US 2006O154991A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0154991 A1 Johnson et al. (43) Pub. Date: Jul. 13, 2006

(54) INHIBITORS OF CANDIDA ALBICANS Related U.S. Application Data (75) Inventors: Douglas I Johnson, Essex Junction, VT (60) Provisional application No. 60/445,314, filed on Feb. (US); Kurt A. Toenjes, Burlington, VT 5, 2003. (US) Publication Classification Correspondence Address: WOLF GREENFIELD & SACKS, PC (51) Int. Cl. NULL A6II 3L/38 (2006.01) FEDERAL RESERVE PLAZA (52) U.S. Cl...... 514/649 6OO ATLANTIC AVENUE BOSTON, MA 02210-2206 (US) (57) ABSTRACT (73) Assignee: University of Vermont and State Agri The invention provides methods for identifying modulators cultural College, Burlington, VT (US) of yeast phenotypic transitions and methods for treating fungal infections with modulators of phenotypic transitions (21) Appl. No.: 10/544,691 of yeast cells. These methods include methods for identify ing inhibitors of the budded-to-hyphal transition of Candida (22) PCT Filed: Feb. 5, 2004 albicans and methods for treating fungal infections with inhibitors of the budded-to-hyphal transition of Candida (86). PCT No.: PCT/USO4/O3208 albicans. Patent Application Publication Jul. 13, 2006 Sheet 1 of 3 US 2006/0154991 A1 Figure 1.

The budded or yeast-like (A), pseudohyphal (B), and hyphal growth forms are inter-convertible and differ in growth properties and cell cycle regulation. Adapted from (12, 15)} Patent Application Publication Jul. 13, 2006 Sheet 2 of 3 US 2006/0154991 A1

Figure 2.

Signals (pH, temp., serum, maerophages) Cdc24 (Cdc24)2- s- N.Go (Gpx2) clad (CL5 Catzoste20) Cyr1 Stei (Stel) cAMP signal Est7 (Stet) Cek (Kiss) protein kinase A

Cphi (Stel2) Efg1 (Phd)

Adapted transvaarov, Eads in Microbiology 7333-338 999 Patent Application Publication Jul. 13, 2006 Sheet 3 of 3 US 2006/0154991 A1

Figure 3.

esults from ...S. f6

C. albicans cells were incubated in YNB media to promote budded growth and then transferred to Spider media to induce the budded-to-hyphal transition and hyphal elongation. The indicated small molecules were added at the indicated concentrations at time 0 and growth was allowed to continue for 4 h at 37'C before the cells were fixed and observed microscopically. US 2006/0154991 A1 Jul. 13, 2006

INHIBITORS OF CANDDA ALBCANS 6, or 7-membered ring; m is an integer varying from 0 to 5: and n is an integer varying from 0 to 5; in an effective FIELD OF THE INVENTION amount. 0001. The invention relates to methods for identifying 0007) In some embodiments, the compound is of the modulators of yeast phenotypic transitions, including meth formula: ods for identifying inhibitors of the budded-to-hyphal tran sition of Candida albicans. The invention also relates to treating fungal infections with modulators of phenotypic RI transitions of yeast cells, including inhibitors of the budded M to-hyphal transition of Candida albicans. s1 (CRR)-N R5 R2 BACKGROUND OF THE INVENTION N N 0002 Candida albicans is the most common and possi X, it --Y bly the most important causative agent of human fungal 2 21 infections (Edmond, M. B., et al. 1999, Clin. Infect. Dis. 29:239-244). Candida albicans, (C. albicans) is a major opportunistic pathogen of immunocompromised hosts, wherein R', R. R. R. R. X., and Y are as defined above including AIDS patients and patients undergoing chemo for Formula 1. In other embodiments, the compound is of the therapy, patients who have had tissue transplants, and formula: patients with central venous catheters. Studies indicate that up to ninety percent of AIDS patients suffer from oropha ryngeal and esophageal candidiasis, in which C. albicans is R1 the major causative agent (Schmidt-Westhausen, A., et al., A 1991, J. Oral Pathol. Med. 20:467-472). 1N1N 2 R5 R SUMMARY OF THE INVENTION N N 0003) The invention is based, in part, on the discovery of Xin it --Y, a method of identifying inhibitors of the budded-to-hyphal 2 21 transition of yeast cells, and the use of the inhibitors to treat fungal infections. Previously, most, if not all, antifungal agents killed yeast cells, and often were toxic to the host wherein R. R. R. X., and Y are as described above for (patient) cells as well. Formula 1. In yet other embodiments, the compound has the formula: 0004 The present invention includes methods for treat ing fungal infections by administering a compound that modulates phenotypic transitions of yeast cells. 0005 According to one aspect of the invention, methods s1N1 /\ for treating fungal infections by administering a compound of Formula I:

R1 M -(CR'R')-N S R2 0008. In some embodiments, the compounds above are R5 provided as a mixture of isomers, which may include N N diastereomers or enantiomers. In other embodiments, the Xin --Y compound is greater than 60%, 70%, 80%, 90%, 95% or 2 21 99% of one isomer. 0009. In another aspect of the invention, methods for 0006 or a pharmaceutically acceptable salt thereof, are treating a fungal infection by administering a compound of provided, wherein R' and R are independently selected Formula II: from the group consisting of hydrogen, C-C alkyl, C-C, alkenyl, C-C alkynyl, aryl, and heteroaryl; R. R. and R' are independently selected from the group consisting of hydrogen, hydroxy, halo, C-C alkoxy, C-C alkyl, C-C, alkenyl, and C-C alkynyl; P is an integer varying from 1 x-S \ sy, to 5; each X and each Y are independently selected from the group consisting of halo, hydroxy, C-C alkoxy, nitro, C-C alkyl, C-C alkenyl, C-C alkynyl, amido, aryl, or a pharmaceutically acceptable salt thereof, are provided heteroaryl, and acyl: or two Xs or two Ys together form a 5. wherein R' and R are independently selected from the US 2006/0154991 A1 Jul. 13, 2006

group consisting of hydrogen, C-C alkyl, C-C alkoxy, In another embodiment, the compound has the formula: hydroxy, and halo; p is an integer ranging from 1 to 5; each X and each Y are independently selected from the group OH consisting of halo, hydroxy, nitro, C-C alkoxy, C-C, alkyl, C-C alkenyl, C-C alkynyl, amido, aryl, heteroaryl, N-- and acyl: or two Xs or two Ys together form a 5, 6, or 7-membered ring; m is an integer varying from 0 to 11; and n is an integer varying from 0 to 11 in an effective amount. 0010) In some embodiments, the compound is of the 0014. In some embodiments, the compounds above are formula: provided as a mixture of isomers, which may include diastereomers or enantiomers. In other embodiments, the compound is greater than 60%, 70%, 80%, 90%, 95% or 99% of one isomer. 0015. In another aspect of the invention, methods for xx()--ice- \ Sy) treating fungal infections comprising administering a com pound of Formula III: 0011 wherein R', R, X, Y are as defined above for Formula II. In some embodiments, m is an integer greater than or equal to 2. In some embodiments, the compound is of the formula:

or a pharmaceutically acceptable salt thereof, are provided, wherein R' and R are independently selected from the group consisting of hydrogen, C-C alkyl, C-C alkenyl, C-C alkynyl, aryl, and heteroaryl; wherein R. R. R. R. and Rare independently selected from the group consisting of hydrogen, hydroxy, C-C alkoxy, halo, C-C alkyl, 0012 wherein R', Y, and Y are as defined above for C-C alkenyl, and C-C alkynyl; or RandR together are Formula II. In other embodiments, the compound is of the a carbonyl oxygen; n is an integer ranging from 1 to 5; each formula: X and each Y are independently selected from the group consisting of halo, hydroxy, C-C alkoxy, nitro, C-C, alkyl, C-C alkenyl, C-C alkynyl, amido, aryl, heteroaryl, and acyl: or two Xs or two Ys together form a 5, 6, or 7-membered ring; m is an integer varying from 0 to 5; and p is an integer varying from 0 to 5 in an effective amount. 0016. In some embodiments, n is 1 or 2. In another embodiment, n is 3. In yet other embodiments, n is 4 or 5. In some embodiments, m and p are each 0, that is the aryl rings are not substituted. In other embodiments, one or both of the aryl rings may be substituted. 0013 wherein X, and Y are as described above for 0017. In some embodiments, R and R together are a Formula II. In certain embodiments, X and Y are each C-C, carbonyl oxygen. In some of these embodiments, the com alkyl. In one embodiment, the compound has the formula: pounds are of the formula:

