WO2012178036A2

WO2012178036A2 - Inhibitors of rtt109 as anti-fungal agents

Info

Publication number: WO2012178036A2
Application number: PCT/US2012/043785
Authority: WO
Inventors: Zhiguo Zhang; Andrew H. Limper; Junhong HAN; Hui Zhou; Theodore J. KOTTOM; Derek Hook; Judith BERMAN; Michael Anthony Walters
Original assignee: Mayo Foundation For Medical Education And Research; Regents Of The University Of Minnesota
Priority date: 2011-06-22
Filing date: 2012-06-22
Publication date: 2012-12-27
Also published as: WO2012178036A3

Abstract

This document relates to materials and methods for identifying chemical inhibitors of fungal targets useful for treating organisms infected with opportunistic fungal pathogens. For example, this document provides the materials and methods for identifying and optimizing chemical inhibitors of Rtt109, a fungal histone acetyltransferase. This document also provides the materials and methods for using high throughput screening technologies to identify candidate chemical inhibitors of Rtt109 as well as the materials and methods for evaluating candidate chemical inhibitors of Rtt109 in secondary screens for their binding efficacy, molecular specificity, and cellular toxicity.

Description

Inhibitors of Rttl09 as Anti-Fungal Agents

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. Provisional Appl. No. 61 /499,940, filed June 22, 201 1 , which is incorporated by reference in its entirety.

TECHNICAL FIELD

This document relates to the materials and methods involved in identifying chemical inhibitors of fungal targets for treating organisms infected with opportunistic fungal pathogens.

BACKGROUND

Fungal infections in humans have increased significantly in the last century, mainly due to the increased number of immunocompromised patients, including individuals infected with HIV, transplant patients, and cancer patients treated with chemotherapeutic agents. Despite significant advances, the mortality rate from opportunistic fungal infections exceeds 50% in most human studies, and can be as high as 90%), as reported for bone marrow transplant recipients infected with Aspergillus species.

At least four major factors have impeded progress towards treating fungal infection. First, fungal pathogens are eukaryotes, and many genes involved in critical biological processes in fungi are conserved in humans. Therefore, many antifungal drugs can be toxic to humans. Second, the kingdom of fungi consists of many species and each has sophisticated strategies to survive the host and evade its immune system. No standardized vaccines have been identified for preventing fungal infections in humans.

Third, it is often difficult to diagnose and confirm invasive fungal infections. Antifungal drugs are often prescribed prophylactically because of the high cost and mortality rate associated with fungal infection. Finally, fungal species can develop resistance to the most frequently used anti-fungal agents. SUMMARY

Provided herein is a method of treating a fungal infection in a patient, the method comprising administering to the patient a therapeutically effective compound of formu la ( 1 ) or formula (2).

A compound of formul

or a pharmaceutical ly acceptable salt form thereof,

wherein :

R¹ is selected from the group consisting of: hydrogen, substituted or unsubstituted

(Ci -C6)alkyl, substituted or unsubstituted (C2-C6)alkenyl, substituted .or unsubstituted

(C2-C6)alkynyl, substituted or unsubstituted (C₃-C₇)cycloalkyl, and substituted or unsubstituted (C3-C] 4)aryl;

R² is selected from the group consisting of: hydrogen, substituted or unsubstituted

(C| -C6)alkyl, substituted or unsubstituted (C2-C6)alkenyl, substituted or unsubstituted (C2-C6)alkynyl, substituted or unsubstituted (C3-C7)cycloalkyl;

R³, R⁴ and R⁵ are independently selected from the group consisting of: hydrogen,

(C, -C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₃-C ₁₄)aryl, -(CH₂)(C₃-C,₄)aryl halogen, -CN, -N0₂, -NR⁸ ₂, and -OR⁸;

R⁶ and R⁷ are independently selected from the group consisting of hydrogen,

(C| -C₆)alkyl, (C₂-C₆)aikenyl, (C₂-C₆)alkynyl, halogen, -CN, -N0₂, -NR⁸ ₂, and -

OR⁸, or R⁶ and R⁷ can come together to form a substituted or unsubstituted fused (C₄-

Ce) ring structure; and

each R⁸ is independently selected from the group consisting of hydrogen and

(C, -C₆)alkyl. In some embod iments, R¹ is selected from the group consisting of hydrogen, substituted or unsubstituted (C | -C6)alkyl, and substituted or unsubstituted (C3-Ci₄)aryl. For example, R ¹ can be a substituted (C3-Ci4)aryl, such as a a para-cumenyl moiety.

I n some embodiments, R² is selected from hydrogen or substituted or

unsubstituted (C | -C6)alkyl. For example, R² can be hydrogen, methyl, or isopropyl.

I n some embodiments, R³ is hydrogen. In some embodiments, R⁴ is selected from hydrogen and halogen. In some embodiments, R⁵ is hydrogen or -OR⁸. For example, R⁵ can be hydrogen, -OH, or -OCH₃. In some embodiments, R⁶ and R⁷ come together to form an unsubstituted fused (C4-C6) ring structure. For example, the fused (C4-C6) ring structure can be an aryl ring.

A non-l im iting example of a compound of formula ( 1 ) includes:

or a pharmaceutical ly acceptable salt form thereof.

A compound of formula (2):

or a pharmaceutically acceptable salt form thereof,

wherein:

X is selected from O, S, and NR' ³ ;

R⁹ is selected from the group consisting of: substituted or unsubstituted (C| -C6)alkyl, substituted or unsubstituted (C₂-C6)alkenyl, substituted or unsubstituted

(C2-C6)alkynyl, substituted or unsubstituted (C3-C7)cycloalkyl, substituted or unsubstituted (C₃-C₇)heterocycloalkyl, substituted or unsubstituted (C₃-C i 4)aryl, and substituted or unsubstituted (C3-Ci4)heteroaryl;

R¹⁰ and R¹ ' are independently selected from the group consisting of: hydrogen,

(C, -C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, halogen, -CN, -N0₂, -NR¹³ ₂, and -

OR¹³, or R¹⁰ and R" can come together to form a substituted or unsubstituted fused

(C4-C6) ring structure;

R¹² is selected from the group consisting of: hydrogen, (Ci-C6)alkyl, (C₂-C6)alkenyl,

(C₂-C₆)alkynyl, halogen, -CN, -N0₂, -NR¹³ ₂, and -OR¹³; and

each R¹³ is independently selected from the group consisting of hydrogen and

(C,-C₆)alkyl.

In some embodiments, X is S. In some embodiments, R⁹ is a substituted or unsubstituted (C3-C₇)heterocycloalkyl or substituted or unsubstituted (C₃-Ci4)heteroaryl. In some embodiments, R¹⁰ and R" come together to form an unsubstituted fused (C4-C₆) ring structure. For example, the fused (C4-C6) ring structure can be an aryl ring. In some embodiments, R¹² is hydrogen.

A non-limiting example of a compound of formula (2) includes:

or a pharmaceutically acceptable salt form thereof.

The compounds and compositions provided herein can also be useful to inhibit inhibiting a fungal infection in a patient, fungal growth in a patient, and Rtt 109 activity in a cell.

In some embodiments, the patient is immunocompromised.

In some embodiments, the fungal infection is caused by a fungal organism selected from C. albicans, P. carinii, and A. f migatus.

Further provided herein is a method for screening a test compound for anti fungal activity, the method comprising determining if the test compound inhibits the histone acety transferase activity of a fungal Rtt 109 polypeptide, wherein the fungal Rtt 109 polypeptide has at least 80% identity to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 -6.

