitor). A sample of a novel phosphinic pseudopeptide inhibitor of ACE-2, Ac-His-ΡΓθΨ (PO₂-CH₂) 2-(3-phenyl-isoxazol-5-ylmethyl)-propionic acid-OH, developed by Dive and colleagues (Mores et al. J Med Chem 51 :2216-2226 2008) to use to further establish the specificity of the ¹²⁵I-MLN-4760 analogs binding has been requested. Varying concentrations of these inhibitors ranging from 1 nM to 100 μΜ (at one log intervals) will be added to the assay system described above with 1 nM of each ¹²⁵I-MLN- 4760 analog. The ability of these enzyme inhibitors to compete for each ¹²⁵I-MLN-4760 analog binding will be assessed using the Prism one-site competition model. In addition, primary screens with a number of other enzyme inhibitors, e.g., amastatin, bestatin, leupeptin, thermolysin, phosphoramidon thiorphan, at 100 μΜ; PMSF, PCMB, o-phenanthroline,

EDTA, at 1 mM, will be performed to assess the ability of these inhibitors to interfere with ¹²⁵I-MLN-4760 analogs binding. The ACE-2 enhancer XNT will be used to determine if this compound, which increases the activity of ACE-2 (Hernandez et al. Hypertension 51 : 1312- 1317 2008; Fraga-Silva et al. Mol Med 16:210-215 2010) will modify the ability of ¹²⁵I- MLN-4760 analogs to bind to ACE-2.

Anticipated results: Only DX-600, Ac-His-Pro (P0₂-CH₂) 2-(3-phenyl-isoxazol-5- ylmethyl)-propionic acid-OH, and the non-radiolabeled MLN-4760 analogs will show micromolar or nanomolar IC5₀ values to compete with each ¹²⁵I-MLN-4760 analog binding in the brain and kidney. This will indicate that this radioligand is not binding to ACE, prolylcarboxypeptidase, carboxypeptidase A or aminopeptidase A. XNT is expected to act allosterically to enhance or decrease the binding affinity of the ¹²⁵I-MLN-4760 analogs for ACE-2.

Autoradiography: The autoradiography of rat and mouse brain and kidney ACE-2 will follow general procedures known in the art (Speth et al. Biol Reprod 38:695-702 1988;

Bourassa et al. Brain Res 1319:83-91 2010; Speth et al. Brain Res 326: 137-143 1985;

Daubert et al. Brain Res 816:8-16 1999). Basically, the brains and kidneys of rats are frozen and stored at -80 °C. They are subsequently sectioned in a cryostat at a thickness of 20 microns and thaw-mounted onto charged microscope slides (Premium Grade, Gorilla Scientific). The slides are stored frozen at -80 °C until the day of the assay. On the day of the assay the sections are thawed and preincubated in 35 ml of assay buffer (without radioligand) in a Coplin jar for 30 minutes to wash away endogenous bound substrate for ACE-2. The sections are then incubated in assay buffer with 500 pM ¹²⁵I-MLN-4760 analog with and without 10 μΜ DX-600 (Phoenix Pharmaceuticals) to saturate ACE-2, for lhour at 20-22 °C. The slides are then quickly dipped in two changes of distilled water and rinsed 4 x 10 seconds in assay buffer without radioligand. The sections are again quickly dipped in two changes of distilled water to remove buffer salts and dried under a stream of cool air for 4 minutes. The slides are then mounted onto cardboard with a standards slide containing known amounts of iodine- 125 (ARI 0133, American Radiolabeled Chemicals) that is used to construct a standard curve for quantitation of radioligand binding to the sections. The cardboard mounted slides are placed in an X-ray cassette apposed to single sided autoradiography film (MR-1, Kodak) for an appropriate exposure period, generally 3 days. The films are then developed in an automated film processor and the images are captured on an image analysis system (MCID, Interfocus Ltd.) for quantitative densitometric analysis. 3-4 brains and kidneys will be analyzed densitometrically. The tissues will be serially sectioned in repeating sets of 3-5 slides to allow for 2 slides/set to be used for autoradiography of ACE-2, one for histology (thionin for brain and hematoxylin and eosin for kidney) and 2 additional slides for another molecule, e.g., ACE (using ¹²⁵I-351A) or ATi receptors (using ¹²⁵I-Sar¹,Ile⁸ Ang II) as described previously (Krebs et al. J Pharmacol Exp Ther 293 :260-267 2000; Bourassa et al. Brain Res 1319:83-91 2010), to allow for comparison of the distribution of ACE-2 with other components of the RAS.

To determine if autoradiography can be paired with procedures requiring in situ fixation of tissues, deeply anesthetized rats will be sacrificed by exsanguination via cardiac perfusion with 0.9% saline followed by perfusion with 4% paraformaldehyde. The brains and kidneys will be postfixed in paraformaldehyde and then cryoprotected in a buffered 20% sucrose solution. The brains are then frozen and used for autoradiography as described above.