US 2006/0154991 A1 Jul. 13, 2006

0.018 wherein R. R. R. X., and Y are as described 0022. In some embodiments, R is hydrogen. In other above for Formula III. embodiments, R is not hydrogen, and the compounds may exist only in the E or Z configuration, or a mixture thereof. 0019. In some embodiments, R is C-C alkoxy and R' In other embodiments, each X and each Y are selected from and R are each alkyl. In some of these embodiments, the C-C alkoxy and hydroxy. In some embodiments, n is 0. In compound is of the formula: other embodiments, n is 1, 2, 3, 4, or 5. 0023. In still other embodiments, m is 0. In other embodi ments, m is 1. In still other embodiments, m is 2, 3, 4, or 5. In one particular embodiment, the compound is:

Nu-N-1N1-

HO

0024. In some embodiments, the compounds above are provided as a mixture of isomers, which may include diastereomers or enantiomers. In other embodiments, the compound is greater than 60%, 70%, 80%, 90%, 95% or 0020. In some embodiments, the compounds above are 99% of one isomer. provided as a mixture of isomers, which may include diastereomers or enantiomers. In other embodiments, the 0025. In another aspect of the invention, methods for compound is greater than 60%, 70%, 80%, 90%, 95% or treating fungal infections by administering a compound of 99% of one isomer. Formula V:

O (CRR), -(CRR), N (CRR). -(CRR), N Xm - l, Y- Z, it 21 R 21 O 2

0021. In another aspect of the invention, methods for or a pharmaceutically acceptable salt thereof are provided, treating fungal infections by administering compounds of wherein each R', each R', and each R are independently Formula IV: selected from from the group consisting of hydrogen, hydroxy, C-C alkoxy, halo, C-C alkyl, C-C alkenyl, and C-C alkynyl; or R' and R together are a carbonyl Yn oxygen; each q is an integer ranging from 0 to 6; each r is an integer ranging from 0 to 6: each S is an integer ranging from 0 to 6; each t is an integer ranging from 0 to 6; each N1 X, each Y, and each Z are independently selected from the group consisting of halo, hydroxy, C-C alkoxy, nitro, &-N- C-C alkynyl, amido, aryl, heteroaryl, and acyl: or two Xs X, it R or two Ys, or two Zs, together form a 5, 6, or 7-membered 2 ring; m is an integer ranging from 0 to 5; n is an integer ranging from 0 to 4; and p is an integer ranging from 0 to 5 in an effective amount. or a pharmaceutically acceptable salt thereof are provided, 0026. In some embodiments, each X, each Y, and each Z wherein R is selected from the group consisting of hydrogen, are selected from halo and C-C alkoxy. In some embodi hydroxy, C-C alkoxy, halo, C-C alkyl, C-C alkenyl, ments, is hydrogen. In other embodiments, at least one R' and C-C alkynyl; each X and each Y are independently and at least one R together are a carbonyl oxygen. selected from the group consisting of halo, hydroxy, C-C, 0027. In some embodiments, q is 1. In other embodi alkoxy, nitro, C-C alkynyl, amido, aryl, heteroaryl, and ments, q is 0. In still other embodiments, q is 2, 3, 4, 5, or acyl: or two Xs or two Ys together form a 5, 6, or 7-mem 6. In other embodiments, r is 0. In still other embodiments, bered ring; m is an integer ranging from 0 to 5; and n is an r is 1, 2, 3, 4, 5, or 6. In some embodiments, s is 0. In other integer ranging from 0 to 5 in an effective amount. embodiments, S is 1, 2, 3, 4, 5, or 6. In some embodiments, US 2006/0154991 A1 Jul. 13, 2006

t is 0. In other embodiments, t is 1, 2, 3, 4, 5, or 6. In a diastereomers or enantiomers. In other embodiments, the particular embodiment, the compound is: compound is greater than 60%, 70%, 80%, 90%, 95% or 99% of one isomer.

C 0032. In one particular embodiment, the compound is other than:

in-QA O OESEO le-N OH O N N N H C 0033 According to some embodiments, the methods of the invention involve administering a compound to a subject matter who is not otherwise indicated for treatment with the 0028. In some embodiments, the compounds above are compound. Similarly, in some embodiments, the methods of provided as a mixture of isomers, which may include the invention involve administering a pharmaceutical prepa diastereomers or enantiomers. In other embodiments, the ration to a subject who is not otherwise indicated for compound is greater than 60%, 70%, 80%, 90%, 95% or treatment with the pharmaceutical preparation. For example, 99% of one isomer. in some embodiments, the methods involve treating patients 0029. In another aspect of the invention, methods for who would not receive the compounds or pharmaceutical treating fungal infections by administering compounds of preparations recited herein for a condition other than a Formula VI: fungal infection. 0034. According to another aspect of the invention, com positions of matter are provided. The compositions of matter R R6 comprise any compound embraced by Formula I, Formula a. N 1. II. Formula III, Formula IV. Formula V, and Formula VI, as well as every embodiment described above, as if specifically R NN -NO restated herein. l R3 0035. According to yet another aspect of the invention, N pharmaceutical compositions are provided. The pharmaceu tical compositions comprise a pharmaceutically acceptable 21 N carrier and any compound embraced by Formula I. Formula X-- R2 II, Formula III, Formula IV. Formula V, and Formula VI, as N N well as every embodiment described above, as if specifically RI restated herein. 0036). In another aspect of the invention, kits are pro or a pharmaceutically acceptable salt thereof are provided, vided. The kits comprise any compound embraced by For wherein R', R, R and Rare each independently selected mula I, Formula II, Formula III, Formula IV. Formula V, and from the group consisting of hydrogen, C-C alkyl, C-C, Formula VI, as well as every embodiment described above, alkenyl, and C-C alkynyl; R and R are each indepen as if specifically restated herein, and instructions for use. dently selected from the group consisting of hydrogen, 0037. In some embodiments, the fungal infection is hydroxy, C-C alkoxy, halo, C-C alkyl, C-C alkenyl, caused by a yeast of the Candida genus. In one embodiment, and C-C alkynyl; each X is independently selected from the yeast is of the Candida albicans species. In other the group consisting of halo, hydroxy, C-C alkoxy, nitro. embodiments, the Candida yeast may be of the Candida C-C alkynyl, amido, aryl, heteroaryl, and acyl; and m is an dubliniensis, Candida parapsilosis, Candida tropicalis, integer ranging from 0 to 4; in an effective amount. Candida kerfir, Candida guilliermondii, Candida incon 0030) In some embodiments, R' is hydrogen. In other spicua, Candida famata, Candida glabrata, Candida krusei, embodiments, R is hydrogen. In another embodiment, R is Candida lusitaniae, or other Candida species. hydrogen. In yet other embodiments, one or both of R and Rare hydrogen. In preferred embodiments, at least one of 0038. In all of the methods described for treating fungal R", R. R. R. R and R is not hydrogen. In some infections, a Subject in need of Such treatment is adminis embodiments, m is 0. In other embodiments, m is 1. In still tered one of the compounds described above in an amount other embodiments, m is 2, 3, or 4. effective to treat the fungal infection. 0031. In some embodiments, the compounds above are 0039 The subject is a human, non-human primate, or provided as a mixture of isomers, which may include other mammal. In important embodiments, the Subject is a US 2006/0154991 A1 Jul. 13, 2006 human. In some embodiments, the subject is an HIV-positive to-hyphal transition described above are provided. In some patient. In other embodiments the Subject is a non-human embodiments, these organisms are eukaryotes. In some vertebrate selected from the group consisting of a dog, cat, embodiments, these organisms are selected from Drosophila horse, cow, pig, turkey, goat, fish, monkey, chicken, rat, melanogaster; Caenorhabditis elegans, and Arabidopsis mouse, and sheep. thaliana. 0040. In another aspect of the invention, methods for 0046. In any of the methods for identifying modulators of identifying modulators of phenotypic transition of Candida phenotypic transitions described above, there is optionally a cells are provided. These methods include providing a step of determining if the test compound is Substantially sample of the cells in a first phenotypic form, adding a test non-toxic to the Subject cells. Test compounds that are compound to the sample of the cells, and observing whether substantially non-toxic to subject cells are preferred. or not there is a measurable change in the cells from a first phenotypic form to a second phenotypic form. The test 0047. Each of the limitations of the invention can encom compound is classified as a modulator of a phenotypic pass various embodiments of the invention. Therefore, it is transition if there is a change or as not being a modulator of anticipated that each of the limitations of the invention the phenotypic transition if there is no change in the cells. In involving any one element or combination of elements, can important embodiments, the change is a morphological be included in each method. change. In some of these embodiments, the change is from 0048. Other aspects, features, and advantages of the a budded form to a hyphal form. In other embodiments, the present invention will become apparent from the following change is from a budded form to a pseudohyphal form. In Detailed Description and Examples. It should be understood, still other embodiments, the change is from a pseudohyphal however, that the Detailed Description and the specific form to a hyphal form. In yet other embodiments, the change Examples, while indicating the embodiments of the inven may be a morphological change from a hyphal form to a tion, are given by way of illustration only, since various budded form, a pseudohyphal form to a budded form, or changes and modifications within the spirit and the scope of even a hyphal form to a pseudohyphal form. the invention will become apparent to those skilled in the art from the Detailed Description. 0041. In some embodiments, the change is an increase in the amount of cells in the first phenotypic form compared to BRIEF DESCRIPTION OF THE DRAWINGS the amount of cells in the second phenotypic form. In other embodiments, the measurable change is a decrease in the 0049) FIG. 1 illustrates the morphological status of C. amount of cells in the second phenotypic form. albicans. 0042. In the methods for identifying modulators of phe 0050 FIG. 2 illustrates the cell signaling pathways of C. notypic transitions described above, the Candida cells may albicans. be Candida albicans cells. However, these methods may 0051) FIG. 3 illustrates results from a small molecule also be applied to Candida dubliniensis, Candida parapsi SCC. losis, Candida tropicalis, Candida kefir, Candida guillier mondii, Candida inconspicua, Candida famata, Candida DETAILED DESCRIPTION OF THE glabrata, Candida krusei, Candida lusitaniae, and other INVENTION species of Candida cells. 0052 The invention is based, in part, on the discovery of 0043. In another aspect of the invention, methods for a method of identifying inhibitors of the budded-to-hyphal identifying a modulator of a phenotypic transition of S. transition of yeast cells, and the use of the inhibitors to treat cerevisiae cells is provided. The method includes providing fungal infections. Previously, most, if not all, antifungal a sample of the cells characterized by a first phenotypic agents were designed to kill yeast cells, and often were toxic form, adding a test compound to the sample of the cells, and to the host (patient) cells as well. observing whether or not there is a measurable change in the cells from the first phenotypic form to a second phenotypic 0053 Fungi are eucaryotic organisms, only a few of form. If there is a change, the test compound may be which cause infection in vertebrate mammals. Because fungi classified as a modulator, but if there is no change, the test are eucaryotic organisms, they differ significantly from compound may be classified as not being a modulator. procaryotic bacteria in size, structural organization, life cycle and mechanism of multiplication. Fungi are classified 0044) In one embodiment of the invention, methods for generally based on morphological features, modes of repro identifying inhibitors of the budded-to-hyphal transition of duction and culture characteristics. Although fungi can Candida albicans cells are provided. The method includes cause different types of disease in Subjects, such as respi providing a sample of Candida albicans cells in the budded ratory allergies following inhalation of fungal antigens, form, adding a test compound to the sample, and observing fungal intoxication due to ingestion of toxic Substances, whether or not there is a measurable change from the budded Such as amatatoxin and phallotoxin produced by poisonous form to the hyphal form. If there is no increase in the hyphal mushrooms and aflotoxins, produced by aspergillus species, form, the test compound is classified as an inhibitor. If there not all fungi cause infectious disease. is no change, the test compound is classified as not being an 0054) Infectious fungi can cause systemic or superficial inhibitor. Additionally, if there is a decrease in the budded infections. Primary systemic infection can occur in normal form, the test compound is classified as an enhancer. healthy Subjects and opportunistic infections, are most fre 0045. In another aspect of the invention, methods of quently found in immuno-compromised subjects. The most studying the morphogenesis of organisms with actin-based common fungal agents causing primary systemic infection morphogenesis using any of the modulators of the budded include blastomyces, coccidioides, and histoplasma. Com US 2006/0154991 A1 Jul. 13, 2006