In some embodiments, the fungal Rtt l 09 polypeptide has at least about 85% sequence identity to a polypeptide comprising an amino acid selected from the group consisting of SEQ I D NOs: 1 -6. In some embodiments, the fungal Rttl 09 polypeptide has at least about 90% sequence identity to a polypeptide comprising an amino acid selected from the group consisting of SEQ ID NOs: 1 -6. In some embodiments, the fungal Rtt l 09 polypeptide has at least about 95% sequence identity to a polypeptide comprising an amino acid selected from the group consisting of SEQ I D NOs: 1 -6. In some embodiments, the fungal Rtt l 09 polypeptide has at least about 98% sequence identity to a polypeptide comprising an amino acid selected from the group consisting of SEQ ID NOs: 1 -6. In some embodiments, the fungal Rttl 09 polypeptide is selected from the group consisting of SEQ ID NOs: 1 -6. In some embodiments, the fungal Rtt 109 polypeptide is complexed with Vps75.

In some embodiments, the determining comprises contacting the test compound with a composition comprising a fungal Rtt l 09, a substrate for a fungal Rtt 109 polypeptide, and acetyl coenzyme A; and determining the level of a de-acetylated coenzyme A.

A suitable substrate for the screening assay includes a fungal histone. For example, a lysine containing histone. In some embodiments, the lysine containing histone is an Asf- 1 -H3-H4 complex or H3K56.

De-acetylated coenzyme A can be detected by labeling either the coenzyme A itsel f or the de-acetylated coenzyme A. For example, the assay solution can further include a label. (e.g., a fluorophore) that reacts with de-acetylated coenzyme A to form a detectable complex.

In some embodiments, the method further comprises determining whether the test compound inhibits a human p300/CBP polypeptide. For example, the test compound can exhibit preferential inhibition of the fungal Rttl 09 activity compared to human p300/CBP activity. In some embodiments, the method further includes determining whether the test compound inhibits a yeast Gcn5 polypeptide. In some embodiments, the test compound inhibits growth of a fungal species. For example, the test compound inhibits growth of a fungal species selected from C. albicans, P. carinii, and A. fumigatus.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

Figures 1 A-C depict the results of spot test experiments. Expression of Rtt 109 from different fungal species suppressed the growth inhibitory effects of camptothecin (CPT). Expression of scRtt l 09 suppressed CPT sensitivity of C. albicans cells lacking Rtt 109 (A). Expression of pcRtt l 09 (B) and afRtt l 09 (C) suppressed the CPT sensitivity of S. cerevisiae cells lacking Rttl 09. Ten-fold serial dilutions of C. albicans (A) or S. cerevisiae cells (B and C) expressing Rttl 09 were spotted onto media with or without CPT. Cell growth was recorded after a two-day incubation period.

Figure 2 depicts the reduced toxicity against human cells by C. albicans cells lacking both copies of Rtt 109 (rttl 09-/-). Three different wild type C. albicans strains, a strain containing one copy of the Rttl 09 gene (rttl 09+/-), and one lacking both copies of the Rtt 109 gene (rtt 109-/-) were used to infect H4 enterocytes. The infected enterocytes' lactate dehydrogenease (LDH) was quantified and used to measure the extent of tissue damage as a result of infection.

Figure 3 depicts how the Rttl 09-Vps75 complex utilizes the Asfl -H3-H4 complex as the substrate for acetylation.

Figures 4 depicts an inhibitor against Rtt 109 preferentially inhibits the activity of Rtt l 09 over p300/CBP (A) and Gcn5 (B) . Using Asf-H3-H4 as the substrate, candidate Rtt l 09 inhibitors were assessed for their ability to inhibit p300, scRttl 09-Vps75 and Gcn5.

Figures 5 A-D depict the results^' of experiments assessing three identified Rtt 1 09 inhibitors against the growth of three fungal species. Each compound ( 100 μΜ) was added to exponentially growing S. cerevisiae (A) and C. albicans (B). After the indicated incubation time (hours), the cell density was determ ined (A-B). Growth effects of these inhibitors against P. Carinii (C) was measured using real-time PCR by detecting fungal spec i fic heat-shock protein 70 mRNA, against A. fumigatus (D) using spot tests.

Figure 6 is a bar graph depicting the results of experiments testing the toxicity of the tt 1 09 inhibitors on human cells. After overnight incubation, the indicated concentration of each Rtt l 09 inhibitor was added to cultures of human lung epithel ial cells (l ine A549). Cell prol iferation and viabi l ity was then measured and quantified.

Figure 7 is a graphical representation outlining the screening strategy for identi fying Rtt 1 09 inhibitors, includ ing secondary screens and SAR compound optimization.

Figure 8 shows the sequence homology of Rttl 09 polypeptides from di fferent fungal species.

DETAILED DESCRIPTION

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary ski ll in the art to which this d isclosure belongs. A ll patents, applications, published appl ications, and other publications cited herein are incorporated by reference in their entirety. In the event that there is a plural ity of definitions for terms cited herein, those in this section prevail unless otherwise stated .

For the terms "for example" and "such as," and grammatical equivalences thereof, the phrase "and without l imitation" is understood to follow unless expl icitly stated otherwise. As used herein, the term "about" is meant to account for variations due to experimental error. A l l measurements reported herein are understood to be modified by the term "about", whether or not the term is explicitly used, unless explicitly stated otherwise. As used herein, the singular forms "a," "an," and "the" include plural referents un less the context clearly d ictates otherwise: A "patient," as used herein, includes both humans and other animals, particularly mammals. Thus the methods are applicable to both human therapy and veterinary applications. In some embodiments, the patient is a mammal, for example, a primate. In some embodiments, the patient is a human.

The terms "treating" and "treatment" mean causing a therapeutically beneficial effect, such as ameliorating existing symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, postponing or preventing the further development of a disorder and/or reducing the severity of symptoms that will or are expected to develop.

A "therapeutically effective" amount of the compounds described herein is typically one which is sufficient to achieve the desired effect and may vary according to the nature and severity of the disease condition, and the potency of the compound. It will be appreciated that different concentrations may be employed for prophylaxis than for treatment of an active disease.

The term "contacting" means bringing at least two moieties together, whether in an in vitro system or an in vivo system.

As used herein, "antifungal activity" refers to inhibition of fungal growth and infection. In some embodiments, the term antifungal activity can include prevention of antifungal infection in a patient.

In general, reference to a certain element such as hydrogen or H is meant to include all isotopes of that element. For example if a R group is defined to represent hydrogen or H, it also includes deuterium and tritium.

The term "alky!" includes straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.) and branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, a straight chain or branched chain alkyl has 6 or fewer carbon atoms in its backbone (e.g., Ci-e for straight chain, C3.10 for branched chain). The term Ci_-6 includes alkyl groups containing 1 to 6 carbon atoms. The term "alkenyl" includes aliphatic groups containing at least one double bond and at least two carbon atoms. For example, the term "alkenyl" includes straight-chain alkenyl groups (e.g. , ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc. ) and branched-chain alkenyl groups. In certain embodiments, a straight chain or branched chain alkenyl group has 6 or fewer carbon atoms in its backbone (e.g. , Ci-β for straight chain, C3.6 for branched chain). The term C2-6 includes alkenyl groups containing 2 to 6 carbon atoms.

The term "alkynyl" includes unsaturated aliphatic groups analogous in length to the alkyls described above, but which contain at least one triple bond and two carbon atoms. For example, the term "alkynyl" includes straight-chain alkynyl groups (e.g. , ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc. ) and branched-chain alkynyl groups. In certain embodiments, a straight chain or branched chain alkynyl group has 6 or fewer carbon atoms in its backbone (e.g. , C2-6 for straight chain, C3.6 for branched chain). The term C₂-6 includes alkynyl groups contain ing 2 to 6 carbon atoms.