Anticipated Results: Based on immunohistochemical (Doobay et al. Am J Physiol Regul Integr Comp Physiol 292:R373-R381 2007; Lin et al. Exp Physiol 93:676-684 2008), PCR (Doobay et al. Am J Physiol Regul Integr Comp Physiol 292:R373-R381 2007; 89 Lin et al. Exp Physiol 93:676-684 2008), and mass spectrometric (Elased et al. Exp Physiol 93:665- 675 2008) studies high ACE-2 binding in the cerebral cortex, hypothalamus and brainstem of the rat and mouse brains, and in the inner cortex of the kidney is expected. A wide range and variation in expression of ACE-2 in these tissues is anticipated and discrete hot spots of ACE-2 expression, e.g., solitary tract nucleus (NTS), and proximal tubules based upon functional studies (Shaltout et al. Am J Physiol Renal Physiol 292:F82-F91 2007; Diz et al. Exp Physiol 93 :694-700 2008) and low overall expression of ACE-2-ir (ACE-2

immunoreactivity) and activity in larger blocks of brain tissue (Feng et al. Circ Res 106:373- 382 2010) is expected. It is not known whether fixation will compromise the binding site of ACE-2 for the radioligand. If 4% paraformaldeyde proves to be too severe to preserve binding lower concentrations of paraformaldehyde to allow for preservation of ACE-2 binding of the radioligand and immunological markers for colocalization studies will be tried.

Third Specific Aim to Test the Hypothesis

To use the methodology developed in Specific Aim 2 to measure the concentration

(Bmax) of ACE-2 molecules in the rat and mouse brain and kidney, specific regions of these tissues, and the localization of ACE-2 to specific brain nuclei and components of the kidney. The degree of over- and under-expression of ACE-2 will be assessed in the following series of experiments:

To determine the expression of ACE-2 in the brains of rats transfected with a viral vector that contains the ACE-2 gene;

To determine the expression of ACE-2 in the brains of rats transfected with a viral vector that contains a shRNA to inhibit translation of ACE-2 mRNA;

To determine the expression of ACE-2 in the brains of transgenic mice (syn-hACE-2) that selectively over-express ACE-2 in neurons; and

To assess the effect of experimental ischemic stroke, with and without angiotensin receptor blocker pretreatment on expression of ACE-2 in the rat brain. Approach

Specific Aim 3: To use the methodology developed in Specific Aim 2 to characterize the distribution and concentrations of ACE-2 in rat/mouse brain and kidney, subjected to experimental treatments aimed at altering ACE-2 expression or pathological conditions which may affect ACE-2 expression.

Viral vector- induced over-expression of ACE-2 in rat brain:

Rationale: Yamazato et al, (Hypertension 49:926-931 2007) showed that administration of a lentivirus containing the ACE-2 gene into the rostral ventrolateral medulla (RVLM) reduced blood pressure in spontaneously hypertensive rats (SHR). To measure an increase in arbitrary ACE-2-immunoreactivity (ACE-2-ir) it was necessary to micropunch the RVLM from 32 rats per group, pool 4 punches per measurement, and analyze them by Western blot analysis with an ACE-2 antibody. Using autoradiography it should be possible to use only one brain per sample, determine the neuroanatomical localization of ACE-2 expression throughout the brainstem and to obtain an absolute value for ACE-2 expression in the RVLM as well as other parts of the brainstem.

Methodology: To use ¹²⁵I-MLN-4760 analogs to assess the extent of over-expression of ACE-2 in the rat brain the effect of viral transfection of ACE-2 in the rat brain will be studied. Briefly, SHR (spontaneously hypertensive rats) and WKY (Wistar Kyoto rats) rats will have lentiviral expression-vector particles containing enhanced green fluorescent protein with and without ACE-2 genes, injected into the RVLM (Yamazato et al. Hypertension

49:926-931 2007). Two-weeks later rats will be sacrificed and evaluated for ACE-2 binding in the brainstem using the autoradiography procedures described above.

Experimental design: Two-way ANOVA (strain: SHR vs WKY) and treatment (EGFP only vs EGFP plus ACE-2), n = 6. Statistical analyses will be carried out using Prism (version 6, Graphpad Software).

Anticipated results: Significant main effects for both strain and treatment are expected to show that WKY rats have greater amounts of ACE-2 in the RVLM and ACE-2 lentiviral expression-vector particle treatment will increase ACE-2 expression in the RVLM of both strains. It is possible that there may be a significant interaction which will show greater enhancement of ACE-2 binding by the ACE-2-containing lentiviral particles in the SHR. Concurrent analysis of ACE-2 expression in the solitary tract nucleus is also expected to show higher expression in the WKY but no change in expression due to different treatments and no strain x treatment interaction. Viral vector-induced inhibition of expression of ACE-2 in rat brain:

Rationale: The SHR has reduced ACE-2-ir expression in the RVLM (Yamazato et al. Hypertension 49:926-931 2007). Administration of lentiviral particles containing ACE-2 into the RVLM reduces blood pressure in the SHR concomitant with increased ACE-2 expression in the RVLM (Yamazato et al Hypertension 49:926-931 2007). Therefore, it is anticipated that inhibition of ACE-2 expression in the RVLM should elevate blood pressure in both SHR and WKY rats.