mon fungi causing opportunistic infection in immuno-com Nystatin is used for the treatment of the cutaneous, muco promised or immunosuppressed subjects include, but are not cutaneous, and allergic diseases. Amphotericin B is useful limited to, candida albicans (an organism which is normally for treating this systemic disease. Other drugs useful for the part of the respiratory tract flora), cryptococcus neoformans treatment include 5-fluorocytosine, fluconazole, itracona (sometimes in normal flora of respiratory tract), and various Zole and Voriconazole. aspergillus species. Systemic fungal infections are invasive infections of the internal organs. The organism usually 0061 Chromoblastomycosis is a chronic infection of the enters the body through the lungs, gastrointestinal tract, or skin and Subcutaneous tissue. Although the infection is intravenous lines. These types of infections can be caused by usually localized, parts can disseminate systemically and in primary pathogenic fungi or opportunistic fungi. particular to the brain. Itraconazole and terbinafine are the drugs used to treat this infection. The principal fungi causing 0.055 Superficial fungal infections involve growth of this infection are cladophialophora, carrionii, fonsecaea, fungi on an external Surface without invasion of internal compacta, fonsecaea pedrosoi, phialophora, verruceosa, tissues. Typical Superficial fungal infections include cutane rhinocladiella, aquasbera. ous fungal infections involving skin, hair, or nails. An example of a cutaneous infection is Tinea infections, such as 0062 Coccidioidomycosis is a fungal disease of the ringworm, caused by dermatophytes, such as microsporum respiratory tract which can be acute, chronic, severe or fatal. or traicophyton species, i.e., microsporum canis, The disease is primarily caused by coccidioides immitis. microsporum gypsum, tricofitin rubrum. Amphotericin B, itraconazole, fluconazole, ketaconazole, and Voriconazole are anti-fungal agents that are used for the 0056. Examples of fungi include: Cryptococcus neofor treatment of this disorder. mans, Histoplasma capsulatum, Coccidioides immitis, Blas tomyces dermatitidis, Chlamydia trachomatis, Candida 0063 Cryptococcosis is a fungal disorder caused by albicans. Other infectious organisms (i.e., protists) include: cryptococcus norformans or filobasidiella neoformans. The Plasmodium Such as Plasmodium falciparum, Plasmodium disease can take the form of a chronic, Subacute, acute, malariae, Plasmodium ovale, and Plasmodium vivax and pulmonary, systemic, or meningitic disease, following pri Toxoplasma gondii. mary infection in the lungs. If the disease spreads from the lungs to the central nervous system, it is usually treated 0057 Diseases associated with fungal infection include immediately with amphotericin B and/or 5-fluorocytosine aspergillosis, blastomycosis, candidiasis, chromoblastomy and in some cases fluconazole. cosis, coccidioidomycosis, cryptococcosis, fungal eye infec tions, fungal hair, nail, and skin infections, histoplasmosis, 0064. Fungal infections of the eye include mycotit kerati lobomycosis, mycetoma, otomycosis, paracoccidioidomy tis, and endogenous or extension occulomycosis. Mycotic cosis, penicilliosis, marneffeii, phaeohyphomycosis, rhino keratitis is caused by a variety of fungi including acremo sporidioisis, sporotrichosis, and Zygomycosis. The com nium, aspergillus, bipolaris, candida albicans, curvularia, pounds of the invention are useful for treating diseases exserohilum, fitsarium, and lasiiodiplodia. Amphotericin B is associated with fungal infection either alone or in combi not used for treatment because it irritates the infected tissue. nation with existing anti-fungal therapies. Drugs useful for treating mycotit keratitis include pimaricin and fluconazole. Occulomycosis is generally caused by 0.058 Aspergillosis is a disease caused by the fungi of the candida albicans or rhizopus, arrhizus. Amphotericin B is genus aspergillus, which can lead to mild or severe disease, the anti-fungal agent used for treatment. generally depending on factors such as the status of the host immune system. Aspergillus frequently arises as an oppor 0065. Fungal infections of the hair, nail, and skin include tunistic infection in patients having immune-suppressive onychomycosis, piedra, pityrisis versiZolor, tinea barbae, diseases, or being treated with chemotherapy. Some forms of tinea capitis, tinea corporis, tinea cruris, tinea faosa, tinea aspergillus can be treated with prednisone, disodium chro nigra, tinea unguium. Onychomycosis, which is generally moglycat, nystatin, amphotericin B, itraconazole, or Vori caused by fungi such as acremonium, aspergillus, candida, conazole. fitsarium, Scopulariopisis, Onychocola, and Scytalidium, can be treated with itraconazole, turbinifine, amphotericin B, 0059 Blastomycosis is a fungal infection arising from the gentian violet, resorcin, iodine, nystatin, thiabendazole, and organism blastomyces dermatitis. The infection initiates in glutarardehyde. Piedra, which is a colonization of the hair the lungs and usually is disseminated to other body sites, shaft to bifungal organisms such as piedraia and trichos especially the skin and bone. It is treated by amphotericin B, porin, can be treated with keratolytic agents, mild fungi hydroxy stilbamidine, itraconazole and Voriconazole. When cides, fluconazole, and itraconazole. The tineas are various amphotericin B is used, at least 1.5 grams must be given to forms of ringworm colonizing different bodily regions. avoid relapse. However, if a drug is to be administered in These diseases are generally caused by fungi such as combination with the compounds of the invention, lower microsporum, trichophyton, and epidermophyton. The tineas doses may be given without a relapse. Generally hydrox can be treated with keratolytic agents, intraconazole, turbin yStilbamidine has been used for treating the cutaneous form ifine, tolnaftate, chlotrimazole, miconazole, econazole, and of the disease but not other forms. When combined with the ketaconzole. compounds of the invention, it can also be used for the 0066 Histoplasmosis (capsulati and duboisii) are fungal treatment of other forms, as well as in lower doses for the infections caused by histoplasma and aiellomyces. Histo cutaneous form. plasmosis capsulati can adequately be treated with ampho 0060 Candidiasis is a fungal infection caused by a mem tericin B, itraconazole or voriconazole. If the subject being ber of the genus candida. The disease can be in the form of treated has AIDS, fluconazole is usually used. Histoplasmo allergic, cutaneous, mucocutaneous, or systemic candidiasis. sis duboisi once it becomes disseminated, especially to the US 2006/0154991 A1 Jul. 13, 2006 liver or spleen, is very difficult to treat. Amphotericin B, that one isomer will be present in an amount ranging from itraconazole, fluconazole, and Voriconazole are used. When 51 to 100%, preferably, more than 80%, more preferably, these compounds are combined with the compounds of the more than 90%, even more preferably, more than 95%, and invention, prognosis may be improved. even more preferably, more than 99% pure with respect to the other isomer or isomers present, but not with respect to 0067 Lobomycosis is a fungal infection caused by laca other impurities or compounds that may be present. Isomer, Zia loboi. Lobomycosis is a cutaneous infection which develops into lesions which can be removed by Surgery. as used herein, may refer to an E or Z isomer, and R or S There are not drugs specifically used for this disorder. isomer, an enantiomer, a diastereomer, or, in the case of Mycetoma is an infection caused by a variety of fungi compounds with several diastereomers, a group of diaste including eumycotic, acromonium, aspergillus, exophiala, reomers, with respect to another group of diastereomers, leptosphaeria, madurella, neotestudina, pseudallesheria, which differ for example, with respect to just one stereo and pyrenochieta. The disease involves lesions of the cuta center of the molecule. neous and Subcutaneous tissues, which can rupture and 0075. Many of the compounds used in the methods spread to Surrounding tissues. The mycetomas can be treated described herein are available from commercial sources as with ketoconazole, in combination with Surgery. pure compounds (for example, from ChemEBridge, Corp., 0068 Otomycosis is a fungal ear infection caused by San Diego, Calif.). Others are not commercially available, aspergillus or candida. The infection is a Superficial infec but may be synthesized using commercially available start tion of the outer ear canal, which is characterized by ing materials and standard synthetic chemistry techniques inflammation, pruritus, Scaling, and sever discomfort. It is a using no more than routine skill in the art. chronic recurring mycosis. 0076. In another aspect of the invention, methods for 0069 Paracoccidioidomycosis is a fungal infection cause identifying modulators of morphological transitions are pro by paracoccidioides brasiliensis. The disease originates as a vided. These methods (or assays) are based, in part, on the pulmonary infection and can disseminate into the nasal, discovery that certain molecules have an inhibitory effect on buccal, and gastrointestinal mucosa. Amphotericin B and the budded-to-hyphal transition of Candida albicans cells. Sulfonamides are generally used to treat the disease. In addition, the methods may be useful to understand the cellular and molecular mechanisms of yeast and other cells. 0070 Phaeohyphomycosis is a fungal infection caused by a variety of fungi including cladophialophora, curvu 0077. The methods for identifying modulators of pheno laria, bipolaris, exserohilum, exophiala, Scedosporium, Och typic transitions include providing a sample of cells char roconis, coniothyrium, phialophora, wangiella, and lasio acterized by a phenotypic form. A phenotypic form is an diplodia. The infection can be localized or can invade observable state. Morphological forms are one type of various tissues including the brain, bone, eyes, and skin. phenotypic form. Phenotypic forms may be an observable Invasion of the brain or bone can be lethal. Generally, state of a cell or collection of cells, such as a budded form, phaeohyphomycosis is treated with amphotericin B and a hyphal form, a pseudohyphal form, and the like. A mea phyfluorocytozine or intaconazole. Rhinosporidiosis is an Surable change from one phenotypic form to another is a infection of the mucus membrane caused by rhinosporidium directly or indirectly observable phenotypic transition, such Seeberi. Local injection of amphotericin B is used as treat as the change of a cell or collection of cells from one ment. phenotypic form to another, the formation of intracellular structure, the formation of extracellular structure, the inhi 0071 Sporotrichosis is a chronic infection of the cutane bition or enhancement of a cellular process which results in ous tissues, Subcutaneous tissues, or lymph system. The a measurable change downstream. Specific examples infection may also spread to tissues such as bone, muscle, include the formation of pedestals on cells, the formation of CNS, lungs, and/or genitourinary system. Usually the fungi mitotic spindles within cells, cell differentiation, centrosome sporothrix schenckii is inhaled or passed through a lesion in duplication, actin assembly, and cell Surface receptor acti the skin. Sporotrichosis is usually treated with oral potas vation (for a review, see Ward, G. E., et al., 2002, Cellular sium iodide, amphotericin B, or 5-fluorocytozine. Microbio. 4(8):471-482). In one embodiment, the pheno 0072 Zygomycosis is a chronic infection caused by typic transition observed is a morphological transition conidobolus and basidiobolus ranarum. The disease is between a cellular budded form, a cellular hyphal form, treated by potassium iodide and/or amphotericin B. and/or a cellular pseudohyphal form. 0073. A compound that modulates phenotypic transitions 0078. The methods for identifying modulators of pheno of yeast cells is a compound that when added to a budded typic transitions include providing a test compound to a cell yeast causes transformation to a hyphal yeast. Preferred or a sample of cells. A test compound, as used herein, is a compounds are those set forth in the Summary and func compound that is added to the cell or cells to query whether tionally equivalent molecules thereof. or not it is capable of inducing a phenotypic transition. Test compounds may be inhibitors or enhancers (activators) of a 0074 According to some aspects of the invention, the phenotypic transition, or may have no effect. Often test compounds used in the methods for treating fungal infec compounds are agonists or antagonists of certain cellular tions or in the methods for identifying modulators of yeast transitions which include or lead to phenotypic transitions. cell phenotypic transitions may exist in different isomeric Test compounds may be added to cell(s) individually, though forms. The compounds may be used in the methods of the often a library approach is used to increase the amount of invention as a Substantially isomerically-pure compound, or test compounds that can be screened for activity in a given as a mixture of isomers. Preferably, isomerically-pure com amount of time. High throughput Screening methods are pounds are used. Isomerically-pure, as used herein, means often employed as well. US 2006/0154991 A1 Jul. 13, 2006