The term "cycloalkyl" includes a cyclic aliphatic group which may be saturated or unsaturated. For example, cycloalkyl groups include cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyi, and cyclooctyl. In some embodiments, cycloalkyls have from 3 - 8 carbon atoms in their ring structure, for example, they can have 3, 4, 5 or 6 carbons in the ring structure.

In general, the term "aryl" includes groups, including 5- and 6-membered single- ring aromatic groups, such as benzene and phenyl. Furthermore, the term "aryl" includes multicyclic aryl groups, e.g., tricyclic, bicyclic, such as naphthalene and anthracene.

The term "heteroaryl" includes groups, including 5- and 6- membered single-ring aromatic groups, that have from one to four heteroatoms, for example, pyrrole, furan, thiophene, thiazole, isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like. Furthermore, the term "heteroaryl" includes multicyclic heteroaryl groups, e.g., tricycl ic, bicycl ic, such as

t

benzoxazole, benzodioxazole, benzoth iazole, benzoimidazole, benzothiophene, methylenedioxyphenyl, quinol ine, isoquinoline, napthyridine, indole, benzofuran, purine, benzofuran, quinazoline, deazapurine, indazole, or indolizine.

The term "heterocycloalkyl" includes groups, including but not lim ited to, 3 - to 10-membered single or multiple rings having one to five heteroatoms, for example, piperazine, pyrrolidine, piperidine, or homopiperazine.

The term "substituted" means that an atom or group of atoms formally replaces hydrogen as a "substituent" attached to another group. For aryl and heteroaryl groups, the term "substituted", unless otherwise indicated, refers to any level of substitution, namely mono, di, tri, tetra, or penta substitution, where such substitution is permitted. The substituents are independently selected, and substitution may be at any chemical ly accessible position. In some cases two sites of substitution may come together to form a 3- 1 0 membered cycloalkyl or heterocycloalkyl ring.

As used herein, "adm inistration" refers to delivery of a compound or composition as described herein by any external route, including, without limitation, IV, intramuscular, SC, intranasal, inhalation, transdermal, oral, buccal, rectal, sublingual, and parenteral adm inistration.

Compounds

Provided herein are compo

or a pharmaceutically acceptable salt form thereof,

wherein:

is selected from the group consisting of: hydrogen, substituted or unsubstituted

(C| -C6)alkyl, substituted or unsubstituted (C2-C6)alkenyl, substituted or unsubstituted (C2-C6)alkynyl, substituted or unsubstituted (C3-C7)cycloalkyl, and substituted or unsubstituted (C₃-C u)aryl ; R² is selected from the group consisting of: hydrogen, substituted or unsubstituted

(C|-C₆)alkyl, substituted or unsubstituted (C₂-C6)alkenyl, substituted or unsubstituted (C2-C₆)alkynyl, substituted or unsubstituted (C3-C₇)cycloalkyl;

R³, R^'1 and R³ are independently selected from the group consisting of: hydrogen,

(C, -C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C3-C, 4)aryl, -(CH₂)(C₃-C, 4)aryl halogen, -CN, ~N0₂, -NR⁸ ₂, and -OR⁸;

R⁶ and R⁷ are independently selected from the group consisting of hydrogen,

(C, -C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, halogen, -CN, -N0₂, -NR⁸ ₂, and - OR⁸, or R⁶ and R⁷ can come together to form a substituted or unsubstituted fused (C4- C6) ring structure; and

each R^s is independently selected from the group consisting of hydrogen and

(C, -C₆)alkyl.

In some embodiments, R¹ is selected from the group consisting of hydrogen, substituted or unsubstituted (Ci -C6)alkyl, and substituted or unsubstituted (QrC^aryl. In some embodiments, R¹ is a substituted (C3-Ci )aryl. For example, R¹ can be a para- cumenyl moiety.

I n some embodiments, R² is selected from hydrogen or substituted or

unsubstituted (C| -C₆)alkyl. For example, R² can be selected from the group consisting of hydrogen, methyl, and isopropyl. In some embodiments, R² is hydrogen.

In some embodiments, R³ is hydrogen. In some embodiments, R⁴ is selected from hydrogen and halogen. For example, R⁴ can be CI.

I n some embodiments, R⁵ is hydrogen or -OR⁸. For example, R⁵ can be selected from the group consisting of hydrogen, -OH, and -OCH₃.

In some embodiments, R⁶ and R⁷ come together to form an unsubstituted fused (C4-C6) ring structure. The ring structure can include cycloalkyi, heterocycloalkyi (e.g., nitrogen containing heterocycloalkyls), aryl, and heteroaryl (e.g., nitrogen containing heteroaryls). For example, the fused (C4-C6) ring structure can be an aryl ring.

A non-limiting example of a compound of formula ( 1 ) includes:

or a pharmaceutically acceptable salt form thereof.

Also provided herein are compounds according to formula (2):

or a pharmaceutically acceptable salt form thereof,

wherein:

X is selected from O, S, and NR.'³;

R⁹ is selected from the group consisting of: substituted or unsubstituted (Ci-C6)alkyl, substituted or unsubstituted (C₂-C6)alkenyl, substituted or unsubstituted

(C₂-C6)alkynyl, substituted or unsubstituted (C₃-C₇)cycloalkyl, substituted or unsubstituted (C3-C₇)heterocycloalkyl, substituted or unsubstituted (C3-C] 4)aryl, and substituted or unsubstituted (C3-Ci4)heteroaryl;

R'° and R " are independently selected from the group consisting of: hydrogen,

(C, -C₅)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, halogen, -CN, -N0₂, -NR^I ₂, and - OR¹³, or R¹⁰ and R^{1 1} can come together to form a substituted or unsubstituted fused (C4-C6) ring structure;

R¹² is selected from the group consisting of: hydrogen, (C| -C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, halogen, -CN, -N0₂, -NR¹³ ₂, and -OR¹³; and

each R¹³ is independently selected from the group consisting of hydrogen and

(C, -C₆)alkyl. In some embodiments, X is S. In some embodiments, R⁹ is a substituted or unsubstituted (C3-C7)heterocycloalkyl or substituted or unsubstituted (C3-C| 4)heteroaryl.

In some embodiments, R'° and R^{1 1} come together to form an unsubstituted fused (C₄-C₆) ring structure. The ring structure can include cycloalkyl, heterocycloalkyl (e.g., nitrogen containing heterocycloalkyls), aryl, and heteroaryl (e.g., nitrogen containing heteroaryls). For example, the fused (C4-C₆) ring structure can be an aryl ring.

I n some embodiments, R^{1 2} is hydrogen.

A non-limiting example of a compound of formula (2) includes:

2- 1 or a pharmaceutically acceptable salt form thereof.

Further provided herein is a compound of formula (3):

or a pharmaceutically acceptable salt form thereof,

wherein:

R¹⁴ is selected from the group consisting of: hydrogen, substituted or unsubstituted (C| -C6)alkyl, substituted or unsubstituted (C2-C6)alkenyl, substituted or unsubstituted (C2-C6)alkynyl, substituted or unsubstituted (C₃-C7)cycloalkyl; and

R¹⁵ and R ¹⁶ are independently a substituted or unsubstituted (C3-C]₄)aryl.