Methodology: To use ¹²⁵I-MLN-4760 analogs to assess the extent of decreased expression of ACE-2 in the rat brain the effect of viral transfection of an ACE-2 shRNA in the rat brain that should inhibit translation of ACE-2 mRNA will be studied. Briefly, SHR and WKY rats will have lentiviral particles containing enhanced green fluorescent protein with and without ACE-2 shRNA, injected into the RVLM using procedures described previously (Yamazato et al. Hypertension 49:926-931 2007) Two-weeks later rats will be sacrificed and evaluated for ACE-2 binding in the brainstem using the autoradiography procedures described above.

Experimental design: Two-way ANOVA (strain: SHR vs WKY) and treatment (EGFP only vs EGFP plus ACE-2 shRNA), n = 6. Statistical analyses will be carried out using Prism (version 6, Graphpad Software).

Anticipated results: Significant main effects for both strain and treatment are expected to show that WKY rats have greater amounts of ACE-2 in the RVLM and ACE-2 shRNA lentiviral particle treatment will decrease ACE-2 expression in the RVLM of both strains. It is possible that there may be a significant interaction which will show a greater reduction of ACE-2 binding by the ACE-2 shRNA-containing lentiviral particles in the WKY rats.

Concurrent analysis of ACE-2 expression in the solitary tract nucleus is also expected to show higher expression in the WKY but no change in expression due to different treatments and no strain x treatment interaction.

Transgenic mice bred to selectively overexpress ACE-2 in neurons:

Rationale: Transgenic mice (syn-ACE-2) in which the ACE-2 gene is coupled to the neuron-specific synapsin promoter, selectively overexpress ACE-2 in neurons (Feng et al. Ore Res 106:373-382 2010; Xia et al. JNeurochem 107: 1482-1494 2008). These mice are resistant to chronic systemic Ang II infusion-induced hypertension (Feng et al. Circ Res 106:373-382 2010). The protective effect is partially reversible by an antagonist of the mas receptor for Ang 1-7 suggesting that increased ACE-2 mediated formation of Ang 1-7 from Ang II is occurring in the brains of these mice. The extent to which ACE-2 is overexpressed throughout the brain is still undetermined.

Methodology: To use ¹²⁵I-MLN-4760 analogs to assess the extent of over-expression of ACE-2 in syn-ACE-2 mouse brains compared to wild-type control mouse brains. For these experiments both homogenate and autoradiography procedures described above will be used. For initial homogenate binding studies whole mouse brains will be used and evaluated for K_D and B_MAX. For follow-up studies, mouse brains will be dissected into cortex/hippocampus, caudate/putamen, hypothalamus, thalamus/midbrain, brainstem and cerebellum. These studies will use a single ~ K_D concentration of radioligand. For autoradiography studies the entire brain will be sectioned and analyzed for ¹²⁵I-MLN-4760 analog binding.

Experimental design: Student's unpaired t test will be used to compare wild-type and syn-ACE-2 mouse brains for KD and B_MAX values. Results will be expressed as mean ± SEM. Significance level will be p < 0.05, n = 6. Statistical analyses will be carried out using Prism (version 6, Graphpad Software).

Anticipated results: There should be a significant global increase in ¹²⁵I-MLN-4760 binding (B_MAX) in the syn-ACE-2 brains compared to the wild-type mouse brains. No change in KD between groups is anticipated. The anticipated increase in ¹²⁵I-MLN-4760 binding (B_MAX) in the syn-ACE-2 brains is expected to be significant in all 6 brain regions. If a difference in K_D is observed between syn-ACE-2 brains and wild-type brains, then comparison of binding in brain regions between groups will require saturation binding assays to determine KD and B_MAX in each brain region for comparisons. Again, it is expected that there will be a significant increase in B_MAX in all 6 brain regions in the syn-ACE-2 brains with no difference in KD between brain regions, but with there still being a difference in KD between the two groups in each brain region.

Effect of experimental ischemic stroke on ACE-2 expression in rat brain:

Rationale: The previous studies indicate that Ang 1-7 can reduce the amount of neuronal damage associated with an experimental ischemic stroke in rat brains (Regenhardt et al, 201 1 Experimental Biology Meeting Abstract 650.10). Additionally, it has been shown that the blockade of brain ATi receptors also reduces the amount of neuronal damage associated with an experimental ischemic stroke in rat brains (Mecca et al. Exp Physiol 94:937-946 2009). Since AT-1 receptor antagonism is associated with increases in ACE-2 activity (Igase et al. Am J Physiol Heart Circ Physiol 289:H1013-H1019 2005) and increased brain angiotensinergic activity is associated with reduced ACE-2 expression (Xia et al. Hypertension 53:210-216 2009). This may indicate that ACE-2 expression is lost in ischemic stroke damaged neurons, but can be rescued, along with Ang 1-7 receptive neurons with ATi receptor blockade.

