WO2001027314A1

WO2001027314A1 - Antiprotozoal histone acetyl transferase inhibitors

Info

Publication number: WO2001027314A1
Application number: PCT/US2000/027337
Authority: WO
Inventors: Dennis M. Schmatz; Anne M. Gurnett; Samantha D. Samaras
Original assignee: Merck and Co Inc
Current assignee: Merck and Co Inc
Priority date: 1999-10-08
Filing date: 2000-10-04
Publication date: 2001-04-19
Anticipated expiration: 2002-04-08
Also published as: EP1222308A4; EP1222308A1; JP2003511054A; AU7992600A; CA2386299A1

Abstract

Histone acetyl transferase inhibition provides a target for identifying potential antiprotozoal compounds. Histone acetyl transferase inhibitors are useful as therapeutic agents against protozoal infections. A method of identifying histone acetyl transferase inhibitors compares i) the acetylation level from a combination of a test compound with a known histone deacetylase inhibitor to ii) a basal acetylation level.

Description

TITLE OF THE INVENTION

ANTIPROTOZOAL HISTONE ACETYL TRANSFERASE INHIBITORS

BACKGROUND OF THE INVENTION This invention is related to a method to identify pharmaceutical antiprotozoal compositions In particular, this invention is directed to a method to identify antiprotozoal compositions that inhibit histone acetyl transferase

Parasitic protozoa are responsible for a wide variety of infections in man and animals Many protozoan infections are life threatening to the host Consequently, such infections pose significant health and economic threats Health threats caused by protozoa include, for example, malaπa and trypanosomiasis Malaria is caused by protozoa of the genus Plasmodium such as P vivax, P falciparum, P ovale and P malariae Trypanosomiasis includes Chagas disease caused by Trypanosoma cruzi and Afπcan sleeping sickness caused by T brucei Further, protozoa such as Pneumocystis carinu, Toxoplasma gondu, and

Cryptosporidium sp can cause opportunistic infections, especially in people with compromised immune systems

Economic threats from protozoa result from the mortality of infected animals and lower production yields from sick animals An economically important protozoan disease of domesticated animals is coccidiosis, which is caused by protozoa of the genus Eimeria In poultry, the infecting protozoa include E tenella, E acervulma, E necattιx, E brunetti, E mιtιs, E praecox and E maxima

Thus, there is a great and continuing need for effective anti-protozoan compounds For some protozoal diseases, such as Chagas disease, there is no satisfactory treatment Further, in other protozoal diseases, drug-resistant strains of the infecting protozoa are decreasing the effectiveness of present treatment drugs Thus, it would be desirable to develop new and effective anti-protozoal compounds that utilize a new mechanism against protozoa - that of inhibiting histone acetyl transferase biological intracellular activity in protozoa Accordingly, it would be desirable to develop a method of identifying such new histone acetyl transferase inhibiting anti -protozoal compounds

It is well known that DNA has the basic structure of a double helix Instead of being meiely a dangling string, however, the DNA double helical structure is further organized by having portions of it wrapped about a core - much as thread is wrapped about a bobbin Histones form the core about which the DNA is wrapped. The DNA wrapped/hi stone structure is known as a nucleosome or chromatm The nucleosomes can be further organized to form higher order structures.

Without being bound to theory, it is believed that various biological activities depend from the geometric conformations of the participating molecules. The core histones have amino terminal regions, πch in lysine, that are ready targets for acetylation. DNA has lower affinity to acetylated histone than to deacetylated histone. As a result, the DNA is held less tightly to the acetylated histone core and the acetylated histone terminal regions are less rigidly held in relation to the DNA. Thus, the local chromatin environment is dramatically changed by acetylation, thereby affecting biological activity.

Acetylation of histone results from the action of the enzyme histone acetyl transferase ("HAT") Substantially acetylated histone is described as being in a state of "hyperacetylation " Deacetylation of histone results from the action of the enzyme histone deacetylase ("HDA"). Histone that is substantially without acetylation is descπbed as being in a state of "hypoacetylation."

Various compounds such as, for example, n-butyrate, tπchostatin, trapoxin or apicidm can inhibit the action of HDA. The HDA inhibiting action of apicidin is descπbed in S. Darkin-Rattray et al., Proc. Natl. Acad. Sci. USA, 93:13143-13147 (1996). Trapoxin is descπbed in H. Itazaki et al., J. Antibiotics, 43: 1524 (1990). Recently, tπchostatin A and trapoxin A have been reported as reversible and irreversible inhibitors, respectively, of mammalian HDA (see e.g., Yoshida et al., BioAssavs, 17(5):423-430 (1995)). Tπchostatin A has also been reported to inhibit partially puπfied yeast HDA (Sanchez del Pino et al., Biochem. J., 303:723-729 (1994)). Tπchostatin A is an antifungal antibiotic and has been shown l) to have anti-tπchomonal activity as well as cell differentiating activity in muπne erythroleukemia cells, and n) the ability to induce phenotypic reversion in sis- transformed fibroblast cells (see e.g., U.S. Patent No. 4,218,478; and Yoshida et al., BioAssays, 17(5):423-430 (1995); and references cited therein). Trapoxin A, a cyclic tetrapeptide, induces moφhological reversion of v-si^'s-transformed NIH3T3 cells (Yoshida and Sugita, Jap. J. Cancer Res., 83(4):324-328 (1992).

HDA inhibition as a target for cancer research is descπbed in Saito et al, Proc. Natl Acad Sci USA, 96:4592-4597(1999); Bernardi et al., Ammo Acids 6:315-318 (1994), and R.E. Shute et al., J. Med. Chem 30:71-78 (1987). Utilizing HDA as a target for antiprotozoal agents is described in U.S. Patent Applications 09/296,834, filed April 22, 1999, and 08/716,978, filed September 20,1996 U.S Patent No 4,218,478 describes the use of tπchostatin as an antiprotozoal agent Antiprotozoal cyclic tetrapeptides, including the HDA inhibitor apicidin, are descπbed in U.S Patent No 5,620,953, incoφorated herein by reference. Apicidm [cvc/o(N-O-methyl-L-Tφ-L-Ile-D-Pιp-L-2-amιno-8-oxo-decanoyl)] is a broad- spectrum antiprotozoal, antifungal and antineoplastic agent isolated from the fermentation culture of Fusaπum fungus The structure of apicidin is shown below:

Nevertheless, there remains a need to develop novel antiprotozoal agents. Particularly desirable would be new antiprotozoal agents utilizing novel properties such as HAT inhibition, as well as a method to identify such HAT inhibiting agents

SUMMARY OF THE INVENTION

This invention provides a method for identifying compounds having antiprotozoal activity The method includes the steps of contacting an histone acetyl transferase with (l) an identifiably labeled compound that interacts with histone acetyl transferases, and (n) a test compound or composition, and quantitating the inhibition of the interaction of the labeled compound, wherein that inhibition is induced by the test compound or composition.