I n some embodiments, R ^{1 4} is a (C| -C6)alkyl. For example, R^{1 4} is CH3. In some embodiments, R ^{1 5} and R ^{1 6} are substituted with electron-withdrawing or electron-donating siibstitiitents. In some embodiments, R^{1 5} and R^{1 6} are an unsubstituted (C3-C i4)aryl. For example, R ^{1 5} and R ^{1 6} are phenyl.

A non-limiting example of a compound of formula (3) is:

or a pharmaceutically acceptable salt form thereof.

Compounds described herein, including pharmaceutically acceptable salts thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes.

The reactions for preparing the compounds described herein can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, e.g., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected by the skilled artisan.

Preparation of compounds can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Protecting Group

Chemistry, l ^sl Ed., Oxford University Press, 2000; and March 's Advanced Organic chemistry: Reactions, Mechanisms, and Structure, 5^th Ed., Wiley-lnterscience

Publication, 2001 (each of which is incorporated herein by reference in their entirety).

Pharmaceutically Acceptable Salts and Compositions

Pharmaceutical ly acceptable salts of the compounds described herein include the acid addition and base salts thereof.

Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the.acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate,

hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, hydrogen phosphate, isethionate, D- and L-lactate, malate, maleate. malonate, mesylate, methylsulphate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen, phosphate/phosphate dihydrogen,

pyroglutamate, saccharate, stearate, succinate, tannate, D- and L-tartrate, l -hydroxy-2- naphthoate tosylate and xinafoate salts.

Suitable base salts are formed from bases which form non-toxic salts. Exam ples include the aluminium, argini ne, benzathine, calcium, chol ine, diethylamine, d iolam ine. glycine, lysine, magnesium, meglum ine, olam ine, potassium, sodium, tromethamine and zinc salts.

Hemisalts of acids and bases may also be formed, for example, hemisu lphate and hem icalcium salts.

Compounds described herein intended for pharmaceutical use may be

adm inistered as crystalline or amorphous products. They may be obtained, for example, as sol id plugs, powders, or fi lms by methods such as precipitation, crystallization, freeze drying, spray drying, or evaporative drying. M icrowave or radio frequency drying may be used for this purpose.

The compounds may be administered alone or in combination with one or more other compounds described herein or in combination with one or more other drugs (or as any combination thereof)- Generally, they will be administered as a formulation in association with one or more pharmaceutically acceptable excipients. The term

"excipient" is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient wi ll to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubi lity and stabil ity, and the nature of the dosage form.

Non-l imiting examples of pharmaceutical excipients suitable for adm in istration of the compounds provided herein include any such carriers known to those ski lled in the art to be su itable for the particular mode of administration. Pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, alum ina, aluminum stearate, lecithin, self-emulsi fying drug delivery systems (SEDDS) such as d-a-tocopherol polyethylene glycol 1 000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other simi lar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium-chloride, zinc salts, colloidal silica, magnesium trisil icate, polyvinyl pyrrol idone, cellulose-based substances, polyethylene glycol, sod ium carboxymethyl cel lulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, and wool fat. Cyclodextrins such as α-, β, and γ-cyclodextrin, or chemical ly mod i fied derivatives such as hydroxyalkylcyclodextrins, including 2- and 3- hydroxypropyl-b-cyclodextrins, or other solubilized derivatives can also be

advantageously used to enhance delivery of compounds of the formulae described herein. I n some embodiments, the excipient is a physiologically acceptable saline solution.

The compositions can be, in one embodiment, formulated into suitable pharmaceutical preparations such as solutions, suspensions, tablets, dispersible tablets, pil ls, capsules, powders, sustained release formulations or elixirs, for oral administration or in steri le solutions or suspensions for parenteral administration, as well as transdermal patch preparation and dry powder inhalers (see, e.g., Ansel Introduction to

Pharmaceutical Dosage Forms, Fourth Edition 1985, 126).

The concentration of a compound in a pharmaceutical composition wi ll depend on absorption, inactivation and excretion rates of the compound, the physicochemical characteristics of the compound, the dosage schedule, and amount admin istered as wel l as other factors known to those of ski ll in the art.

The pharmaceutical composition may be administered at once, or may be divided into a number of smal ler doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular patient, specific dosage regimens should be adjusted over time according to the ind ividual need and the professional judgment of the person admin istering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to lim it the scope or practice of the claimed compositions.

The pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, steri le parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the compounds or pharmaceutical ly acceptable derivatives thereof. The pharmaceutically therapeutically active compounds and derivatives thereof are, in one embodiment, formulated and administered in unit- dosage forms or multiple-dosage forms. Unit-dose forms as used herein refers to physical ly discrete un its suitable for human and animal patients and packaged ind ividual ly as is known in the art. Each unit-dose contains a predetermined quantity of the therapeutically active compound sufficient to produce the desired therapeutic effect, in association with the requ ired pharmaceutical carrier, vehicle or diluent. Examples of unit-dose forms include ampoules and syringes and individually packaged tablets or capsu les. Unit-dose forms may be admin istered in fractions or multiples thereof. A multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.

Liqu id pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension. I f desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxi l iary substances such as wetting agents, emu lsi fying agents, solubi l izing agents, pH buffering agents and the like, for example, acetate, sod ium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolam ine sodium acetate, triethanolamine oleate, and other such agents.

Dosage forms or compositions containing.a compound as described herein in the range of 0.005% to 100% with the balance made up from non-toxic carrier may be prepared . Methods for preparation of these compositions are known to those ski lled in the art. The contemplated compositions may contain 0.001 %- 100% active ingredient, in one embodiment 0. 1 -95%o, in another embodiment 75-85%.

Pharmaceutical compositions suitable for the delivery of compounds described herein and methods for their preparation wi ll be readily apparent to those skil led in the art. Such compositions and methods for their preparation may be found, for example, in Remington's Pharmaceutical Sciences, 1 9th Edition (Mack Publ ishing Company, 1 995).

Methods of Use

The compounds and compositions provided herein can be used in a method o f treating and/or inhibiting a fungal infection in a patient. For example, the method can include adm inistering to the patient a therapeutically effective amount of a compound as provided herein. In some embodiments, inhibition of a fungal infection can include inh ibiting fungal growth in the patient.

A patient as provided herein can include a patient that is immunocompromised. For example, a patient infected with HIV/AIDS, transplant patients (e.g., patients undergoing introduction of al logenic and/or autologous stem cells, patients undergoing bone marrow transplantation, and patients undergoing organ transplatation), and cancer patients treated with chemotherapeutic agents.

A fungal infection can be caused by any fungal organism that causes infection in a patient. For example, the fungal infection can be caused by a fungal organism selected from C. albicans, non-albicans Candida species, P. carinii, and A. fumigatus.

In some embodiments, a compound or composition provided herein inh ibits the transferase activity of a fungal tt l 09 polypeptide.

A lso provided herein is a method of inhibiting a fungal Rtt l 09 polypeptide in a cel l . The method comprising contacting the cel l with an effective amount of a compound as provided herein. Uses of such in vitro methods include, but are not limited to, use in a screening assay (for example, wherein the compound is used as a positive control or standard compared to compounds of unknown activity or potency in any of the methods provided herein).

Screening: target compounds for antifungal activity

A lso provided herein are materials and methods involved in identi fying chemical inhibitors of fungal targets for treating organisms infected with opportunistic fungal pathogens. For example, this document provides the materials and methods for identi fyi ng and optim izing chemical inhibitors of Rtt l 09 polypeptide, a fungal histone acetyltransferase (see Fig. 7). This document also provides the materials and methods for using high throughput screen ing (HTS) technologies to identify candidate chemical inhibitors of fungal Rtt l 09 as well as the materials and methods for evaluating cand idate chemical inh ibitors of Rtt l 09 in secondary screens for their binding efficacy, molecular speci ficity, and cel lular toxicity.