Methodology: Rats will be subjected to endothelin- induced ischemia to simulate a middle cerebral artery occlusion with and without prior treatment with candesartan as described previously (Mecca et al. Exp Physiol 94:937-946 2009). 24 hours later rats are sacrificed with isoflurane overdose and the brains removed and frozen for autoradiography for ACE-2 binding as described above. The portions of forebrain 2 mm caudal and rostral to the injection site will be coronally sectioned to assess alterations in ACE-2 binding in the hemispheres ipsilateral and contralateral to the site of endothelin injection. A collateral experiment will be done to determine if it is possible to do TTC staining on brain slices to assess the infarct size prior to using the brain slice for autoradiographic analysis of ACE-2 binding. Three control 1 mm thick slices of fresh normal rat brain will be cut. One slice will be subjected to TTC staining as described previously (Mecca et al. Exp Physiol 94:937-946 2009) except that the slice will be digitally imaged without fixation. Another slice will be incubated in the same medium minus the TTC and digitally imaged, while the third slice will immediately be digitally imaged and frozen following dissection. After TTC or sham incubation and digital imaging the first two slices will be frozen similar to the third section. The slices are then mounted on a cryostat plate for sectioning at 20 micron for

autoradiography as described above.

Experimental design: For the experimental stroke study, a repeated measures two-way ANOVA (side: ipsi- vs contralateral is matched) and pretreatment (vehicle or candesartan) is used. Results will be expressed as mean ± SEM. Significance level will be p < 0.05, n = 6. For the TTC study a one-way ANOVA design will be used with Bonferroni comparisons of the 3 different groups. Results will be expressed as mean ± SEM. Significance level will be p < 0.05, n = 6. Statistical analyses will be carried out using Prism software.

Anticipated results: A main effect for hemisphere, with the lesion side showing less ¹²⁵I- MLN-4760 binding on the side ipsilateral to the endothelin injection compared to the contralateral side may occur if the lesion causes a large reduction in binding. A main effect for treatment is also expected with an increase in ¹²⁵I-MLN-4760 binding in the candesartan pretreated brain. A significant interaction effect is predicted with the candesartan pretreatment causing a substantial increase in ¹²⁵I-MLN-4760 binding on the ipsilateral side compared to that of the vehicle control brains. It is predicted that there will be a small reduction in ¹²⁵I-MLN-4760 binding in the TTC and control incubated sections compared to the quickly frozen brain slices. If the reduction is less than 20%, future studies will evaluate infarct size with TTC prior to autoradiography to enable a direct comparison of the ¹²⁵I- MLN-4760 binding in the infarcted area with that in the penumbra and in the undamaged brain regions.

Future Experiments: Experiments for follow-up studies include determination of the effect of DOCA-salt treatment of uninephrectomized mice on brain ACE-2 expression in wild-type and neuron-specific ACE-2 overexpressing mice, in mice with conditional and tissue-specific ACE-2 deletions, in mice with various components of the RAS deleted, e.g. AT i receptor and ACE knockouts, and in db/db diabetic mice transfected with a viral vector containing the ACE-2 gene or a control vector using ¹²⁵I-MLN-4760 analog. Other studies include determining the bioavailability of this inhibitor to assess its utility for in vivo studies of the physiological importance of ACE-2.

EXPERIMENTAL EXAMPLES

Radioligand binding assays for hormones have been in use for 40 years (Cuatrecasas,

P. Proc Natl Acad Sci USA 68: 1264-1268 1971), while receptor autoradiography of hormone receptors dates back more than 50 years (Stumpf, W. E. Journal Histochem Cytochem 18:21- 29 1970). The best example of the use of a high affinity enzyme inhibitor to radiolabel an enzyme is 351A, a radioiodinatable analog of the widely used ACE inhibitor lisinopril, first used to measure serum ACE (Fyhrquist et al. Clin Chem 30:696-700 1984). As noted by these authors (Fyhrquist et al), "the advantages of this new principle include simplicity, specificity, lack of interference from other enzymes or immunologically by similar substances, obviation of simple blanks, and adaptability to automated analytical systems." In addition to measurement of serum ACE, other investigators have adapted this method to assess ACE in a variety of tissues including brain, (Walters et al. Brain Res 507:23-27 1990), kidney, lung, and testis (Perich et al. Mol Pharmacol 42:286-293 1992). The ¹²⁵I-351A has also been used for autoradiographic localization of ACE in brain, vasculature, kidney, prostate, and ovary (Mendelsohn et al. J Hypertension 2(suppl 3):41-44 1984; Rogerson et al. J Chem Neuroanat 8:227-243 1995; Correa et al. Braz J Med Biol Res 25:515-519 1992; Zhuo et al. J Cardiovasc Pharmacol 29:297-310 1997; Nassis et al. J Pathology 195:571-579 2001 ; Speth et al. Biol Reprod 38:695-702 1988; Krebs et al. J Pharmacol Exp Ther 293:260- 267 2000; Bourassa et al. Brain Res 1319:83-91 2010).