In another method, this invention identifies compounds having histone acetyl transferase inhibiting activity by the steps of l) determining a basal histone acetylation level in a histone/histone acetyl transferase/histone deacetylase system; π) determining a refeience histone hyperacetylation level in a histone/histone acetyl transferase/histone deacetylase/histone deacetylase inhibitor system; in) determining a test histone acetylation level in a histone/histone acetyl transferase/histone deacetylase/histone deacetylase inhibitoi/test composition system; and IV) comparing the test level to the basal and reference levels.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig la is a schematic diagram of the action of uninhibited HAT and uninhibited HDA on the acetylation of histone.

Fig. lb is a schematic diagram of the action of inhibited HDA and uninhibited HAT on the acetylation of histone Fig lc is a schematic diagram of the action of inhibited HDA and inhibited HAT on the acetylation of histone.

DETAILED DESCRIPTION OF THE INVENTION

As descπbed above, the action of HDA and HAT together control the net level of acetylation of histones Inhibition of the action of HDA (for example, by the action of apicidin) results in the accumulation of hyperacetylated histones from the unopposed action of HAT Such hyperacetylation accumulation can be measured. For example, an ELISA (Enzyme-linked immunosorbent assay) of apicidin treated HeLa cells can directly quantify hyperacetylated histone buildup. The ELISA can utilize antibodies such as, for example, one raised against an acetylated peptide which has a sequence identical to the amino terminus of human histone H3 or H4 Such ELISA serves as a control expeπment that provides a baseline against which test compounds can be screened for each compound's effectiveness at inhibiting the action of HAT. An inhibition of the action of HAT would result in a lessening of the accumulation of hyperacetylated histones That is, the inhibition of the action of HDA in the baseline control is counterbalanced by any inhibition of the action of HAT provided by the test candidate.

In one aspect the present invention provides a method for identifying compounds having HAT inhibition activity by adding a test compound to the above- descπbed control expeπment. After a specific duration of time, the amount of hyperacetylation is compared to the control. The lower the build-up of hyperacetylated histone, the more effective is the inhibiting action of the test compound to the action of HAT

Referring to Fig la, the basal condition of a histone/HAT/HDA system is shown in which there is an equihbnum level of acetylated histone on the right and deacetylated histone on the left as a result of the actions of HAT and HDA This equilibrium level of acetylation can be measured as a basal level By the initial addition of an amount of an HDA inhibitor, referπng to Fig lb, an augmented concentration of acetylated histone is caused by the decreased action of the inhibited HDA Thus, in the histone/HAT/HDA/HDA inhibitor system a condition of hyperacetylation exists This hyperacetylation level of acetylation can be measured as a reference or control level of acetylation (hyperacetylation) Referπng to Fig lc, when an active test candidate (one that effectively inhibits HAT) is added together with the HDA inhibitor to provide a histone/HAT/HDA/HDA inhibitor/test compound system, both HDA and HAT are inhibited and the acetylation condition resembles the basal equihbπum

Therefore, an ineffective HAT inhibiting test candidate would lead to an acetylation condition closer to Fig lb than to Fig la (thus resembling hyperacetylation or the reference level of acetylation), while an effective HAT inhibiting test candidate would lead to an acetylation condition closer to Fig la (thus resembling the basal level of acetylation) Accordingly, the present invention generally provides for l) measuπng a basal acetylation condition, n) measuπng an inhibited histone deacetylase reference condition, in) measuπng a test acetylation condition of histone deacetylase inhibition in combination with histone acetyl transferase inhibition from a HAT inhibition candidate, and IV) compaπng the test measurement to the other two (basal and reference) measurements An active HAT inhibiting candidate will provide a measurement substantially similar to the basal condition

Thus, in one aspect the present invention identifies compounds having histone acetyl transferase inhibiting activity by a method that includes the steps of I) determining a basal histone acetylation level in a histone/histone acetyl transferase/histone deacetylase system, n) determining a reference histone hyperacetylation level in a histone/histone acetyl transferase/histone deacetylase/histone deacetylase inhibitor system, in) determining a test histone acetylation level in a histone/histone acetyl transferase/histone deacetylase/histone deacetylase inhibitor/test composition system, and iv) compaπng the test level to the basal and reference levels

Techniques to determine histone acetylation levels are well known in the art Any convenient technique may be used such as, for example, measuring the optical density of ELISA plates or measuπng the radioactivity level of tritium marked reagents One in the art readily understands the applicability of particular techniques in given situations.

Accordingly, a method of this invention includes the steps of

A) contacting histone acetyl transferase with (1) an identifiably labeled compound that interacts with histone acetyl transferases; and (n) a test compound or composition; and

B) quantitating the labeled compound to determine a level of histone acetyl transferase that is induced by the test compound.

That is, the test compound or composition, by affecting the action of HAT, affects the quantifiable amount of the labeled compound Thus, quantification of the labeled compound serves as a diagnostic surrogate for determining any inhibiting of HAT by the test compound.

One embodiment of the present invention provides a method for identifying compounds having HAT inhibition activity compπsing the steps of a) contacting with histone l) a known amount of histone deacetylase, n) a known amount of histone acetyl transferase, and in) a known amount of a labeled compound that interacts with histone acetyl transferase; b) measuπng the level of histone acetylation and setting the measured level as a baseline (zero, basal or background) level; c) contacting with histone l) a known amount of histone deacetylase, n) a known amount of histone acetyl transferase, in) a known amount of a labeled compound that interacts with histone acetyl transferase, and iv) a known amount of an histone deacetylase inhibitor, wherein (c)(ι), (c)(n), and (c)(nι) are substantially in the same proportions to each other as (a)(ι), (a)(n), and (a)(ιn) above respectively; d) quantitating the inhibition of the HDA by measuπng the level of histone acetylation (hyperacetylation) after a set duration of time and setting the measured level as a hyperacetylation or reference level; e) contacting with histone l) a known amount of histone deacetylase, n) a known amount of histone acetyl transferase, in) a known amount of a labeled compound that interacts with histone acetyl transferase, iv) a known amount of an histone deacetylase inhibitor, and v) a known amount of a test compound, wherein

(e)(ι), (e)(n), (e)(uι), and (e)(ιv) are substantially in the same proportions to each other as (c)(ι), (c)(n), (c)(ιu), and (c)(ιv) above respectively, f) quantitating the affect of the test compound by measuπng the level of histone acetylation after a set duration of time substantially the same duration as that of (d) and setting the measured level as a test level; and g) quantitating the HAT inhibiting effect of the test compound by compaπng the results of (f) with (d) and (b).