As described herein, the histone acetyltransferase, Rttl 09, is a lysine

acetyltransferase (KAT) highly conserved in fungal species that exhibits no obvious sequence homology to other mammalian ATs. Compared to other histone

acetyltransferases (HATs) discovered in yeast or other organisms, Rtt l 09 is unique in several ways. First, Rtt l 09 does not appear to have a classic acetyl-CoA binding moti f found in other known HATs despite the fact that it utilizes acetyl-CoA as a co-factor. Second, i n contrast to most HATs that utilize free histones or nucleosomal histones as substrates, Rtt l 09 can use a heterotrimeric complex Asfl -H3-H4; Rtt l 09 can form a complex with Vps75 that uti lizes the Asfl -H3-H4 complex as the substrate during acetylation. Third, most HATs are conserved from yeast to humans, and yet the sequence homologs of Rtt l 09 appear not be present in humans. Finally, Rtt l 09 is critical for yeast cel ls to survive fol lowing treatment with DNA damaging agents. Taken together, Rtt l 09 may be a fungal-speci fic target that can be used to identify chemical inhibitors for treating organisms infected with opportunistic fungal pathogens. Using tt 109, chemical agents can be screened for their ability to inhibit or attenuate the function of the fungal target, which may compromise the health or survival of the fungal pathogen. Chemical agents can also disrupt the target's interactions with other proteins or protein complexes, which may compromise the health or survival of the fungal pathogen. As described herein, Rtt 109 forms a complex with Vps75 and uses another complex consisting of Asfl -H3-H4 as a substrate during acetylation in

Saccharomyces cerevisiae, which is required for DNA replication and cellular proli eration. In some cases, chemical agents can be screened for their ability to inhibit directly the function of Rtt 109 or for their ability to disrupt the interactions between Rtt l 09-Vps75 and its substrate Asfl -H3-H4.

In some embodiments, a HTS screening assay can be developed based on the activity of the fungal target. For example, the fungal target can be an enzyme that acts post-translationally in biochemical processes, including acetylation, phosphorylation, methylation. and ubiquitination. These modifications regulate a variety of essential cellular processes including gene transcription, cell proliferation and differentiation. The HTS screening assay can be designed to detect the presence or absence of the products of the enzymatic reaction catalyzed by the fungal target. The HTS screening assay can also be designed to detect the presence or absence of the by-products of the enzymatic reaction catalyzed by the fungal target.

For example, a screening assay can be used to identify a test compound having antifungal activity, the assay can include determining if the test compound inhibits the histone acetyltransferase activity of a fungal Rttl 09 polypeptide. A fungal Rtt l 09 polypeptide can include any polypeptide that has at least 80% identity (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, and at least about 99%) to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ I D NOs: 1 -6 (see Fig. 9). In some embodiments, a fungal Rtt l 09 polypeptide is selected from the group consisting of SEQ ID NOs: 1 -6. In some embodiments, a fungal Rtt l 09 is complexed with Vps75.

I n some embodiments, if the test compound inhibits the histone acetyltransferase activity of a fungal Rtt 109 polypeptide can be determined by contacting the test compound with a composition comprising a fungal Rtt 109 polypeptide, a substrate for a fungal Rtt 1 09 polypeptide, and acetyl coenzyme A; and determ ining the level of a de- acetylated coenzyme A.

A substrate for a fungal Rtt l 09 polypeptide can be any substrate that facil itates the activ ity of the polypeptide. For example, the substrate is a fungal histone, such as a lysine containing histone. I n some embodiments, the lysine containing histone is selected from an Asf- 1 -H3-H4 complex or H3 56.

Determining the level of a de-acetylated coenzyme A can be done by any method known to those of ski ll in the art. In some embodiments, a second reaction can be used in the HTS screening assay to detect the presence or absence of the products or by-products of the enzymatic reaction catalyzed by the fungal target (e.g., deacetylated coenzyme A).

For example, the presence or absence of the products or by-products of the enzymatic reaction catalyzed by the fungal target can be monitored or detected using fluorescence, chemi lum inescence, radioactivity, colorimetrics, and photometries. In some

embodiments, the coenzyme A is labeled. For example, the label can be a radioisotope or a fluorophore.

Suitable radionuclides include but are not limited to ²H (deuterium), ³H (tritium), " C, ^{l 3}C, ^UC, ^{, 3}N, ^{, 5}N, ^{, 5}0, ^{, 7}0, ^{l 8}0, ^{l 8}F, ³⁵S, ³⁶C1, ⁸²Br, ⁷⁵Br, ⁷Br, ⁷⁷Br, ^{, 23}I, ^{l 24}I, ^{, 25}I and ¹³¹ 1. I n some embodiments, the label is a radioisotope, such as ³H, C, ³⁵S, and ^{l 2i}I . I n some embodiments, the acetyl coenzyme A is ³H-acetyl coenzyme A.

Suitable fluorophores include, for example, xanthene derivatives (e.g., fluorescein, rhodam ine, Oregon green, eosin, Texas red, and Cal Fluor dyes), cyan ine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, and Quasar dyes), naphthalene derivatives (e.g., dansyl and prodan derivatives), coumarin derivatives, oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole), pyrene derivatives (e.g., cascade blue), oxazine derivatives (e.g., Nile red, Nile blue, cresyl violet, oxazine 1 70), acridine derivatives (e.g., proflavin, acridine orange, acridine yellow), arylmethine derivatives (e.g., auramine, crystal violet, malachite green), and tetrapyrrole derivatives (e.g., porphin, phtalocyanine, bi l irubin). In some embodiments, the assay solution can further ■ include a label that reacts with de-acetylated coenzyme A to form a detectable complex. For example, the assay solution can include 7-diethyIamino-3-(4'-maleimidylphenyl)-4- methylcoumarin).

In some cases, a HTS screening assay can be developed in which candidate chemical inhibitors of the fungal target are identified from among hundreds of thousands of chemical agents contained in chemical libraries. The candidate chemical inhibitors of fungal targets contained in chemical libraries can have known or unknown biological effects or activities both in vitro and in vivo. In some cases, the HTS screening assay can be performed on an automated or semi-automated platform capable of screening thousands of candidate chemical inhibitors daily. For example, automated or semi- automated screening platforms can detect or monitor the readout of a HTS screening assay within individual wells of a 96 or 384-well screening plate. The readout of the HTS can be captured and processed electronically using computers and computerized robotics.

In some embodiments, once a candidate chemical inhibitor of a fungal target has been identified, the candidate chemical inhibitor can be screened in one or more secondary assays that assess the compounds binding efficacy, molecular specificity, and cellular toxicity. In some embodiments, the secondary assay is a repeat of the primary screen. In some embodiments, the secondary screen is designed to evaluate a separate feature of the candidate chemical inhibitors. For example, secondary screens can be designed to evaluate the binding kinetics of candidate chemical inhibitors, including the rates at which substrates are bound and released, or whether the candidate chemical inhibitors are reversible or non-reversible inhibitors. Secondary screens can also be designed to evaluate the effects of candidate chemical inhibitors on similar enzymes from similar or di fferent species. Secondary screens can also be designed to evaluate candidate chemical inhibitors for their toxic effects on cells of similar or different species.