Because of the high homology (42% with the testis ACE variant; AF 118569_2, per NCBI BLAST) between ACE and ACE-2, Millennium Pharmaceuticals, Inc. began development of ACE-2 inhibitors as potential therapeutic agents for diseases attributable to ACE-2 (Dales et al. J Am Chem Soc 124: 11852-11853 2002). Millennium Pharmaceuticals, Inc. developed a large number of inhibitors for use as cardiovascular therapeutics; U.S. Patent 6,632,830 Bl . When it became apparent that ACE-2 did not cause adverse effects on the cardiovascular system, Millennium Pharmaceuticals, Inc. focused on these inhibitors as possible weight-loss agents; U.S. Patent 7,045,532 B2. Now that it is known that ACE-2 has beneficial effects on the cardiovascular system clinical interest in ACE-2 inhibitors has waned.

One of the compounds (Millennium Pharmaceuticals, Inc.; U.S. Patent 6,632,830 B l), MLN-4760 (Formula I), has extremely high affinity (IC₅₀ = 440 picomolar) for ACE-2, yet it causes less than 50% inhibition of ACE at 100 μΜ concentration and its IC₅₀ for

carboxypeptidase A is 27 μΜ (Dales et al. J Am Chem Soc 124: 11852-1 1853 2002). The chemical structural formula for MLN-4760 is shown in FIG. 1 (Formula I).

Evaluation of a series of compounds with different substituents on the basic framework of the inhibitor suggests that the R² moiety is not critical to the inhibitory activity of MLN-4760. The structural framework of the ACE-2 inhibitor is shown in FIG. 2 (Dales et al. J Am Chem Soc 124: 1 1852- 11853 2002). Substitution of a phenyl moiety for a methyl moiety at R² had a small deleterious effect on affinity of the R¹ phenyl-substituted compound (Dales et al. J Am Chem Soc 124: 11852-1 1853 2002).

Therefore, in order to develop an ACE-2 inhibitor which can be radioiodinated, the instant inventor developed an analog of MLN-4760 that has a phenol substituent in the R² position. The chemical structural formula for the radioiodinatable MLN-4760 analog is shown in FIG.3 (Formula III).

Example 1 : Enzyme Autoradiography of ACE: Proof of Concept. Previous studies have validated the concept of using a potent enzyme inhibitor to radio label and measure an enzyme. 351 A has been radioiodinated using the chloramine T procedure, purified by high performance liquid chromatography (HPLC), and used to measure the concentration of ACE in rat brains subjected to putative cholinergic neurotoxin AF-64 (Walters et al. Brain Res 507:23-27 1990). The ¹²⁵I-351A has also been used to demonstrate the presence and localization of ACE in the rat ovary and brain by receptor autoradiography (Speth et al. Biol Reprod 38:695-702 1988; Bourassa et al. Brain Res 1319:83-91 2010). A chromatogram (HPLC) of purification of ¹²⁵I-351A from uniodinated 351A is shown in FIG. 4. Line R indicates ¹²⁵Iodine. Line B is UV absorbance at 210 nm. 351A eluted at 2.7 minutes and mono¹²⁵I-351A at 6.2 minutes. Arrow 1 indicates mono¹²⁵I-351 A; Arrow 2 indicates 0.16; Arrow 3 indicates 351 A; and Arrow 4 indicates 1.28. Chromatogram notes: 0-2000; 18% MeCN; ¹²⁵I-351A; 30 μΐ of lmM; ¹²⁵I scale; 82% TEAP, pH 3.0; 060720; and 7 μΐ of Na¹²⁵I (Ref. Date 060712).

Example 2:Synthesis of ACE-2 Inhibitor Analogs. A small test batch of two stereoisomers of the ACE-2 inhibitor was synthesized by Irvine Chemistry (July 201 1). The steps for synthesis of the two stereoisomers, JFS101 (R conformation) and JFS102 (S conformation), is shown in FIG. 5. JFS101 and JFS 102 are radioiodinatable MLN-4760 analogs that are in the R and S conformation at the carbon from which the phenolic residue branches.

Example 3 : Radioiodination of JFS101 and JFS102. The R analog (JFS 101) and the S analog (JFS 102) were radioiodinated using the chloramine T procedure of Hunter and Greenwood (Nature 194:495-496 1962). The radioiodinated compounds were then applied to a Ci₈ reverse phase high performance liquid chromatography (HPLC) column and resolved from the un-iodinated compounds (FIGS. 6-7).

Example 4: Purification of ¹²⁵I-JFS101 by HPLC (shown in FIG. 6). The reaction mixture was applied to a Cis column and eluted with 23% acetonitrile 77% triethylamine phosphate; 83 mM H₃PO₄ adjusted to pH 3.0 with triethylamine at a flow rate of 1.8 ml/minute. Line R indicates elution of ¹²⁵Iodine. The largest peak corresponds to the expected elution profile of ¹²⁵I-JFS101. Line B indicates elution of compounds absorbing light at 210 nm. Elution of the mono¹²⁵I-JFS101 peak was at ~3.6 minutes and the un- iodinated JFS 101 peak was at ~2.8 minutes. *used the front and back of the ¹²⁵I peak. Arrow 1 indicates 500 μΐ H₂0: Arrow 2 indicates 50% MeCN; Arrow 3 indicates chloramines T; Arrow 4 indicates JFS 101; Arrow 5 indicates ¹²⁵IJFS101 ; and Arrow 6 indicates elution. Chromatogram notes; 0-9999; 23% MeCN; ¹²⁵IJFS 101; 3 μΐ; 1.77 mg/ml; ¹²⁵I scale; 77% TEAP; 1 10724; and 4 μΐ of Na ¹²⁵I Ref. date 110720.