In still another embodiment, the present invention provides a method for identifying compounds having HAT inhibition activity compπsing the steps of aa) contacting with histone 1) a known amount of histone deacetylase, n) a known amount of histone acetyl transferase, in) a known amount of a labeled compound that interacts with histone acetyl transferase, and iv) a known amount of an histone deacetylase inhibitor, bb) quantitating the inhibition of the HDA by measuπng the level of histone acetylation (hyperacetylation) after a set duration of time and setting the measured level as a reference level; cc) contacting with histone l) a known amount of histone deacetylase, n) a known amount of histone acetyl transferase, in) a known amount of a labeled compound that interacts with histone acetyl transferase, iv) a known amount of an histone deacetylase inhibitor, and v) a known amount of a test compound, wherein (cc)(ι), (cc)(n), (cc)(ιn), and (cc)(ιv) are substantially in the same proportions to each other as (aa)(ι), (aa)(n), (aa)(nι), and (aa)(ιv) above respectively; dd) quantitating the affect of the test compound by measuπng the level of histone acetylation after a set duration of time substantially the same duration as that of (bb) and setting the measured level as a test level; and ee) quantitating the HAT inhibiting effect of the test compound by compaπng the results of (dd) with (bb)

Another method of this invention includes the steps of: A) contacting histone with l) a histone acetyl transferase, (n) a histone deacetylase, (in) a histone deacetylase inhibitor, (iv) a test compound or composition, and (in) an antibody that interacts with acetylated histone; and B) quantitating the antibody to determine a level of acetylated histone induced by the test compound.

That is, by affecting the action of HAT, the test compound or composition affects the level of histone acetylation - which can be monitored by quantifying the antibody Thus, quantification of the antibody serves as a diagnostic suιτogate for determining any inhibiting of HAT by the test compound In an embodiment, the present invention provides a method for identifying compounds having HAT inhibition activity compπsing the steps of aaa) contacting with histone 1) a known amount of histone deacetylase, n) a known amount of histone acetyl transferase, and in) a known amount of an antibody that interacts with acetylated histone; bbb) measuring the level of histone acetylation and setting the measured level as a baseline (zero, basal or background) level; ccc) contacting with histone 1) a known amount of histone deacetylase, n) a known amount of histone acetyl transferase, in) a known amount of an antibody that interacts with acetylated histone, and iv) a known amount of an histone deacetylase inhibitor, wherein (ccc)(ι), (ccc)(n), and (ccc)(nι) are substantially the same proportions to each other as (aaa)(ι), (aaa)(n), and (aaa)(m) above respectively; ddd) quantitating the inhibition of the HDA by measuπng the level of histone acetylation (hyperacetylation) after a set duration of time and setting the measured level as a hyperacetylation or reference level; eee) contacting with histone l) a known amount of histone deacetylase, n) a known amount of histone acetyl transferase, in) a known amount of an antibody that interacts with acetylated histone, iv) a known amount of an histone deacetylase inhibitor, and v) a known amount of a test compound, wherein (eee)(ι), (eee)(n), (eee)(ιn), and (eee)(ιv) are substantially in the same proportions to each other as (ccc)(ι), (ccc)(n), (ccc)(ιn), and (ccc)(ιv) above respectively; fff) quantitating the affect of the test compound by measuπng the level of histone acetylation after a set duration of time substantially the same duration as that of (ddd) and setting the measured level as a test level; and ggg) quantitating the HAT inhibiting effect of the test compound by compaπng the results of (fff) with (ddd) and (bbb).

In another embodiment, the present invention provides a method for identifying compounds having HAT inhibition activity compπsing the steps of:

AA) contacting with histone I) a known amount of histone deacetylase, n) a known amount of histone acetyl transferase, in) a known amount of an antibody that interacts with acetylated histone, and iv) a known amount of an histone deacetylase inhibitor;

BB) quantitating the inhibition of the HDA by measuring the level of histone acetylation (hyperacetylation) after a set duration of time and setting the measured level as a hyperacetylation or reference level; CC) contacting with histone 1) a known amount of histone deacetylase, n) a known amount of histone acetyl transferase, in) a known amount of an antibody that interacts with acetylated histone, iv) a known amount of an histone deacetylase inhibitor, and v) a known amount of a test compound, wherein (CC)(ι), (CC)(n), (CC)(ιn), and (CC)(ιv) are substantially in the same proportions to each other as (AA)(ι), (AA)(n), (AA)(nι), and (AA)(ιv) above respectively;

DD) quantitating the affect of the test compound by measuring the level of histone acetylation after a set duration of time substantially the same duration as that of (BB) and setting the measured level as a test level; and EE) quantitating the HAT inhibiting effect of the test compound by compaπng the results of (DD) with (BB).

The amount of histone deacetylase and histone acetyl transferase above can be those amounts found in a specific cell line, as well as extracellular known amounts. That is, the enzyme amounts can be that found intracellularly. The enzyme amounts can also be that amount mixed extracellularly.