Suitable secondary screens can include, for example, determining whether the test compound inhibits a human p300/CBP polypeptide. In some embodiments, a desirable test compound exhibits preferential inhibition of the fungal Rttl 09 activity compared to human p300/CBP activity. Preferential inhibition of fungal Rtt l 09 over p3007CBP activity can indicate low toxicity of the compound to human patients. Another example of a secondary screen can include determining whether the test compound inhibits a yeast Gcn5 polypeptide.

The test compounds can also be examined to determine how each compound affects growth of one or more specific fungal species. For example, various doses of a target compound can be tested to determine how a particular does of the compound affects the growth of a fungal species. For example, the fungal species can be selected from C. albicans, P. carinii, and A. f migatus.

In general, once primary and secondary screens for chemical inhibitors of a fungal target have been performed, the candidate chemical inhibitors can be used as the basis for further optimization. For example, candidate chemical inhibitors can be organized into groups based on structural and binding properties. This information can be used to synthesize other candidate chemical inhibitors that incorporate or enhance the features necessary for efficient binding, potency, specificity, and reduced toxicity. The optimized candidate chemical inhibitors can then be evaluated in the same primary and secondary screening assays for their ability to modify fungal targets. In some cases, candidate or optimized candidate chemical inhibitors can be used on mammals as a therapeutic agent for treating opportunistic fungal pathogens.

EXAMPLES

Example 1 - Identifying and optimizing inhibitors for the histone acetyltransferase lt 109 complex

In vitro enzymatic assays for screening compounds that inhibit the activity of Rtt 109 were developed based on the biochemical function of Rttl 09, as schematized in Fig. 3. Using these assays, about 90,248 compounds were screened and 2 1 3 compounds were identified as having reasonable dose response. Eighteen compounds were counter- screened for inhibition of human p300 lysine acetyltransferase, and three lead compounds exhibited selective activity against scRtt ! 09. Example 2 - Complementation experiments using Rttl09 from different fungal species

The sequence homologs of scRttl09 have been identified from many fungal species (see Fig.8). In C. albicans, cells lacking both copies of tt 109 are less virulent than wild type controls (Fig.2). It was determined that expressing scRtt!09 in rtt 109-/- C. albicans cells rendered the mutant cells resistant to CPT (Fig.1 A) to a similar degree as expressing caRttl09. It was also determined that expressing pcRttl09 or afRttl09 in S. cerevisiae cells lacking scRttl09 led to resistance to CPT and restoration of H3K56Ac (Fig. IB). These results indicate that Rttl09 from these fungal species have similar functions and suggest that small molecule inhibitors against scRttl09 will also inhibit Rtt 109 in other species.

Example 3 - Enzymatic assay to identify small molecule inhibitors of Rtt 109

In S. cerevisiae, Rtt 109 is the catalytic subunit of the Rttl09-Vps75 that utilizes Asfl-H3-H4 complexes as substrates. Rtt 109 from other fungal species likely functions similarly. Therefore, to identify inhibitors against scRttl09, the Rttl09-Vps75 and Asf-1- H3-H4 complexes were purified and used as enzyme and substrate, respectively, for histone acetyltransferase assays (See Scheme 1).

Scheme 1

Assays conditions described in Trievel et al. were used with slight modifications (Trievel, R.C. et al.2000. Anal Biochem, 287, 319-29). Briefly, 15 nL DMSO or 10 μΜ test compound were added to wells of Corning 384-well plate using an ECHO 550 contactless liquid handler. Controls were added to the appropriate wells using the same dispensing method. Then 5 \xL IX HAT buffer (50 mM Tris-HCl, 0.1 mM EDTA, 50 in KG, pH 8.0) was added using a Multidrop 384 bulk reagent liquid dispenser.

Following this, Rttl09-Vsp75 (200 ng/well) and then Asfl-H3-H4 (800 ng/well), was added using the Multidrop. To initiate the reaction, 10 15 μΜ Acetyl-Coenzyme A was added to the entire plate. Following incubation at 30°C for 1 -2 hours, 5 of 80 μΜ CPM (final concentration 20μΜ) was added. The fluorescence intensity was measured using a M2E plate reader (Molecular Devices) with excitation at 405 nm and emission at 530 nm. The assay was validated for top-to-bottom and edge-to-edge variability using a control plate where either Rtt l 09-Vps75, Acetyl-CoA, or Asfl -H3-H4 were not added and using duplicated assay runs of the LOPAC compound collection (Sigma-Aldrich, St. Louis, MO) on two separate days. Using this method, 90,248 compounds were screened and a 1 .8% hit rate was achieved when 35% inhibition was set as the cutoff. Following the primary screen, 3 14 compounds were cherry-picked from the primary screening and 1 35 compounds were exhibited dose response. Dose-response confirmed hits were fi ltered to remove compounds that had low solubility (high molecular weight and/or high cLogP values), contained synthetically inaccessible functionalities, possessed known toxicophores, or were likely to be pan-assay interference compounds (PAINS). After this analysis, 50 compounds were purchased, 18 of which were selected and tested for inhibition of p300.

Example 4 - Testing candidate compounds for inhibition of p300/CBP

While scRtt l 09 does not share sequence homology with human p300/CBP, the structure of the catalytic domain of scRttl 09 is similar to that of p300/CBP despite the fact that scRtt l 09 and p300 likely utilize distinct mechanisms for catalysis. In addition, p300/CBP can acetylate H3 56 in human cells. Therefore, commercially available human p300 was obtained and a counter screening assay was developed to determine whether the compounds identified in the primary assay inhibit the activity of p300/CBP.

Eighteen compounds with lower ICso_s were chosen for counter-screening against human p300 using the same assay methodology for scRtt ! 09-Vps75 as described in Example 3.

Six compounds exhibited selective inhibition of scRtt l 09 over p300, one of which is shown in Fig. 4A.

Example 5 - Testing candidate compounds for inhibition of Gcn5

In addition to p300/CBP, human Gcn5 is another HAT that targets human H3 K56. I n S. cerevisiae, it was determined that cells lacking both Gcn5 and Rtt l 09 exhibited more severe growth defects than cells lacking scRtt l 09 alone. Six compounds that inh ibited the activity of scRtt l 09-Vps75 preferentially over human p300 were exam ined for their abi lity to inhibit yeast Gcn5 using an independent assay in which radioactive ³H- labled-acetyl-CoA was used. This assay detected reaction products of acetylated H3 directly using a scinti l lation counter. It was found that two compounds preferential ly inh ibited the activity of scRt l 09-Vps75 over Gcn5, one of which is shown in Fig. 4B .

Example 6 - Three compounds inhibit fungal pathogen growth but are not toxic to human cel ls

The effects on the growth of four fungal species, S. cerevisiae, C. albicans, P. carinii, and A. f igat s were examined. Dose response experiments were performed with each compound using liquid cultures. Concentrations ranging from 10- 100 μΜ of each compound inhibited growth. All three compounds inhibited the growth of S.

cerevisiae to a sim i lar extent. Two compounds (Compound 3- 1 and Compound 1 - 1 ) inhibited growth of C. albicans, whereas one compound (Compound 2- 1 ) exhibited partial activities (Fig. 5A-B).