Example 5: Purification of ¹²⁵I-JFS102 by HPLC (shown in FIG. 7). The reaction mixture was applied to a Cis column and eluted with 23% acetonitrile 77% triethylamine phosphate; 83 mM H₃PO₄ adjusted to pH 3.0 with triethylamine at a flow rate of 1.8 ml/minute. Line R indicates elution of ¹²⁵Iodine. The largest peak corresponds to the expected elution profile of ¹²⁵I-JFS102. Line B indicates elution of compounds absorbing light at 210 nm. Elution of the mono¹²⁵I-JFS102 peak was at ~4.5 minutes and the un- iodinated JFS101 peak at ~3.1 minutes. Arrow 1 indicates ¾0 wash; Arrow 2 indicates Chloramine T; Arrows 3-4 indicate ¹²⁵IJFS102; Arrow 5 indicates JFS 102; and Arrow 6 indicates elution. Chromatogram notes: 0.9999; 25% MeCN; "TJFSIOI A-2; 3 μΐ; 2 mg/ml; ¹²⁵ 1 scale; 75% TEAP; 110724; and 4 μΐ Na ¹²⁵ 1 Ref. Date 1 10720.

Example 6: Autoradiographic Labeling of ACE-2 in rat kidney and liver.

Autoradiography of rat liver and kidney ACE-2 were carried out using general procedures known in the art (Speth et al. Biol Reprod 38:695-702 1988; Bourassa et al. Brain Res

1319:83-91 2010; Speth et al. Brain Res 326: 137-1431985; Daubert et al. Brain Res 816:8-16 1999). The livers and kidneys of rats were frozen and stored at -80°C. The livers and kidneys were subsequently sectioned in a cryostat at a thickness of 20 μιη and thaw-mounted onto charged microscope slides (Premium Grade, Gorilla Scientific). The slides were stored frozen at -80°C until the day of the assay. On the day of the assay, the sections were thawed and pre-incubated in 37 ml of assay buffer (without radioligand) in a Coplin jar for 30 minutes to wash away endogenous bound substrate for ACE-2. The sections were then incubated in assay buffer with 200 pM ¹²⁵I-JFS101 or ¹²⁵I-JFS 102 with and without ~ 400 nM DX-600 (Phoenix Pharmaceuticals) to saturate ACE-2 for one hour at 20-22 °C. The slides were then quickly dipped in two changes of distilled water and rinsed 4 x 15 seconds in assay buffer without radioligand. The slides were again quickly dipped in two changes of distilled water to remove buffer salts. The slides were then dried under a stream of cool air for 4 minutes. The slides were then mounted onto cardboard with a standards slide containing known amounts of iodine- 125 (ARI 0133, American Radiolabeled Chemicals) that is used to construct a standard curve for quantitation of radioligand binding to the sections. The cardboard mounted slides were placed in an X-ray cassette exposed to single sided autoradiography film (MR-1, Kodak) for a 17 hour exposure period. The films were then developed in an automated film processor and the images captured on an image analysis system (MCID, Interfocus Ltd.) for quantitative densitometric analysis.

The "total" and "non-specific" (binding in the presence ~ 400 nM DX-600) binding of both radioligands in both the liver and kidneys were determined by quantitative densitometry to assess "specific" ("total" minus "non-specific binding) binding. In both the liver and the kidney sections that were assayed "total" binding was greater than "non-specific" binding indicating that both ¹²⁵I-JFS 101 and ¹²⁵I-JFS 102 radiolabeled ACE-2 in these tissues.

Aliquots of ¹²⁵I-JFS101 and ¹²⁵I-JFS102 were assessed for their ability to inhibit

ACE-2 using an established fluorometric assay (Vickers et al. J Biol Chem 277: 14838-14843 2002; Elased et al. Exp Physiol 93 :665-675 2008). Both compounds were capable of inhibiting ACE-2 activity at micromolar concentrations. Example 7: Matrix-Assisted Laser Desorption/Ionization (MALDI) mass

spectrometry (MS) imaging of renal Angiotensin II (Ang II) metabolism. An imaging approach using MALDI imaging was used to characterize and localize renin-angiotension system (RAS) enzymes metabolizing Ang II in the kidney. Incubation of murine kidney sections (FIG. 8A) with Ang II (FIG.8B) resulted in the formation of Ang (1-7) and Ang II (FIGS. 8C-D). Product generation was destroyed by heat treatment confirming that the measured peptide formation was of enzymatic origin. Ang (1-7) was mainly detected in the renal cortical region while Ang II was predominantly localized in the medulla (FIG. 8C) and (FIG. 8D), respectively. An overlay of the MS signals obtained for both peptide products shows their distinct spatial distribution patterns (FIG. 8E).