0079 The test compounds are optionally assayed for in the adherence and invasion of multiple cell types (Brown, toxicity against the Subject cells. In one embodiment, the A. J. P., et al., 1999. Trends Microbiol. 7:333-338; Mitchell, toxicity of a test compound against human cells is deter A. P. 1998, Curr. Opin. Microbiol. 1:687-692; Odds, F. C. mined. A step of selecting test compounds that are Substan 1994, J. Am. Acad. Dermatol. 31:S2-S5). C. albicans exists tially non-toxic to subject cells is provided. “Substantially within immunocompetent individuals in a commensal rela non-toxic' means that the test compound can be adminis tionship on mucosal linings of the oral cavity, esophagus, tered to a Subject with an acceptable amount of damage to vagina, gastrointestinal tract, etc. Oropharyngeal, esoph the host (Subjects) cells. An acceptable amount of damage ageal, Vulvovaginal, and cutaneous candidiasis leads to can be determined by one of skill in the art with no more significant morbidity. Lethality is often associated with than routine skill. Acceptable amounts may depend on route systemic infections in immunocompromised patients. Sys of administration, risk of side effects versus benefit of temic infections arise from colonization of mucosal Surfaces administration, etc. by adherence to epithelial cells, followed by the penetration 0080. In addition to the powerful assay to identity yeast of epithelial and endothelial cell barriers and dissemination form modulators, understanding the input signals and output throughout the body (Filler, S. G. et al., 1995, Infect. responses that lead to the budded-to-hyphal transition will Immun. 63:976-983). lead to insight into virulence mechanisms and may ulti 0084. It is believed that the vascular endothelium has a mately lead to new anti-fungal therapeutic targets and drugs. key role in the dissemination of C. albicans which leads to New anti-fungal therapeutic targets and drugs are needed establishment of systemic candidiasis. Endothelial cells are because there are serious side effects due to renal and liver sensitive to invasion and damage by C. albicans, which dysfunction associated with the polyenes (e.g., amphotericin requires the attachment of germinated C. albicans to the B, nystatin) that are usually used to treat C. albicans endothelial cells, their phagocytosis, and induction of infections. In addition, a significant increase in resistance to hyphal-specific genes. This damage can be mitigated by C. the less toxic azole drugs (e.g., fluconoazole) has occurred albicans strains with mutations in the proteinase Sap2p within the patient population, especially HIV-positive (Ibrahim, A. S., et al., 1998, Infect. Immun. 66:3003-3005) patients (reviewed in White, T. C., et al., 1998, Clin. and the adhesin Hwplp Tsuchimori, N., et al., 2000, Infect. Immun. 68:1997-2002). Both budded and hyphal forms of Microbiol. Rev. 11:382–402). C. albicans have been identified in infections and are 0081. C. albicans can exist in different morphological important for virulence. The budded form is found predomi states including budded or yeast-like cells and hyphal forms. nantly attached to epithelial cells and the hyphal form is FIG. 1 illustrates the morphological states of C. albicans, found disseminated, presumably due to its enhanced ability including the budded or yeast-like (A), pseudohyphal (B), to invade cells through the production and secretion of and hyphal growth forms. proteinases and hydrolases. 0082 The morphological states of C. albicans include 0085 Since C. albicans cells respond to a variety of round to ovoid budded or yeast-like cells and filamentous different growth and environmental signals to induce the forms including pseudohyphae (chains of ellipsoidal cells budded-to-hyphal transition, it is believed that multiple with constricted septa between mother and daughter cells) signaling pathways function in hyphal development, shown and hyphae (long filaments with no constriction at septa). in FIG. 2 (Brown, A. J. P. 2002. p. 95-106. In R. A. Hyphal cells are morphologically distinct from budded and Calderone (ed.), Candida and Candidiasis. ASM Press, pseudohyphal cells, with differences in septation patterns, Washington, D.C). Mutational analyses indicate that the actin dynamics, and growth properties. Hyphal growth is the Tuplp, Cphlp, and Efglp transcriptional regulatory proteins result of alterations in polarized growth patterns and in function independently in promoting (Cphlp and Efg1p) or transit through the cell cycle. The budded-to-hyphal transi inhibiting (Tup1p) hyphal development (Braun, B. R. et al., tion occurs in response to environmental stress signals such 2000, Genetics 155:57-67). The Cst20p-Hst7p-Ceklp MAP as temperature above 35°C., pH above 6.5, nitrogen and/or kinase pathway, which is homologous to the Ste20p-Ste7p carbon starvation, low oxygen concentration, and changes in KSSlp pathway in S. cerevisiae, signals to the Cphlp tran cell density, growth in serum or other chemicals such as scription factor (S. cerevisiae Ste12p) to induce hyphal N-acetylglucosamine, proline and other amino acids, or specific genes (Csank, C., et al., 1998, Infect. Immun. alcohols, and the presence of host macrophages (Brown, A. 66:2713-2721; Köhler, J. R., et al., 1996, Proc. Natl. Acad. J. P., 2002. p. 87-93. In R. A. Calderone (ed.), Candida and Sci. USA 93:13223-13228; Leberer, E., et al., 1996, Proc. Candidiasis. ASM Press, Washington, D.C.). In response to Natl. Acad. Sci. USA 93:13217-13222; Liu, H., et al., 1994, these hyphal-inducing signals, the expression of a variety of Science 266:1723-1726). Two Cdc42p-interacting proteins, hyphal-specific genes increases, including genes that encode the p21-activated kinase (PAK) CaCla4p and a phosphati cell Surface adhesins and secreted proteinases. Induced dylinositol 3-kinase CaVps34p, are also involved in hyphal genes ALS3, ALS8, and HWP1 (adhesins), HYR1 (cell development (Bruckmann, et al. 2000. Microbiol.-UK surface glycoprotein), SAP4, SAP5, SAP 6 (secreted aspar 146:2755-2764; Leberer, E., et al., 1997, Curr. Biol. 7:539 tyl proteinases), and ECE.1 (unknown function) are believed 546). In a parallel pathway, the Efg1p transcription factor to be important reporters for examining the effects of the functions downstream of Tpk2p, the catalytic component of Cdc42p signaling pathway on the budded-to-hyphal transi protein Icinase A, and the Ras GTPase to integrate a cAMP tion. signal into hyphal development (Feng, Q. H., et al., 1999, J Bacteriol 181:6339-6346; Lo, H. J., et al., 1997, Cell 0083. The pathogenicity of C. albicans can be attributed 90:939-949; Sonneborn, A., et al., 2000, Mol. Microbiol. to its ability to survive and thrive in multiple microenviron 35:386-396; Stoldt, V. R., et al., 1997, EMBO J. 16:1982 ments within a host, including multiple organs, the mucosa, 1991). Recently, the Coslp? Niklp two-component histidine and the bloodstream, as well as to virulence factors that aid kinase (Alex, et al., 1998. Proc. Natl. Acad. Sci. USA US 2006/0154991 A1 Jul. 13, 2006