To determine the effect of each of the three compounds on the growth of P.

carinii, about 1 ^χ 1 07 P. carinii cel ls were incubated in Ham's F 12 media in one wel l of a 6-wel l plate. After incubation at 37°C for 24 hours, 1 μg/mL Pentam idine (a known inhibitor of P. carinii growth) or each compound at different concentrations ( 1 0, 50, and 1 00 μ ) was added. After 24 hours, total RNA was isolated with Trizol reagents and cDNA was produced. The viability of organisms after treatment was monitored by measuring the mRNA of heat shock 70 protein (Hsp70) by quantitative real-time PCR. Compound 3- 1 inhibited the growth of P. carinii at all three concentrations tested, and

Compound 1 - 1 exhibited limited activity, whereas Compound 2- 1 had no apparent e ffect on the growth . It was also observed that Compound 3- 1 inhibited growth of A. fumigatus at 1 00 μΜ concentrations in vitro (Fig. 5C-D). Finally, none of these compounds exhibited signi ficant toxicity against human cells at concentrations that can inhibit growth of fungal species (Fig. 6). Example 7 - Deletion of Rttl 09 from C. albicans resulted in reduced virulence

C. albicans lacking Rtt 109 was tested for reduced virulence in a mammalian tissue culture model. This assay mimics the tissue injuries caused by C. albicans infection and takes advantage of the fact that epithelial cells damaged by C. albicans infection release lactate dehydrogenease (LDH) into the supernatant. Briefly, H4 enterocytes were cultured at a concentration of 2x 104 cells per well and grown to approximately 80% confluence in a 96-well flat-bottomed tissue culture plate. The enterocytes were infected with wild type C. albicans and a C. albicans mutant lacking one or both copies of Rtt 109 genes. The released LDH was quantified

spectrophotometrically using the Cyto-Tox-96® assay (Promega). C. albicans lacking both copies of the Rtt l 09 gene caused significantly less damage to enterocytes compared to C. albicans lacking just one copy of the Rtt 109 gene or compared different wild type yeast strains. This reduction was not due to slow growth of the rttl 09 -/- cells.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a fungal infection in a patient, the method comprising

administering to the patient a therapeutically effective compound of formula (1):

or a pharmaceutically acceptable salt form thereof,

wherein:

R¹ is selected from the group consisting of: hydrogen, substituted or unsubstituted (Ci-Ce)alkyl, substituted or unsubstituted (C₂-Ce)alkenyl, substituted or unsubstituted (C₂-Ce)alkynyl, substituted or unsubstituted (C₃-Cy)cycloalkyl, and substituted or unsubstituted (C₃-Ci₄)aryl;

R² is selected from the group consisting of: hydrogen, substituted or unsubstituted (Ci-Ce)alkyl, substituted or unsubstituted (C₂-Ce)alkenyl, substituted or unsubstituted (C₂-Ce)alkynyl, and substituted or unsubstituted (C₃-Cy)cycloalkyl;

R³, R⁴ and R⁵ are independently selected from the group consisting of: hydrogen, (Ci-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₃-d₄)aryl, -(CH₂)(C₃-Ci₄)aryl halogen, -CN, -N0₂, -N(R⁸)₂, and -OR⁸;

R⁶ and R⁷ are independently selected from the group consisting of hydrogen,

(Ci-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, halogen, -CN, -N0₂, -N(R⁸)₂, and -OR⁸, or R⁶ and R⁷ can come together to form a substituted or unsubstituted fused (C₄-C₆) ring structure; and

each R⁸ is independently selected from the group consisting of hydrogen and

(Ci-C₆)alkyl.

2. The method of claim 1, wherein R¹ is selected from the group consisting of

hydrogen, substituted or unsubstituted (Ci-Ce)alkyl, and substituted or unsubstituted (C₃-Ci₄)aryl.

3. The method of claim 2, wherein R¹ is a substituted (C₃-Ci₄)aryl.

4. The method of claim 3, wherein R¹ is a para-cumenyl moiety.

5. The method of claim 1, wherein R² is selected from hydrogen or substituted or unsubstituted (Ci-C6)alkyl.

6. The method of claim 5, wherein R² is selected from the group consisting of

hydrogen, methyl, and isopropyl.

7. The method of claim 6, wherein R² is hydrogen.

8. The method of claim 1, wherein R³ is hydrogen.

9. The method of claim 1, wherein R⁴ is selected from hydrogen and halogen.

10. The method of claim 1, wherein R⁵ is hydrogen or -OR⁸.

11. The method of claim 10, wherein R⁵ is selected from the group consisting of hydrogen, -OH, and -OCH₃.

12. The method of claim 1, wherein R⁶ and R⁷ come together to form an unsubstituted fused (C4-C₆) ring structure.

13. The method of claim 12, wherein the fused (C₄-C₆) ring structure is an aryl ring.

14. The method of claim 1, wherein the compound of formula (1) is:

or a pharmaceutically acceptable salt form thereof.

15. The method of claim 1 , wherein the patient is immunocompromised.

16. The method of claim 1, wherein the fungal infection is caused by a fun

organism selected from C. albicans, P. carinii, and A. fumigatus .

A method of inhibiting a fungal infection in a patient, the method comprising administering to the patient a therapeutically effective compound of formula (1):

or a pharmaceutically acceptable salt form thereof,

wherein:

R¹ is selected from the group consisting of: hydrogen, substituted or unsubstituted (Ci-C₆)alkyl, substituted or unsubstituted (C₂-C₆)alkenyl, substituted or unsubstituted (C₂-C6)alkynyl, substituted or unsubstituted (C3-Cy)cycloalkyl, and substituted or unsubstituted (C₃-Ci₄)aryl;

R² is selected from the group consisting of: hydrogen, substituted or unsubstituted (Ci-C₆)alkyl, substituted or unsubstituted (C₂-C₆)alkenyl, substituted or unsubstituted (C₂-C₆)alkynyl, and substituted or unsubstituted (C₃-C₇)cycloalkyl; R³, R⁴ and R⁵ are independently selected from the group consisting of: hydrogen, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₃-C₁₄)aryl, -(CH₂)(C₃-C₁₄)aryl halogen, -CN, -N0₂, -N(R⁸)₂, and -OR⁸;

R⁶ and R⁷ are independently selected from the group consisting of hydrogen,

each R⁸ is independently selected from the group consisting of hydrogen and

(Ci-C₆)alkyl.

18. A method of inhibiting fungal growth in a patient, the method comprising

or a pharmaceutically acceptable salt form thereof,

R¹ is selected from the group consisting of: hydrogen, substituted or unsubstituted (Ci-C₆)alkyl, substituted or unsubstituted (C₂-C₆)alkenyl, substituted or unsubstituted (C₂-Ce)alkynyl, substituted or unsubstituted (C₃-Cy)cycloalkyl, and substituted or unsubstituted (C₃-Ci₄)aryl;

R² is selected from the group consisting of: hydrogen, substituted or unsubstituted (Ci-C₆)alkyl, substituted or unsubstituted (C₂-C₆)alkenyl, substituted or unsubstituted (C₂-Ce)alkynyl, and substituted or unsubstituted (C₃-Cy)cycloalkyl;

R³, R⁴ and R⁵ are independently selected from the group consisting of: hydrogen, (Ci-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₃-Ci₄)aryl, -(CH₂)(C₃-Ci₄)aryl halogen, -CN, -N0₂, -N(R⁸)₂, and -OR⁸;

R⁶ and R⁷ are independently selected from the group consisting of hydrogen,

each R⁸ is independently selected from the group consisting of hydrogen and

(Ci-C₆)alkyl.