The matrix assisted laser desorption/ionization (MALDI) imaging method was also used to localize and characterize Ang II conversion in murine kidney (Grobe N, Elased KM, Cool DR, Speth RC, Morris M. Localization and Characterization of Renal Angiotensin II Metabolism using Mass Spectrometry, abstract submitted to High Blood Pressure Council meeting, September 2011). Kidney sections (12 μιη) obtained from C57B16 mice were incubated with 100 μΜ Ang II for 3 minutes at 37 °C. Formation of Ang (1-7) and Ang III was verified using MALDI-TOF/TOF (matrix assisted laser desorption/ionization-time of flight/time of flight). Enzyme activities were dose and time dependent and absent in heat treated kidney sections. Formation of Ang III (m/z 931) through hydrolysis of the Asp^x-Arg² bond of Ang II was mainly found in the medullary region and was inhibited 33.3 ± 12.2 % (0.16 of 0.48 ratio of Ang III/Ang II) by 3 μΜ glutamate phosphonate, aminopeptidase A inhibitor. Ang (1-7) (m/z 899) was predominantly generated in the renal cortex via cleavage of the Pro⁷-Phe⁸ bond of Ang II. Inhibitor studies delineated enzyme activities to peptidases known to generate Ang (1-7) from Ang II: angiotensin converting enzyme 2 (ACE-2), prolyl carboxypeptidase (PCP) and prolyl endopeptidase (PEP). Incubation with Ang II and 1 μΜ ACE2 inhibitor, MLN-4760, or 100 μΜ PCP inhibitor, Z-pro-prolinal, reduced cortical Ang (1-7) formation to 39.7 ± 1.2% (0.31 of 0.78 ratio of Ang (l-7)/Ang II) or 44.4 ± 8.7% (0.36 of 0.81 ratio of Ang (l-7)/Ang II), respectively. Addition of 100 μΜ PEP inhibitor, Z-pro- pro-aldehy de-dimethyl acetal, showed no significant effects on cortical Ang (1-7) generation (80.9 ± 14.8%, 0.72 of 0.89 ratio of Ang (l-7)/Ang II).

Example 8: Viral Transfecti on-Induced Protein Expression. To investigate the role of the angiotensin II receptor (AT2R) in rostro ventrolateral medulla (RVLM) of adult Sprague- Dawley (SD) rats by mediated gene transfer technology for overexpression of this Ang II receptor subtype, an adeno-associated virus type 2 (AAV2) AT2R recombinant AAV2 vector (AAV2-CBA-AT2R) containing the full-length AT2R cDNA under the control of a CBA promoter was constructed. The effectiveness of this vector in transducing neurons was first studied in vitro. This vector efficiently transduced the AT2R into primary neuronal cells in culture, resulting in the expression of high levels of AT2R that were primarily localized to neurons. Microinjection of AAV2-CBA-AT2R (1 μΐ of lxlO⁹ particles) into the RVLM of adult SD rats produced a high level of AT2 receptor expression within 7-14 days, which was sustained to the end of the experiment (4.5 months), as evidenced by autoradiography (FIGS. 9A-D).

Receptor autoradiography with ¹²⁵I-SarI-Ile8-Ang II (500 pM) reveals overexpression of AT2R in the RVLM of AAV2-CBA-ATR2-WPRE-transduced rats. FIG. 9A shows nonspecific binding (3 μΜ Ang II). FIG. 9B shows AT-1 receptor binding (in the presence of 10 μΜ PD 123319, a selective AT -2 receptor antagonist. FIG. 9C shows AT-2 receptor binding (in the presence of 10 μΜ losartan, a selective AT-1 receptor antagonist) highly expressed in the RVLM. In non-AT-2 Receptor transfected rats no AT-2 receptor binding can be seen. FIG. 9D shows a thionin stain of a RVLM section adjacent to the sections shown in FIGS. 9A-C.

All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. It is to be understood that while a certain form of the invention is illustrated, it is not intended to be limited to the specific form or arrangement herein described and shown. It will be apparent to those skilled in the art that various changes may be made without departing from the scope of the invention and the invention is not to be considered limited to what is shown and described in the specification. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objectives and obtain the ends and advantages mentioned, as well as those inherent therein. The compositions, inhibitors, radioligands, methods, procedures, and techniques described herein are presently representative of the preferred embodiments, are intended to be exemplary and are not intended as limitations on the scope. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention. Although the invention has been described in connection with specific, preferred embodiments, it should be understood that the invention as ultimately claimed should not be unduly limited to such specific embodiments. Indeed various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the invention.

Claims

THE CLAIMS What is claimed is: Claim 1. An angiotensin-converting enzyme-2 (ACE-2) inhibiting compound, the compound comprising a formula:

wherein R² is a phenol moiety.

Claim 2. The ACE-2 inhibiting compound according to claim 1, further comprising a radiolabel on the phenol moiety.