In another embodiment, l) the basal acetylation of histone is first measured by collecting subject cells, extracting their histones, placing the extractions on AUT gels, followed by quantitating by Western blots or with protein stains the amount of histone acetylation in a first measurement, n) Second, the control amount of histone hyperacetylation is measured by dosing the same type subject cells with a control amount of a control HDA inhibitor, collecting the cells, extracting their histones, placing the extractions on AUT gels, and quantitating by Western blots or with protein stains the amount of acetylation (hyperacetylation) in a second measurement, in) Third, under the same conditions as (n), a known amount of a test compound is added together with the control amount of the control HDA inhibitor, and the amount of hyperacetylation of histone is quantitated by Western blot or with protein stains in a third measurement The third measurement is compared to the first and second measurements to quantitate the inhibiting property of the test compound to HAT activity. In another aspect, another method of this invention determines the

HAT inhibiting property of a test candidate by l) adding a specific amount of an HDA inhibitor together with a lesser known amount of a test candidate to a histone/HAT/HDA system and measuπng the histone acetylation level, u) adding a specific amount of an HDA inhibitor together with an augmented known amount of a test candidate to a histone/HAT/HDA system and measuπng the histone acetylation level, and in) comparing the measured histone acetylation levels An active HAT inhibitor will yield a lower histone acetylation level from the addition of an augmented amount than from the addition of a lessor amount of test candidate

Any of the above methods can be used to compare the effect of test candidates on protozoal HAT and on host HAT It is apparent that the test candidates that more potently inhibit protozoal HAT, compared to their potency at inhibiting host HAT, are likely effective antiprotozoal compounds Test candidates that affect protozoal and host HAT approximately the same, however, are also antiprotozoal compounds Without being bound by theory, it is believed that the reproductive rate of protozoa being usually much greater than that rate of the host cells causes protozoa to be more susceptible to HAT inhibition Therefore, compounds showing any substantial protozoal HAT inhibition properties are antiprotozoal compounds It is preferred that the test compounds inhibit protozoal and host HAT about equally It is more preferred that the test compounds inhibit protozoal HAT greater than host HAT. In another aspect, the present invention provides a method for identifying compounds having antiprotozoal activity, by HAT inhibition, compπsing the steps of:

I) determination of HAT inhibition activity:

(a) contacting an intact host or protozoal cell with a test compound and a known amount of an HDA inhibiting compound;

(b) disrupting the host or cell to obtain histones;

(c) determining the level of histone acetylation; and

(d) compaπng the level of histone acetylation to (I) the level of acetylation from the action of the HDA inhibiting compound without the presence of the test compound and to (π) the level of acetylation of the cell without any HDA inhibiting or test compound to determine if the test compound is an effective (active) HAT inhibitor.

II) determination of selective HAT inhibition by taking active candidates and testing against parasites such as E. tenella for anti -protozoal activity by any convenient method, such as descnbed in D.M. Schmatz, et al., J Protozoology, 33(1) 109-114 (1986):

(a) in parallel expeπments each having known amounts of host cells and parasite, a screen experiment adds a candidate active HAT inhibitor composition to the host/parasite while a control expeπment does not include such added composition,

(b) in the first screening decision, a candidate that shows a continuing presence of the host in companson to the decreasing presence of the host in the control passes to the next screening decision,

(c) the viability of the host is determined after a specified amount of time and compared to the viability of the parasite after the specified amount of time;

(d) the viabilities determined are compared; a good selective candidate is one that kills the parasite and not the host.

Thus, this invention provides facile and specific assays to screen compounds as HAT inhibitors and as potential antiprotozoal drugs.

In the present invention, the HDA can be any convenient protein or mixture of proteins known in the art such as, for example, a native protein in whole cells, a purified or partially purified native enzyme, a cloned histone deacetylase or an engineered vaπant thereof, a crude preparation of the enzyme, or an extract containing histone deacetylase activity The enzyme can be from a mammalian (e g. human cervical carcinoma, HeLa cell), avian (e.g chicken liver or erythrocyte nuclei) or protozoal (e.g. Eimeria tenella or P. Berghei) source. Preferably, a protozoal histone deacetylase is used. Fragments of histone deacetylase that retain the desired enzyme activity are also within the scope of this invention.

In the present invention, the HAT can be any convenient protein or mixture of proteins known in the art such as, for example, a native protein in whole cells, a puπfied or partially puπfied native enzyme, a cloned histone acetyl transferase or an engineered vaπant thereof, a crude preparation of the enzyme, or an extract containing histone acetyl transferase activity. Fragments of histone acetyl transferase that retain the desired enzyme activity are also within the scope of this invention. In the present invention, the HDA inhibitor can be any convenient compound known in the art including those that act as substrates for the histone deacetylase enzyme, those that bind the enzyme at its active site, or those that otherwise act to alter enzyme activity by binding to an alternate site.

The test compound can be a synthetic compound, a puπfied preparation, crude preparation, or an initial extract of a natural product obtained from plant, microorganism, or animal sources. One embodiment of the present method is based on test compound induced inhibition of histone acetyl transferase activity as measured by scintillation counting to determine the histone acetylation level Compounds that inhibit histone acetyl transferase induced incoφoration of 3H acetyl CoA into histones - using any enzyme source (protozoan/mammahan/avian/etc — would be considered histone acetyl transferase inhibitors The level of incoφoration can also be determined using fluorography or autoradiography following SDS polyacryl amide gel electrophoresis

In a preferred embodiment, the method of the present invention utilizes a histone acetyl transferase or an extract containing histone acetyl transferase obtained from a protozoal source, such as Eimeria or Plasmodium sp

Another method, similar to that descπbed above, determines the test compound's inhibiting properties directed selectively to the parasite Compounds that inhibit parasite histone acetyl transferase induced incoφoration of H acetyl CoA into histones — with little or no effect on chicken or mammalian histone acetyl transferase induced incoφoration of H acetyl CoA into histones — would be considered selective or specific parasitic histone acetyl transferase inhibitors Similarly, the level of incoφoration can also be determined using fluorography or autoradiography following SDS polyacrylamide gel electrophoresis

In a more preferred embodiment, the method of the present invention further comprises determining the IC50 of test compounds against host histone acetyl transferase in the enzyme inhibition assay as descπbed above, to identify those compounds that have selectivity for parasitic histone acetyl transferase over that of a host The assays are the same as previously described, with the histone acetyl transferase activity obtained from a host of protozoa, for example the host histone acetyl transferase may be obtained from a mammalian source, e g human, or an avian source, e g chicken