A method of inhibiting Rttl09 in a cell, the method comprising contacting the cell with an effective amount of a compound of formula (1):

or a pharmaceutically acceptable salt form thereof,

wherein:

R¹ is selected from the group consisting of: hydrogen, substituted or unsubstituted (Ci-Ce)alkyl, substituted or unsubstituted (C₂-Ce)alkenyl, substituted or unsubstituted (C₂-Ce)alkynyl, substituted or unsubstituted (C3-Cy)cycloalkyl, and substituted or unsubstituted (C₃-Ci₄)aryl;

R² is selected from the group consisting of: hydrogen, substituted or unsubstituted (Ci-C₆)alkyl, substituted or unsubstituted (C₂-C₆)alkenyl, substituted or unsubstituted (C₂-Ce)alkynyl, and substituted or unsubstituted (C₃-Cv)cycloalkyl;

R⁶ and R⁷ are independently selected from the group consisting of hydrogen,

each R⁸ is independently selected from the group consisting of hydrogen and

(Ci-C₆)alkyl. A method of treating a fungal infection in a patient, the method comprising administering to the patient a therapeutically effective compound of formula (2)

or a pharmaceutically acceptable salt form thereof,

wherein:

X is selected from O, S, and NR 13.

R⁹ is selected from the group consisting of: substituted or unsubstituted (Ci-C₆)alkyl, substituted or unsubstituted (C₂-C₆)alkenyl, substituted or unsubstituted

(C₂-C₆)alkynyl, substituted or unsubstituted (C₃-Cy)cycloalkyl, substituted or unsubstituted (C₃-Cy)heterocycloalkyl, substituted or unsubstituted (C₃-Ci₄)aryl, and substituted or unsubstituted (C₃-Ci₄)heteroaryl;

R¹⁰ and R¹¹ are independently selected from the group consisting of: hydrogen,

(Ci-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, halogen, -CN, -N0₂, -NR¹³ ₂, and -OR¹³, or R¹⁰ and R¹¹ can come together to form a substituted or unsubstituted fused (C4-C₆) ring structure;

R¹² is selected from the group consisting of: hydrogen, (Ci-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, halogen, -CN, -N0₂, -NR¹³ ₂, and -OR¹³; and

each R¹³ is independently selected from the group consisting of hydrogen and

(Ci-C₆)alkyl.

21. The method of claim 20, wherein X is S.

22. The method of claim 20, wherein R is a substituted or unsubstituted

(C₃-C₇)heterocycloalkyl or substituted or unsubstituted (C₃-Ci₄)heteroaryl.

23. The method of claim 20, wherein R¹⁰ and R¹¹ come together to form an unsubstituted fused (C₄-C₆) ring structure.

24. The method of claim 23, wherein the fused (C₄-C₆) ring structure is an aryl ring.

25. The method of claim 20, wherein R¹² is hydrogen.

26. The method of claim 20, wherein the compound of formula (2) is:

or a pharmaceutically acceptable salt form thereof.

27. The method of claim 20, wherein the patient is immunocompromised.

28. The method of claim 20, wherein the fungal infection is caused by a fungal organism selected from C. albicans, P. carinii, and A. fumigatus .

29. A method of inhibiting a fungal infection in a patient, the method comprising administering to the patient a therapeutically effective compound of formula (2):

or a pharmaceutically acceptable salt form thereof,

wherein:

X is selected from O, S, and NR ; R⁹ is selected from the group consisting of: substituted or unsubstituted (Ci-C₆)alkyl, substituted or unsubstituted (C₂-C₆)alkenyl, substituted or unsubstituted

(Ci-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, halogen, -CN, -N0₂, -NR¹³ ₂, and -OR¹³, or R¹⁰ and R¹¹ can come together to form a substituted or unsubstituted fused (C₄-C₆) ring structure;

each R¹³ is independently selected from the group consisting of hydrogen and

(Ci-C₆)alkyl.

A method of inhibiting fungal growth in a patient, the method comprising administering to the patient a therapeutically effective compound of formula (2)

or a pharmaceutically acceptable salt form thereof,

wherein:

X is selected from O, S, and NR¹³;

(C₂-C₆)alkynyl, substituted or unsubstituted (C₃-Cy)cycloalkyl, substituted or unsubstituted (C₃-Cy)heterocycloalkyl, substituted or unsubstituted (C₃-Ci₄)aryl, and substituted or unsubstituted (C₃-Ci₄)heteroaryl; R¹⁰ and R¹¹ are independently selected from the group consisting of: hydrogen, (Ci-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, halogen, -CN, -N0₂, -NR¹³ ₂, and -OR¹³, or R¹⁰ and R¹¹ can come together to form a substituted or unsubstituted fused (C4-C₆) ring structure;

each R¹³ is independently selected from the group consisting of hydrogen and

(Ci-C₆)alkyl.

31. A method of inhibiting Rttl09 in a cell, the method comprising contacting the cell with an effective amount of a compound of formula (2):

or a pharmaceutically acceptable salt form thereof,

wherein:

X is selected from O, S, and NR 13.

R¹² is selected from the group consisting of: hydrogen, (Ci-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, halogen, -CN, -N0₂, -NR¹³ ₂, and -OR¹³; and each R is independently selected from the group consisting of hydrogen and

(Ci-C₆)alkyl.

32. A method for screening a test compound for antifungal activity, the method

comprising determining if the test compound inhibits the histone acetyltransferase activity of a fungal Rttl09 polypeptide, wherein the fungal Rttl09 polypeptide has at least 80% identity to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6.

33. The method of claim 32, wherein the fungal Rttl09 polypeptide has at least about 85% sequence identity to a polypeptide comprising an amino acid selected from the group consisting of SEQ ID NOs: 1-6.

34. The method of claim 33, wherein the fungal Rttl09 polypeptide has at least about 90%) sequence identity to a polypeptide comprising an amino acid selected from the group consisting of SEQ ID NOs: 1-6.

35. The method of claim 34, wherein the fungal Rttl09 polypeptide has at least about

95%o sequence identity to a polypeptide comprising an amino acid selected from the group consisting of SEQ ID NOs: 1-6.

36. The method of claim 35, wherein the fungal Rttl09 polypeptide has at least about 98%) sequence identity to a polypeptide comprising an amino acid selected from the group consisting of SEQ ID NOs: 1-6.

37. The method of claim 32, wherein the fungal Rttl09 polypeptide is selected from the group consisting of SEQ ID NOs: 1-6.

38. The method of claim 32, wherein the determining comprises contacting the test compound with a composition comprising a fungal Rttl09, a substrate for a fungal Rttl09 polypeptide, and acetyl coenzyme A; and determining the level of a de- acetylated coenzyme A.

39. The method of claim 38, wherein the substrate is a fungal histone.

40. The method of claim 39, wherein the fungal histone is a lysine containing histone.

41. The method of claim 40, wherein the lysine containing histone is an Asf-1-H3-H4 complex.

42. The method of claim 40, wherein the lysine containing histone is H3K56.

43. The method of claim 38, wherein the coenzyme A is labeled.

44. The method of claim 38, wherein the de-acetylated coenzyme A is labeled.

45. The method of claim 32, wherein the fungal Rttl09 polypeptide is complexed with Vps75.

46. The method of claim 38, wherein the assay solution further comprises a label that reacts with de-acetylated coenzyme A to form a detectable complex.

47. The method of claim 46, wherein the label is a fluorophore.

48. The method of claim 32, wherein the method further comprises determining

whether the test compound inhibits a human p300/CBP polypeptide.

49. The method of claim 48, wherein the test compound exhibits preferential

inhibition of the fungal Rttl09 activity compared to human p300/CBP activity.

50. The method of claim 32, wherein the method further comprises determining whether the test compound inhibits a yeast Gcn5 polypeptide.

51. The method of claim 32, wherein the test compound inhibits growth of a fungal species.

52. The method of claim 51 , wherein the fungal species is selected from C. albicans, P. carinii, and A. fumigatus .