Claim 3. The ACE-2 inhibiting compound according to claim 2, wherein the radiolabel is iodine- 125.

Claim 4. The ACE-2 inhibiting compound according to claim 2, further comprising an alkyl linker/spacer chain extending the phenol moiety with the radiolabel away from other parts of the compound.

Claim 5. A method for studying a sample containing or suspected of containing angiotensin-converting enzyme-2 (ACE-2) protein, the method comprising:

a) contacting the sample with an inhibitor of ACE-2; and

b) quantifying binding of said inhibitor to ACE-2 present in the sample.

Claim 6. The method according to claim 5, wherein the sample is a biological sample.

Claim 7. The method according to claim 5, wherein the inhibitor of ACE-2 comprises a formula:

wherein R² is a radiolabled phenol moiety.

Claim 8. The method according to claim 7, wherein the phenol moiety is radiolabeled with iodine- 125.

Claim 9. The method according to claim 5, wherein the quantifying includes carrying out autoradiography.

Claim 10. A method for quantifying angiotensin-converting enzyme-2 (ACE-2) protein in a biological sample containing or suspected of containing ACE-2 protein, the method comprising:

a) obtaining at least one biological sample;

b) contacting the sample with an inhibitor of ACE-2;

c) quantifying binding of said inhibitor to ACE-2 present in the sample; and d) quantifying amount of the ACE-2 protein detected in the biological sample.

Claim 11. The method according to claim 10, wherein the biological sample is a tissue sample.

Claim 12. The method according to claim 10, wherein the method further comprises determining anatomical location and distribution of the ACE-2 protein quantified.

Claim 13. The method according to claim 10, wherein the inhibitor of ACE-2 comprises a formula:

wherein R² is a radiolabled phenol moiety.

Claim 14. The method according to claim 13, wherein the phenol moiety is radiolabeled with iodine- 125.

Claim 15. A pharmaceutical composition comprising a pharmaceutically - acceptable carrier and an effective amount of an angiotensin-converting enzyme-2 (ACE-2) inhibitor.

Claim 16. The pharmaceutical composition according to claim 15, wherein the ACE-2 inhibitor is a com ound comprising a formula:

Claim 17. A method for modulating activity of an angiotensin-converting enzyme-2 (ACE-2) protein comprising contacting said protein with an angiotensin-converting enzyme-2 (ACE-2) inhibitor.

Claim 18. The method according to claim 17, wherein the ACE-2 inhibitor is a compoun comprising a formula:

Claim 19. A method of treating an angiotensin-converting enzyme-2 (ACE-2) associated condition in a subject in need thereof comprising administering a therapeutically- effective amount of an ACE-2 inhibitor to the subject such that the ACE-2 associated condition is treated.

Claim 20. The method according to claim 19, wherein the ACE-2 inhibitor is a compoun comprising a formula:

Claim 21. The method according to claim 19, wherein the angiotensin-converting enzyme-2 (ACE-2) associated condition is severe acute respiratory syndrome (SARS).

Claim 22. Use of an angiotensin-converting enzyme-2 (ACE-2) inhibitor for studying a sample containing or suspected of containing an angiotensin-converting enzyme-2 (ACE-2) protein.

Claim 23. Use according to claim 22, wherein the sample is a biological sample.

Claim 24. Use according to claim 22, wherein the inhibitor of ACE-2 comprises a formula:

wherein R² is a radiolabled phenol moiety.

Claim 25. Use according to claim 24, wherein the phenol moiety is radiolabeled with iodine- 125.

Claim 26. Use of an angiotensin-converting enzyme-2 (ACE-2) inhibitor for quantifying angiotensin-converting-enzyme-2 (ACE-2) protein in a biological sample containing or suspected of containing ACE-2 protein.

Claim 27. Use according to claim 26, wherein the biological sample is a tissue sample.

Claim 28. Use according to claim 26, wherein the inhibitor of ACE-2 comprises a formula:

wherein R² is a radiolabled phenol moiety.

Claim 29. Use according to claim 28, wherein the phenol moiety is radiolabeled with iodine- 125.

Claim 30. Use of an angiotensin-converting enzyme-2 (ACE-2) inhibitor for determining anatomical location and distribution of an ACE-2 protein quantified in a sample.

Claim 31. Use according to claim 30, wherein the sample is a biological sample.

Claim 32. Use according to claim 31, wherein the biological sample is a tissue sample.

Claim 33. An angiotensin-converting enzyme-2 (ACE-2) inhibitor for use in the manufacture of a composition for modulating activity of an ACE-2 protein.

Claim 34. Use according to claim 33, wherein the ACE-2 inhibitor is a compound comprising a formula:

Claim 35. An angiotensin-converting enzyme-2 (ACE-2) inhibitor for use in the manufacture of a composition for treating an angiotensin-converting enzyme-2-associated condition in a subject in need thereof.

Claim 36. Use according to claim 35, wherein the inhibitor of ACE-2 is a compound comprising a formula:

Claim 37. Use according to claim 35, wherein the angiotensin-converting enzyme-2-associated condition is severe acute respiratory system (SARS).