Where the enzyme inhibition or ELISA utilizes a crude preparation or an extract or a whole cell containing histone acetyl transferase, the target of the test compound may be veπfied by examining the level of histone acetylation Thus, the Eimeπa infected host cell containing the enzyme is treated with Apicidin (to induce hyperacetylation of histones) and the test compound The cells are lysed and the level of histone acetylation is determined using an antibody raised against an acetylated peptide the sequence of which is identical to amino terminus of human histone H3 or H4 This is pei formed using an ELISA based assay A histone acetyl transferase inhibitor will cause inhibition of apicidin induced hyperacetylation of histones Since this method uses intact cells treated with the test compound, this technique may also be used to identify prodrugs that may be converted to histone acetyl transferase inhibitor within the cellular environment, but may not be so identified by assay based on the enzyme itself Another method useful to identify inhibitors that are selective for parasitic histone acetyl transferase is the use of acid urea tπon (AUT) gel electrophoresis to determine the level of acetylation of histones. Cells are treated as descπbed in preparation for the ELISA assay but the level of hyperacetylation is determined by any convenient method such as, for example, by protein stains or on AUT gels. In the AUT system the histones of host and parasite can be separated and can be detected, following transfer to nitrocellulose, using an antibody which is specific for certain acetylated lysme residues in the amino terminal region of histone H3 or H4 as described in B.M. Turner, et al , FEBS Lett., 253, 141-145 (1989). These antibodies were raised against an acetylated peptide the sequence of which is identical to the amino terminus of human histone H3 or H4. Thus compounds that inhibit the apicidin induced hyperacetylation of parasite histone, with no or little inhibition of apicidin induced hyperacetylation of host histone, would be considered selective parasitic histone acetyl transferase inhibitors.

In another aspect the present invention provides a method for the treatment of protozoal infections compπsing administeπng to a host suffenng from a protozoal infection a therapeutically effective amount of a compound which inhibits histone acetyl transferase A therapeutically effective amount may be one that is sufficient to inhibit histone acetyl transferase of the causative protozoa

An example of a compound which has shown histone acetyl transferase inhibiting properties and therefore useful in the treatment of protozoal diseases is represented by the chemical structure (I) below

(I) Compound I can be prepared using techniques descπbed in V.I. Saloutin, et al., J. Fluorine Chemistry, 56(3}.325-334 (1992); K.C. Joshi, et al., Indian J. Chem , 14: 1004 (1976); V.Y. Sosnovskykh, et al., Geterotskikl Soedin, 6.847-849 (1998), Q -F Wang, et al , Tetrahedron Lett., 39(16).2377-2380 (1998); and S. Eguchi, et al., Heterocvcles, 42(l).333-339 (1996). For example, 0 08mol of ethylenediamine is added to 0.02mol CF₃C(O)CH₂C(O)-2-[(4-NO₂)thιenyl] in 30mL of glacial acetic acid at RT. The resulting solution is heated to reflux for 2h. The solution is then cooled to RT, poured into 250mL water and extracted with three

50mL ahquots of diethyl ether to remove unreacted beta-diketone. The aqueous layer is neutralized with a 20% NaOH solution and is then extracted with benzene. The 1,4-dιazepme base thus obtained is crystallized from benzene or puπfied by flash chromatography on silica gel Active HDA inhibiting compounds include TAN- 1746, HC-toxin, chlamydocin, WF-3161, trapoxin A, Cly-2 and analogues thereof. However, analogues of such compounds are good sources of test compounds for screening by the methods of this invention.

Apicidin la, lb, Ic are descπbed in pending applications USSN 08/281,325 filed July 27, 1994 and 08/447,664 filed May 23, 1995. They are produced from a strain of Fusanum as disclosed in the above mentioned applications. Histone acetyl transferase inhibitors are useful as antiprotozoal agents. As such, they can be used in the treatment and prevention of protozoal diseases in human and animals, including poultry. Examples of protozoal diseases against which histone acetyl transferase inhibitors may be used, and their respective causative pathogens, include 1) amoebiasis (Dientamoeba sp., Entamoeba histolytica); 2) giardiasis (Giardia lambha), 3) malaria (Plasmodium species including P vivax, P falciparum, P malanae and P. ovale); 4) leishmaniasis (Leishmania species including L. donovam, L tropica, L mexicana, and L. brazihensis); 5) trypanosomiasis and Chagas disease (Trypanosoma species including T brucei, T theile , T. rhodesiense, T gambiense, T. evansi, T. equiperdum, T. equinwn, T. congolense, T. vivax and T cnizi); 6) toxoplasmosis (Toxoplasma gondu); 7) neosporosis (Neospora caninwn); 8) babesiosis (Babesia sp.); 9) cryptospoπdiosis (Cryptosporidium sp ); 10) dysentary (Balantidium coli); 1 1) vaginitis (Trichomonas species including T.vaginitis, and T. foetus); 12) coccidiosis (Eimeria species including E. tenella, E. necatrix, E. acervuhna, E. maxima and E. brunetti, E. mitis, E bovis, E. melagramatis, and Isospora sp.); 13) enterohepatitis (Histomonas gallinarum), and 14) infections caused by Anaplasma sp., Besnoitia sp., Leucocytozoan sp., Microspoήdia sp., Sarcocystis sp., Theileria sp., and Pneumocystis carinii.

Histone acetyl transferase inhibitors are preferably used in the treatment or prevention of protozoal infections caused by a member of the sub- phylum Apicomplexans. More preferably histone acetyl transferase inhibitors are preferably used in the treatment or prevention of malaria, toxoplasmosis, and cryptospoπdiosis in humans and animals; and in the management of coccidiosis, particularly in poultry, either to treat coccidial infection or to prevent the occurrence of such infection. Further, although not caused by an Apicomplexan, trypanosomiasis may be treated by histone acetyl transferase inhibitors.

In the case that a histone acetyl transferase inhibitor is expected to be administered on a chronic basis, such as in the prevention of coccidiosis in poultry, the histone acetyl transferase inhibitor preferably is selective for protozoal over the host histone acetyl transferase. Long term administration of such a selective inhibitor would minimize adverse effects to the host due to histone acetyl transferase inhibition.

Two specific examples of using histone acetyl transferase inhibitors to prevent the establishment of parasitic infections in humans and animals are 1) the prevention of Plasmodium (malaπa) infection in humans in endemic areas and 2) the prevention of coccidiosis in poultry by administeπng the compound continuously in the feed or drinking water Malaπa is the number one cause of death in the world. The disease is transmitted by mosquitoes in endemic areas and can very rapidly progress to a life threatening infection. Therefore, individuals living in or visiting areas where malaπa carrying mosquitoes are present routinely take prophylactic drugs to prevent infection The histone acetyl transferase inhibitor would be administered oi ally or parenterally one or more tιme(s) a day. The dose would range from about 0 01 mg/kg to about lOOmg/kg The compound could be administered for the entire period during which the patient or animal is at risk of acquiπng a parasitic infection. Coccidiosis is a disease that can occur in animals and is caused by several genera of coccidia The most economically important occurrence of coccidiosis is the disease in poultry Coccidiosis in poultry is caused by protozoan parasites of the genus Eimeria. The disease can spread quite rapidly throughout flocks of birds via contaminated feces. The parasites destroy gut tissue, thereby impaiπng nutπent absoφtion. An outbreak of coccidiosis in a poultry house can cause such dramatic economic losses for poultry producers that it has become standard practice to use anticoccidial agents prophylactically in the feed A histone acetyl transferase inhibitor would be administered in the feed or drinking water for, a portion of, or the entire life of the birds. The dose would range from about O.lppm to about 500ppm in the feed or water.