95:7069-7073; Nagahashi, S., et al. 1998, Microbiol. not affect the transition in response to other stimuli such as 144:425-432: Srikantha, T., et al., 1998, Microbiol. changes in pH, nitrogen and/or carbon starvation, growth in 144:2715-2729), a Hog1p MAP kinase (Monge, R. A., et al., N-acetylglucosamine growth in proline, or growth in serum. 1999, J. Bacteriol. 181:3058-3068), and the Crklp cyclin Therefore, the molecules are tested for inhibitory or stimu dependent kinase (Chen, et al., 2000, Mol. Cell. Biol. latory effects under these growth conditions using standard 20:8696-8708) have also been shown to be involved in protocols. The effects of these molecules under different hyphal development. It is believed that these different sig hyphal-inducing conditions may provide clues to the signal naling pathways are activated by separate but overlapping ing pathways impacted by the molecules. environmental signals, eventually leading to rearrangements of the actin- and tubulin-based cytoskeleton (Akashi, et al., 0089. In the methods for treating fungal infections pro 1994. Microbiol. 140:271-280; Yokoyama, K., et al., 1994, vided herein, the compounds described above are adminis Microbiol. 140:281-287). For example, the Efg1p pathway tered in effective amounts. An effective amount means that can be triggered by pH, N-acetylglucosamine, proline, and amount necessary to delay the onset of inhibit the progres nitrogen and/or carbon starvation while the Cphlp pathway sion of halt altogether the onset or progression of, or can be stimulated by nitrogen starvation but not in response diagnose the particular infection being treated. When admin to N-acetylglucosamine, amino acids, or serum (Brown, A. istered to a subject, effective amounts will depend on the J. P., 2002. p. 95-106. In R. A. Calderone (ed.), Candida and particular condition being treated, the severity of the con Candidiasis. ASM Press, Washington, D.C.). In addition, the dition, individual patient parameters including age, physical hyphal-specific genes ALS8, ECE1, HWP1, and HYR1 are condition, size and weight, concurrent treatment, frequency regulated primarily by the Efg1p pathway with the Cph1p of treatment, and the mode of administration. These factors pathway being less important (Braun, B. R., et al., 2000. are well known to those of ordinary skill in the art and can Genetics 155:57-67: Brown, A. J. P. 2002. p. 95-106. In R. be addressed with no more than routine experimentation. It A. Calderone (ed.), Candida and Candidiasis. ASM Press, is preferred generally that a maximum dose be used, that is, Washington, D.C.). However, the molecular mechanisms the highest safe dose according to some medical judgment. that underlie these different signaling pathways and their 0090. As used herein, the term treat, treated, or treating potential cross-talk are still unclear. when used with respect to an disorder Such as an infectious 0.086 To identify molecules that stimulate the budded disease refers to a prophylactic treatment which increases to-hyphal transition, cells are incubated in the absence of the resistance of a subject to development of the disease Spider media; stimulatory molecules are identified by rap (e.g., to infection with a pathogen) or, in other Words, idly screening a picture of each well for the presence of decreases the likelihood that the subject will develop the hyphae in the absence of added Spider media. Secondary disease (e.g., become infected with the pathogen) as well as screens with stimulatory molecules are carried out as a treatment after the subject has developed the disease in described below. order to fight the disease (e.g., reduce or eliminate the infection) or prevent the disease from becoming worse. 0087. The structure of the inhibitory molecules identified are used to search molecule libraries to identify structurally 0091 Generally, daily doses of active compounds will be related molecules. The use of chemical libraries for screen from about 0.01 milligrams/kg per day to 1000 milligrams/ ing is known (see, for example, Ward, G. E., et al., 2002, kg per day. It is expected that oral doses in the range of 0.5 Cellular Microbio. 4(8):471-482). Chemical libraries are to 50 milligrams/kg, or even 1-10 milligrams/kg per day, in readily available from numerous sources, such as Chemical one or several administrations per day, will yield the desired Diversity (San Diego, Calif.), NeoGenesis (Cambridge, results. Dosage may be adjusted appropriately to achieve Mass.), and Chembank (National Cancer Institute). Deriva desired drug levels, local or systemic, depending upon the tives of the inhibitors (similar molecules to the inhibitors) mode of administration. For example, it is expected that are also designed using the inhibitors. These related mol intravenous administration would be from an order to sev ecules are tested for their effects in the assay, which allows eral orders of magnitude lower dose per day. It is expected for identification of key structures of the bioactive molecules that intravenous and other parenteral forms of administration that are required for biological activity as well as to identify will yield the desired results in the range of 0.1 to 10 related molecules that may have better efficacy (e.g., show milligrams/kg per day. In the event that the response in a effects at a lower dose). These more efficient bioactive Subject is insufficient at Such doses, even higher doses (or molecules for each of the sets of related molecules identified effective higher doses by a different, more localized delivery from the database searches are further analyzed for struc route) may be employed to the extent that patient tolerance tural similarity to known anti-fungal compounds and for permits. Multiple doses per day are contemplated to achieve phenotypes in secondary assays (as described below). appropriate systemic levels of compounds. 0088. Inhibitory or stimulatory bioactive compounds 0092. When administered, the formulations of the inven identified in this screen are likely to function through a tion are applied in pharmaceutically acceptable amounts and diverse set of mechanisms. As a first step in deciphering in pharmaceutically acceptable compositions. Such prepa these mechanisms, the effects of these molecules under rations may routinely contain salts, buffering agents, pre different hyphal-inducing conditions are tested. Subse servatives, compatible carriers, and optionally other thera quently, several secondary screens are performed to identify peutic ingredients. When used in medicine, the salts should the cellular processes impacted by these molecules. As be pharmaceutically acceptable, but non-pharmaceutically mentioned above, Hyphae can be induced in response to a acceptable salts may conveniently be used to prepare phar variety of environmental stimuli that act through different maceutically acceptable salts thereof and are not excluded signaling pathways. Molecules that affect the budded-to from the scope of the invention. Such pharmacologically hyphal transition on Spider media in the screen may or may and pharmaceutically acceptable salts include, but are not US 2006/0154991 A1 Jul. 13, 2006