For treatment of established parasitic infections in humans or animals, the histone acetyl transferase inhibitor could be administered orally or parenterally once the infection is suspected or diagnosed. The treatment period would vary according to the specific parasitic disease and the seventy of the infection. In general the treatment would be continued until the parasites were eradicated and/or the symptoms of the disease were resolved. Two specific examples are the treatment of a 1) Cryptosporidium parvum infection in an animal or human and treatment of acute Plasmodium falciparum malaπa in humans Cryptosporidium parvum is a protozoan parasite that infects and destroys cells lining the intestinal tract of humans and animals. The infection establishes quite rapidly and has acute effects on the patient. In the case of humans, patients get severe dysentery for a period of 5-7 days. In immune compromised patients C. parvum infections can persist and can be life threatening. In animals C. parvum infection is the number one cause of death in young dairy calves. A C. parvum infection can be easily diagnosed by symptoms and examination of a stool sample. Once the disease is suspected and/or diagnosed treatment with a histone acetyl transferase inhibitor can be initiated. The dose would vary from about O.Olmg/kg to about 500mg/kg. Treatments would be one or more tιme(s) a day, orally or parenterally until the infection is eliminated Routinely this dosing peπod would be 1-3 weeks P falciparum causes acute life threatening malarial infections in humans The infection if left untreated can quite often result in death of the patient. A malaria infection can be easily diagnosed by symptoms and/or examination of a blood sample from the patient Treatment would be initiated following diagnosis. A histone acetyl transferase inhibitor would be administered one or more tιme(s) a day, orally or parenterally, until the infection was eliminated. The dose would range from about 0 01 mg/kg to about 200mg/kg.

Histone acetyl transferase inhibitors may be administered to a host in need of treatment in a manner similar to that used for other antiprotozoal agents; for example, they may be administered parenterally, orally, topically, or rectally. The dosage to be administered will vary according to the particular compound used, the infectious organism involved, the particular host, the seventy of the disease, physical condition of the host, and the selected route of administration; the appropriate dosage can be readily determined by a person skilled in the art For the treatment of protozoal diseases in human and animals, the dosage may range from about

O.Olmg/kg to about 500mg/kg For prophylactic use in human and animals, the dosage may range from about O.Olmg kg to about lOOmg/kg. For use as an anticoccidial agent, particularly in poultry, the compound is preferably administered in the animals' feed or dnnkmg water. The dosage ranges from about O.lppm to about 500ppm.

The compositions of the present invention compnse a histone acetyl transferase inhibitor and an inert earner. The compositions may be in the form of pharmaceutical compositions for human and vetennary usage, or in the form of feed composition for the control of coccidiosis in poultry. The term "composition" is intended to encompass a product compπsing the active ιngredιent(s), and the inert ιngredιent(s) that make up the earner, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions of one or more of the ingredients. The composition of the present invention thus includes a composition when made by admixing a histone acetyl transferase inhibitor and inert earner.

The pharmaceutical compositions of the present invention comprise a histone acetyl transferase inhibitor as an active ingredient, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients The compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administrations, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered The pharmaceutical compositions may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

In practical use, a histone acetyl transferase inhibitor can be combined as the active ingredient in intimate admixture with a pharmaceutical earner according to conventional pharmaceutical compounding techniques The earner may take a wide vaπety of forms depending on the form of preparation desired for administration, e.g , oral or parenteral (including intravenous).

In prepanng the compositions for oral dosage form, any of the usual pharmaceutical media may be employed. For example, in the case of oral liquid preparations such as suspensions, elixirs and solutions, water, glycols, oils, alcohols, flavoring agents, preservatives, coloπng agents and the like may be used, or in the case of oral solid preparations such as powders, capsules and tablets, earners such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubπcants, binders, disintegrating agents, and the like may be included. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical earners are obviously employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques. In addition to the common dosage forms set out above, histone acetyl transferase inhibitors may also be administered by controlled release means and/or delivery devices.

Pharmaceutical compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bπnging into association the active ingredient with the earner, which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid earners or finely divided solid earners or both, and then, if necessary, shaping the product into the desired presentation For example, a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be piepared by compressing, in a suitable machine, the active ingredient in a free- flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent Desirably, each tablet contains from about lmg to about 500mg of the active ingredient and each cachet or capsule contains from about lmg to about 500mg of the active ingredient.

Pharmaceutical compositions of the present invention suitable for parenteral administration may be prepared as solutions or suspensions of these active compounds in water suitably mixed with a surfactant such as hydroxypropylcellulose Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms

The pharmaceutical forms suitable for injectable use include steπle aqueous solutions or dispersions and steπle powders for the extemporaneous preparation of steπle injectable solutions or dispersions In all cases, the form must be stenle and must be fluid to the extent that easy synngabihty exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteπa and fungi. The earner can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

Suitable topical formulations include transdermal devices, aerosols, creams, ointments, lotions, dusting powders, and the like These formulations may be prepared via conventional methods containing the active ingredient. To illustrate, a cream or ointment is prepared by mixing sufficient quantities of hydrophilic matenal and water, containing from about 5-10% by weight of the compound, in sufficient quantities to produce a cream or ointment having the desired consistency.

Pharmaceutical compositions suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositoπes. Suitable earners include cocoa butter and other mateπals commonly used in the art, and the suppositones may be conveniently formed by admixture of the combination with the softened or melted carπer(s) followed by chilling and shaping moulds.

It should be understood that in addition to the aforementioned earner ingredients the pharmaceutical formulations described above may include, as appropnate, one or more additional earner ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the puφose of rendering the formulation isotonic with the blood of the intended recipient.