limited to, those prepared from the following acids: hydro chemical modification contemplated is the attachment of at chloric, hydrobromic, Sulfuric, nitric, phosphoric, maleic, least one moiety to the component molecule itself, where acetic, Salicylic, p-toluenesulfonic, tartaric, citric, methane said moiety permits (a) inhibition of proteolysis; and (b) Sulfonic, formic, Succinic, naphthalene-2-sulfonic, pamoic, uptake into the blood stream from the stomach or intestine. 3-hydroxy-2-naphthalenecarboxylic, and benzene Sulfonic. Also desired is the increase in overall stability of the Also, pharmaceutically acceptable salts can be prepared as component or components and increase in circulation time alkaline metal or alkaline earth salts, such as Sodium, in the body. Examples of such moieties include: polyethyl ammonium, magnesium, potassium or calcium salts of the ene glycol, copolymers of ethylene glycol and propylene carboxylic acid group. glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. Abuchowski and 0093 Suitable buffering agents include: acetic acid and Davis, 1981, “Soluble Polymer-Enzyme Adducts” In: salts thereof (1-2% W/V); citric acid and salts thereof (1-3% Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley W/V); boric acid and salts thereof (0.5-2.5% W/V); and Interscience, New York, N.Y., pp. 367-383: Newmark, et al., phosphoric acid and salts thereof (0.8-2% W/V). Suitable 1982, J. Appl. Biochem. 4:185-189. Other polymers that preservatives include benzalkonium chloride (0.003-0.03% could be used are poly-1,3-dioxolane and poly-1,3,6-tioXo W/V); chlorobutanol (0.3-0.9% W/V); parabens (0.01 cane. Preferred for pharmaceutical usage, as indicated 0.25% W/V) and thimerosal (0.004-0.02% W/V). above, are polyethylene glycol moieties. 0094) A variety of administration routes are available. 0097. For the component (or derivative) the location of The particular mode selected will depend, of course, upon release may be the stomach, the Small intestine (the duode the particular combination of drugs selected, the severity of num, the jejunum, or the ileum), or the large intestine. One the condition or disorder being treated, or prevented, the skilled in the art has available formulations which will not condition of the patient, and the dosage required for thera dissolve in the stomach, yet will release the material in the peutic efficacy. The methods of this invention, generally duodenum or elsewhere in the intestine. Preferably, the spealing, may be practiced using any mode of administration release will avoid the deleterious effects of the stomach that is medically acceptable, meaning any mode that pro environment, either by protection of the compound (or duces effective levels of the active compounds without derivative) or by release of the biologically active material causing clinically unacceptable adverse effects. Such modes beyond the stomach environment, Such as in the intestine. of administration include oral, rectal, topical, transdermal, Sublingual or intramuscular, infusion, intraparenteral, intra 0098. To ensure full gastric resistance a coating imper venous, intramuscular, intracavity, as a feed additive, as an meable to at least pH 5.0 is essential. Examples of the more aerosol, aurally (e.g., via eardrops), intranasal, inhalation, or common inert ingredients that are used as enteric coatings subcutaneous. Direct injection could also be preferred for are cellulose acetate trimelitate (CAT), hydroxypropylm local delivery to the site of infection. Oral administration ethylcellulose phthalate (HPMCP), HPMCP50, HPMCP55, may be preferred for prophylactic treatment because of the polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aqua convenience of the Subject (patient) as well as the dosing teric, cellulose acetate phthalate (CAP), Eudragit L. schedule. Eudragit S, and Shellac. These coatings may be used as mixed films. 0.095 For oral administration, the compounds of the invention can be formulated readily by combining the active 0099. A coating or mixture of coatings can also be used compound(s) with pharmaceutically acceptable carriers well on tablets, which are not intended for protection against the known in the art. Such carriers enable the compounds of the stomach. This can include Sugar coatings, or coatings which invention to be formulated as tablets, pills, dragees, cap make the tablet easier to Swallow. Capsules may consist of Sules, liquids, gels, syrups, slurries, Suspensions and the like, a hard shell (such as gelatin) for delivery of dry therapeutic for oral ingestion by a subject to be treated. Pharmaceutical i.e. powder; for liquid forms, a soft gelatin shell may be preparations for oral use can be obtained as Solid excipient, used. The shell material of cachets could be thick starch or optionally grinding a resulting mixture, and processing the other edible paper. For pills, lozenges, molded tablets or mixture of granules, after adding Suitable auxiliaries, if tablet triturates, moist massing techniques can be used. desired, to obtain tablets or dragee cores. Suitable excipients 0.100 The therapeutic can be included in the formulation are, in particular, fillers such as Sugars, including lactose, as fine multi-particulates in the form of granules or pellets of Sucrose, mannitol, or Sorbitol; cellulose preparations such particle size about 1 mm. The formulation of the material for as, for example, maize starch, wheat starch, rice starch, capsule administration could also be as a powder, lightly potato starch, gelatin, gum tragacanth, methyl cellulose, compressed plugs or even as tablets. The therapeutic could hydroxypropylmethyl-cellulose, sodium carboxymethylcel be prepared by compression. lulose, and/or polyvinylpyrrolidone (PVP). If desired, dis integrating agents may be added, such as the cross-linked 0101 Colorants and flavoring agents may all be included. polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof For example, the compound (or derivative) may be formu Such as Sodium alginate. Optionally the oral formulations lated (such as by liposome or microsphere encapsulation) may also be formulated in saline or buffers, i.e. EDTA for and then further contained within an edible product, such as neutralizing internal acid conditions or may be administered a refrigerated beverage containing colorants and flavoring without any carriers. agents. 0096. Also specifically contemplated are oral dosage 0102 One may dilute or increase the volume of the forms of the above component or components. The compo therapeutic with an inert material. These diluents could nent or components may be chemically modified so that oral include carbohydrates, especially mannitol, a-lactose, anhy delivery of the derivative is efficacious. Generally, the drous lactose, cellulose, Sucrose, modified dextrans and US 2006/0154991 A1 Jul. 13, 2006