For use in the management of coccidiosis in poultry, a histone acetyl transferase inhibitor may be conveniently administered as a component of a feed composition. Suitable poultry feed composition will typically contain from about lppm to about lOOOppm, preferably from about 0.01% to about 0.1% percent, by weight of a histone acetyl transferase inhibitor. The optimum levels will naturally vary with the species of Eimeria involved, and can be readily determined by one skilled in the art. Levels of in poultry feed of from about 0.01% to about 0.1% by weight of the diet are especially useful in controlling the pathology associated with E. tenella, while the preferred concentration for similar control of mtestinal-dwellmg species is from about 0.01% to about 0.1% by weight of the diet Amounts of about 0.01% to about 0.1% by weight are advantageous in reducing the pathogenic effects of both cecal and intestinal coccidiosis.

In the preparation of poultry feed, a histone acetyl transferase inhibitor may be readily dispersed by mechanically mixing the same in finely ground form with the poultry feedstuff, or with an intermediate formulation (premix) that is subsequently blended with other components to prepare the final poultry feedstuff that is fed to the poultry. Typical components of poultry feedstuff include molasses, fermentation residues, corn meal, ground and rolled oats, wheat shorts and middlings, alfalfa, clover and meat scraps, together with mineral supplements such as bone meal, calcium carbonate and vitamins.

Compositions containing a compound of formula I may also be prepared in powder or liquid concentrate form. In accordance with standard veteπnary formulation practice, conventional water soluble excipients, such as lactose or sucrose, may be incoφorated in the powders to improve their physical properties. Thus particularly suitable powders of this invention compπse about 50 to 100% w/w, and preferably 60% to 80% w/w of the combination together with 0 to 50% w/w and preferably 20% to 40% w/w of conventional vetennary excipients. These powders may either be added to animal feedstuff, for example by way of an intermediate premix, or diluted in animal dπnking water.

Liquid concentrates of this invention suitably contain a water-soluble compound combination and may optionally include a veteπnanly acceptable water miscible solvent, for example polyethylene glycol, propylene glycol, glycerol, glycerol formal or such a solvent mixed with up to 30% v/v of ethanol. The liquid concentrates may be administered to the dπnking water of animals, particulaily poultry.

The following non-limiting examples are provided to illustrate the invention. In the following Examples, anti-histone H4 sera were prepared according to B.M Turner, et al., FEBS Lett., 253, 141-145 (1989). Apicidin was prepared according to the procedure descnbed in U.S. Patent No 5,620,953. All temperatures expressed herein are in °C unless specifically stated otherwise. All percentages expressed herein are in percent by weight unless specifically stated otherwise.

EXAMPLE 1 ELISA test to detect inhibition of Apicidin induced hyperacetylation

Madin Derby Chick Kidney (MDBK) cells were seeded at

25,000cells/mL in MEM (Gibco) with the following additions- I) 10% fetal calf serum (FCS), π) ImM Sodium Pyruvate, in) lOmM HEPES, iv) 0.1 mM MEM non-essential Ammo Acids Solution, v) lOOunits/ml Penicillin, and vi) lOOμg/ml Streptomycin. The cells were seeded into 96 well plates at lOOμL per well. Freshly prepared E. tenella sporozoites (20,000/well) were added to the host cells and after 3h the infected cells were washed to remove extracellular sporozoites. Fresh media as descnbed above but with 2% heat-inactivated FCS (instead of 10%) added The infected cultures were incubated at 41°C for 48h.

After 48 hours, the cells were treated with a mixture of 250ng/mL Apicidin and the test compound in a suitable solvent such as DMSO. The resulting treated cells were then incubated at 41°C for 5 hours, and washed with PBS (phosphate buffered saline) 3 times. The washed cells were lysed by 3 successive rounds of freeze thawing followed by being blocked with 5% fat free dπed milk in PBS with 0.5% Tween® 20 (polyoxyethylene (20) sorbitan monolaurate) at 200μL/well for 1 hour at 37°C with shaking. The blocking solution was removed and primary antibodies added (anti-hyperacetylated Histone H4 diluted 1:250 in 1% milk/PBS with 0.5% Tween® 20 at lOOμLΛvell) for 1 hour at 37°C with shaking. The wells were then washed 6 times with a solution of 0.5% Tween® 20 in PBS and a secondary antibody added The secondary antibody was donkey anti-rabbit conjugated to hoiseradish peroxidase, diluted at 1 10,000 in 1% milk in PBS containing 0.5% Tween® 20 lOOμL of the diluted secondary antibody was added per well followed by incubation for 1 hour at 37°C with shaking Cells were then washed 3 times with a 0 5% Tween® 20 solution in PBS, followed by 3 more washings with PBS alone To each plate was added 150μL of a K-blue substrate solution available from Ehsa Technologies. The plates were then incubated for 30 minutes at room temperature. The optical density at 570nm was determined, which provided a measure of acetylation

EXAMPLE 2 Histone Acetyl Transferase Inhibition Assay

HAT assay on unsporulated oocysts Preparation of the lysate.

E tenella oocysts Approximately 2 x 109 E tenella oocysts were suspended in 5mL of 50 mM HΕPΕS pH 7 4. Next, 4mL of an equal mixture (vol/vol) of 4.0mm and 1.0mm glass beads was added The resulting glass bead oocyst mixture was then shaken for 20 minutes to cause disruption of the oocysts. The efficiency of breakage was checked microscopically. The resulting homogenate was separated from the glass beads and centnfuged at 3000 x g for lOmin The pellet was then mixed with 20mL 50mM Tπs (Trιs(hydroxymethyl)amιnomethane) pH 8, 0 25M sucrose, 0 5M NaCl, ImM dithiothreitol (DTT), 15mM MgCl2, and 0 ImM phenylmethylsulfonyl fluoride (PMSF). The resulting mixture was stirred for 30mιn on ice, centnfuged at 100,000 x g for lhr at 4°C and the supernatant dialyzed overnight at 4°C against 50mM Tπs pH 8, ImM DTT, 15mM MgCl2, and O.lmM PMSF. The dialysate was made 10% in glycerol, ahquoted, and was then frozen at -80°C or used immediately.

The assay:

About lOμL of extract containing HAT was mixed with Buffer A, which was the combination of l) 50mM Tπs pH 8, n) 0.25M sucrose, in) ImM DTT, iv) 15mM MgCb, and v) 0 ImM PMSF to a volume of 40μL. To the resulting mixture was added 5μL of 200mM butyric acid, followed by adding 25μg calf thymus histones (5μL of 5mg/ml histones in Buffer A) Next, the test compound was added at a known concentration in a suitable solvent such as DMSO A dilute label ( H acetyl CoA) was made at a dilution of 1.10 in buffer A to a level of O.OlμCi/μL Reaction was started by adding 5μL of H acetyl CoA (50nCι, 3Cι/mmol) After lOmin at 30°C the resulting mixture was then spotted onto Whatman™ P81 filters The filters were washed with lOOmL 50mM NaH₂CO₃/Na2HCθ3 pH 9 2 for 30 minutes, followed by washing sequentially with acetone then 1.2 v/v chloroform methanol Then, they were dried and radioactivity was measured by liquid scintillation counting

Alternatively, after completion of the incubation, the sample was mixed with an equal volume of Tns-Glycine SDS Sample buffer available from Novex and analyzed by SDS gel electrophoresis followed by detection of labeled histones by fluorography or autoradiography, as known in the art Inhibition of the histone acetyl transferase was measured by a reduction in the number of counts per minute associated with histones either trapped on the filter or in a protein of appropπate molecular weight.

Gel electrophoresis:

Acid urea tnton (AUT) polyacryl amide gels were performed according to the methods of Alfageme, et al., J. Bwl Chem. 249, 3729, (1974) with modifications as descπbed in Lennox and Cohen, Methods in Enzymology, 170, 532- 549 (1989). For optimal resolution of MDBK cell and E. tenella histones, the separation gel contained I) 7.5M urea, n) 12% acrylamide, in) 0.38% Triton® X 100 available from Aldnch Chemical Company, Milwaukee, Wisconsin, iv) 0.8% bis acrylamide, and v) 0 87M acetic acid The loading gels contained I) 7.5M urea, n) 0.37% Tnton® X 100, in) 6% acrylamide, iv) 0 04% bis acrylamide and v) 0.87M acetic acid Gels were run m 1M acetic acid Gels were pre-electrophoresed at 350V for 1 hour and run at 450V for 3.5 hours. Gels were either stained with Coomassie® Brilliant Blue R available from Aldnch Chemical Company, Milwaukee, Wisconsin in 7% acetic acid, 20% methanol and then destained in 7% acetic acid and 20% methanol, or treated with Enlightening (from New England Nuclear), dned and radiolabel detected by fluorography.

Claims

WHAT IS CLAIMED IS:

1. A method for identifying compounds having antiprotozoal activity, said method comprising the steps of: contacting a first amount of an histone acetyl transferase with (l) a second amount of an identifiably labeled compound that interacts with histone acetyl transferases, and (n) a third amount of a test compound or composition; and quantitating said labeled compound effective to determine a level of histone acetyl transferase activity, wherein said level is induced by said third amount

2. The method of Claim 1 wherein said labeled compound binds to histone acetyl transferase.

3. The method of Claim 1 wherein said labeled compound is a substrate of histone acetyl transferase

4. The method of Claim 1 wherein said histone acetyl transferase is a protozoal histone acetyl transferase.

5 The method of Claim 4, further comprising the steps of: contacting a fourth amount of a host histone acetyl transferase with (l) a fifth amount of said identifiably labeled compound; and (n) a sixth amount of said test compound or composition; and quantitating said labeled compound effective to determine a level of host histone acetyl transferase activity, wherein said level is induced by said sixth amount

6. A method for identifying compounds having antiprotozoal activity, said method comprising the steps of: contacting histone with I) a first amount of an histone acetyl transferase, (n) a second amount of a histone deacetylase, (in) a third amount of a histone deacetylase inhibitor, (iv) a fourth amount of a test compound or composition, and (in) a fifth amount of an antibody that interacts with acetylated histone; and quantitating said antibody effective to determine a level of acetylated histone, wherein said level is induced by said fourth amount.

7. The method according to Claim 6, further including the pπor step of. determining a hyperacetylation level against which is compared said level of acetylated histone.

8. The method of Claim 6 wherein said histone acetyl transferase is a protozoal histone acetyl transferase.

9. The method of Claim 8, further comprising the steps of: contacting histone with l) a sixth amount of a host histone acetyl transferase, (u) a seventh amount of a histone deacetylase, (in) an eighth amount of a histone deacetylase inhibitor, (iv) a ninth amount of a test compound or composition, and (in) a tenth amount of an antibody that interacts with acetylated histone; and quantitating said antibody effective to determine a level of acetylated histone, wherein said level is induced by said ninth amount.

10. A method for identifying compounds having histone acetyl transferase inhibiting activity, said method compπsing the steps of: determining a reference histone hyperacetylation level in a histone/histone acetyl transferase/histone deacetylase/histone deacetylase inhibitor system; determining a test histone acetylation level in a histone/histone acetyl transferase/histone deacetylase/histone deacetylase inhibitor/test composition system; and companng the test level to the reference level.

11. The method of Claim 10, further including a step of: determining a basal histone acetylation level in a histone/histone acetyl transferase/histone deacetylase system; and wherein said comparing step includes comparing the test level to the basal level

12. A method for identifying compounds having histone acetyl transferase inhibiting activity, said method comprising the steps of. determining a first histone acetylation level in a histone/histone acetyl transferase/histone deacetylase/test compound system at a first concentration of test compound; determining a second histone acetylation level in a histone/histone acetyl transferase/histone deacetylase/test compound system at a second concentration of test compound; and comparing said first and second histone acetylation levels.

13. A method for the treatment or prevention of a protozoal disease compπsing a step of administeπng to a host a therapeutically or prophylactically effective amount of a compound which inhibits histone acetyl transferase of the disease protozoa.

14. The method of Claim 13 wherein said compound inhibits protozoal histone acetyl transferase to a greater extent than host histone acetyl transferase.

15. The method of Claim 13 wherein said protozoal disease is caused by a protozoan belonging to the sub-phylum Apicomplexans.

16. The method of Claim 13 wherein said protozoal disease is coccidiosis, malana, cryptospondiosis or toxoplasmosis.

17. The method of Claim 13 wherein said compound is represented by chemical structure (I):

(I)

18. A method for identifying compounds having antiprotozoal activity compπsing

(a) contacting an intact host or protozoal cell with a test compound,

(b) disrupting said cell to obtain histones; and

(c) determining the level of histone acetylation

19. The method of Claim 18 wherein said test compound is a natural product extract.

20. The method of Claim 18 wherein the level of histone acetylation is determined using acid urea tnton gel electrophoresis.

21 A composition useful for the prevention or treatment of protozoal diseases which compnses an inert earner and an effective amount of a histone acetyl transferase inhibitor.