starch. Certain inorganic salts may be also be used as fillers in Suitable liquids, such as fatty oils, liquid paraffin, or liquid including calcium triphosphate, magnesium carbonate and polyethylene glycols. In addition, stabilizers may be added. sodium chloride. Some commercially available diluents are Microspheres formulated for oral administration may also be Fast-Flo, Emdex, STA-RX 1500, Emcompress and Avicell. used. Such microspheres have been well defined in the art. 0103 Disintegrants may be included in the formulation All formulations for oral administration should be in dos of the therapeutic into a solid dosage form. Materials used ages Suitable for Such administration. as disintegrates include but are not limited to starch, includ 0.109 For buccal administration, the compositions may ing the commercial disintegrant based on Starch, Explotab. take the form of tablets or lozenges formulated in conven Sodium starch glycolate, Amberlite, Sodium carboxymeth tional manner. ylcellulose, ultramylopectin, Sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural Sponge 0110 For administration by inhalation, the compounds and bentonite may all be used. Another form of the disin for use according to the present invention may be conve tegrants are the insoluble cationic exchange resins. Pow niently delivered in the form of an aerosol spray presentation dered gums may be used as disintegrants and as binders and from pressurized packs or a nebulizer, with the use of a these can include powdered gums such as agar, Karaya or Suitable propellant, e.g., dichlorodifluoromethane, trichlo tragacanth. Alginic acid and its sodium salt are also useful rofluoromethane, dichlorotetrafluoroethane, carbon dioxide as disintegrants. or other Suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to 0104 Binders may be used to hold the therapeutic agent deliver a metered amount. Capsules and cartridges of e.g. together to form a hard tablet and include materials from gelatin for use in an inhaler or insufflator may be formulated natural products such as acacia, tragacanth, starch and containing a powder mix of the compound and a suitable gelatin. Others include methyl cellulose (MC), ethyl cellu powder base Such as lactose or starch. lose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose 0111. Also contemplated herein is pulmonary delivery of the compounds. The compound of the invention is delivered (HPMC) could both be used in alcoholic solutions to granu to the lungs of a mammal while inhaling and traverses across late the therapeutic. the lung epithelial lining to the blood stream. Examples of 0105. An anti-frictional agent may be included in the reports of inhaled molecules include Adjei et al., 1990, formulation of the therapeutic to prevent Sticking during the Pharmaceutical Research, 7:565-569; Adjei et al., 1990, formulation process. Lubricants may be used as a layer International Journal of Pharmaceutics, 63:135-144 (leupro between the therapeutic and the die wall, and these can lide acetate); Braquet et al., 1989, Journal of Cardiovascular include but are not limited to; Stearic acid including its Pharmacology, 13(suppl. 5): 143-146 (endothelin-1); Hub magnesium and calcium salts, polytetrafluoroethylene bard et al., 1989, Annals of Internal Medicine, Vol. III, pp. (PTFE), liquid paraffin, vegetable oils and waxes. Soluble 206-212 (al—antitrypsin); Smith et al., 1989, J. Clin. Invest. lubricants may also be used such as sodium lauryl Sulfate, 84:1145-1146 (a-1-proteinase); Oswein et al., 1990, “Aero magnesium lauryl Sulfate, polyethylene glycol of various solization of Proteins’. Proceedings of Symposium on Res molecular weights, Carbowax 4000 and 6000. piratory Drug Delivery II, Keystone, Colo., March, (recom binant human growth hormone); Debs et al., 1988, J. 0106 Glidants that might improve the flow properties of Immunol. 140:3482-3488 (interferon-g and tumor necrosis the drug during formulation and to aid rearrangement during factor alpha) and Platz et al., U.S. Pat. No. 5.284,656 compression might be added. The glidants may include (granulocyte colony stimulating factor). A method and com starch, talc, pyrogenic silica and hydrated silicoaluminate. position for pulmonary delivery of drugs for systemic effect 0107 To aid dissolution of the therapeutic into the aque is described in U.S. Pat. No. 5,451,569, issued Sep. 19, 1995 ous environment a surfactant might be added as a wetting to Wong et al. agent. Surfactants may include anionic detergents such as 0112 Contemplated for use in the practice of this inven Sodium lauryl Sulfate, dioctyl Sodium sulfo Succinate and tion are a wide range of mechanical devices designed for dioctyl sodium sulfonate. Cationic detergents might be used pulmonary delivery of therapeutic products, including but and could include benzalkonium chloride or benzethomium chloride. The list of potential non-ionic detergents that could not limited to nebulizers, metered dose inhalers, and powder be included in the formulation as Surfactants are lauromac inhalers, all of which are familiar to those skilled in the art. rogol 400, polyoxyl 40 Stearate, polyoxyethylene hydroge 0113 Some specific examples of commercially available nated castor oil 10, 50 and 60, glycerol monostearate, devices suitable for the practice of this invention are the polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. methyl cellulose and carboxymethyl cellulose. These sur Louis, Mo.; the Acorn II nebulizer, manufactured by Mar factants could be present in the formulation of the compound quest Medical Products, Englewood, Colo.; the Ventolin either alone or as a mixture in different ratios. metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, N.C.; and the Spinhaler powder inhaler, 0108) Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as manufactured by Fisons Corp., Bedford, Mass. soft, sealed capsules made of gelatin and a plasticizer, Such 0114 All such devices require the use of formulations as glycerol or Sorbitol. The push-fit capsules can contain the Suitable for the dispensing of compounds of the invention. active ingredients in admixture with filler such as lactose, Typically, each formulation is specific to the type of device binders such as starches, and/or lubricants such as talc or employed and may involve the use of an appropriate pro magnesium Stearate and, optionally, stabilizers. In soft cap pellant material, in addition to the usual diluents, adjuvants Sules, the active compounds may be dissolved or Suspended and/or carriers useful in therapy. Also, the use of liposomes, US 2006/0154991 A1 Jul. 13, 2006 microcapsules or microspheres, inclusion complexes, or 0121 The compounds, when it is desirable to deliver other types of carriers is contemplated. Chemically modified them systemically, may be formulated for parenteral admin compounds may also be prepared in different formulations istration by injection, e.g., by bolus injection or continuous depending on the type of chemical modification or the type infusion. Formulations for injection may be presented in unit of device employed. dosage form, e.g., in ampoules or in multi-dose containers, 0115 Formulations suitable for use with a nebulizer, with an added preservative. The compositions may take Such either jet or ultrasonic, will typically comprise the com forms as Suspensions, Solutions or emulsions in oily or pound dissolved in water at a concentration of about 0.1 to aqueous vehicles, and may contain formulatory agents such 25 mg of biologically active compound per mL of solution. as Suspending, stabilizing and/or dispersing agents. The formulation may also include a buffer and a simple 0.122 Pharmaceutical formulations for parenteral admin Sugar (e.g., for stabilization and regulation of osmotic pres istration include aqueous solutions of the active compounds Sure). The nebulizer formulation may also contain a surfac in water-soluble form. Additionally, suspensions of the tant, to reduce or prevent Surface induced aggregation active compounds may be prepared as appropriate oily caused by atomization of the Solution in forming the aerosol. injection Suspensions. Suitable lipophilic solvents or 0116 Formulations for use with a metered-dose inhaler vehicles include fatty oils such as sesame oil, or synthetic device will generally comprise a finely divided powder fatty acid esters, such as ethyl oleate or triglycerides, or containing the compound Suspended in a propellant with the liposomes. Aqueous injection Suspensions may contain Sub aid of a Surfactant. The propellant may be any conventional stances which increase the viscosity of the Suspension, Such material employed for this purpose, such as a chlorofluoro as sodium carboxymethyl cellulose, Sorbitol, or dextran. carbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or Optionally, the Suspension may also contain Suitable stabi a hydrocarbon, including trichlorofluoromethane, dichlo lizers or agents which increase the solubility of the com rodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2- pounds to allow for the preparation of highly concentrated tetrafluoroethane, or combinations thereof. Suitable surfac Solutions. tants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant. 0123. Alternatively, the active compounds may be in 0117 Formulations for dispensing from a powder inhaler powder form for constitution with a Suitable vehicle, e.g., device will comprise a finely divided dry powder containing sterile pyrogen-free water, before use. compound and may also include a bulking agent, such as 0.124. The compounds may also be formulated in rectal or lactose, Sorbitol. Sucrose, or mannitol in amounts which vaginal compositions such as suppositories or retention facilitate dispersal of the powder from the device, e.g., 50 to enemas, e.g., containing conventional Suppository bases 90% by weight of the formulation. The compound of the Such as cocoa butter or other glycerides. invention should most advantageously be prepared in par 0.125. In addition to the formulations described previ ticulate form with an average particle size of less than 10 ously, the compounds may also be formulated as a depot mm (or microns), most preferably 0.5 to 5 mm, for most preparation. Such long acting formulations may be formu effective delivery to the distal lung. lated with suitable polymeric or hydrophobic materials (for 0118 Nasal delivery of a pharmaceutical composition of example as an emulsion in an acceptable oil) or ion the present invention is also contemplated. Nasal delivery exchange resins, or as sparingly soluble derivatives, for allows the passage of a pharmaceutical composition of the example, as a sparingly soluble salt. present invention to the blood stream directly after admin istering the therapeutic product to the nose, without the 0.126 The pharmaceutical compositions also may com necessity for deposition of the product in the lung. Formu prise Suitable Solid or gel phase carriers or excipients. lations for nasal delivery include those with dextran or Examples of Such carriers or excipients include but are not cyclodextran. limited to calcium carbonate, calcium phosphate, various 0119 For nasal administration, a useful device is a small, Sugars, starches, cellulose derivatives, gelatin, and polymers hard bottle to which a metered dose sprayer is attached. In Such as polyethylene glycols. one embodiment, the metered dose is delivered by drawing 0.127 Suitable liquid or solid pharmaceutical preparation the pharmaceutical composition of the present invention forms are, for example, aqueous or saline Solutions for solution into a chamber of defined volume, which chamber inhalation, microencapsulated, encochleated, coated onto has an aperture dimensioned to aerosolize and aerosol microscopic gold particles, contained in liposomes, nebu formulation by forming a spray when a liquid in the chamber lized, aerosols, pellets for implantation into the skin, or dried is compressed. The chamber is compressed to administer the onto a sharp object to be scratched into the skin. The pharmaceutical composition of the present invention. In a pharmaceutical compositions also include granules, pow specific embodiment, the chamber is a piston arrangement. ders, tablets, coated tablets, (micro)capsules, Suppositories, Such devices are commercially available. syrups, emulsions, Suspensions, creams, drops or prepara 0120 Alternatively, a plastic squeeze bottle with an aper tions with protracted release of active compounds, in whose ture or opening dimensioned to aerosolize an aerosol for preparation excipients and additives and/or auxiliaries Such mulation by forming a spray when Squeezed is used. The as disintegrants, binders, coating agents, Swelling agents, opening is usually found in the top of the bottle, and the top lubricants, flavorings, Sweeteners or solubilizers are custom is generally tapered to partially fit in the nasal passages for arily used as described above. The pharmaceutical compo efficient administration of the aerosol formulation. Prefer sitions are suitable for use in a variety of drug delivery ably, the nasal inhaler will provide a metered amount of the systems. For a brief review of methods for drug delivery, see aerosol formulation, for administration of a measured dose Langer, Science 249:1527-1533, 1990, which is incorpo of the drug. rated herein by reference. US 2006/0154991 A1 Jul. 13, 2006

0128. The compositions may conveniently be presented described. The kits also may contain instructions for mixing, in unit dosage form and may be prepared by any of the diluting, and/or administrating the compounds. The kits also methods well known in the art of pharmacy. All methods can include other containers with one or more solvents, include the step of bringing the compounds of the invention Surfactants, preservative and/or diluents (e.g., normal saline into association with a carrier which constitutes one or more (0.9% NaCl), or 5% dextrose) as well as containers for accessory ingredients. In general, the compositions are pre mixing, diluting or administering the components to the pared by uniformly and intimately bringing the compounds sample or to the patient in need of Such treatment. of the invention into association with a liquid carrier, a finely 0.134. The compositions of the kit may be provided as any divided solid carrier, or both, and then, if necessary, shaping Suitable form, for example, as liquid solutions or as dried the product. powders. When the composition provided is a dry powder, 0129. Other delivery systems can include time-release, the powder may be reconstituted by the addition of a suitable delayed release or sustained release delivery systems. Such solvent, which may also be provided. In embodiments where systems can avoid repeated administrations of the com liquid forms of the composition are Sued, the liquid form pounds of the invention, increasing convenience to the may be concentrated or ready to use. The solvent will subject and the physician. Many types of release delivery depend on the compound and the mode of use or adminis systems are available and known to those of ordinary skill in tration. Suitable solvents for drug compositions are well the art. They include polymer based systems such as poly known and are available in the literature. The solvent will lactic and polyglycolic acid, polyanhydrides and polycapro depend on the compound and the mode of use or adminis lactone, nonpolymer systems that are lipids including sterols tration. Such as cholesterol, liposomes; phospholipids; hydrogel release systems; Silastic systems; peptide based system; 0.135 The kit, in one set of embodiments, may comprise implants and the like. Specific examples include, but are not a carrier means being compartmentalized to receive in close limited to: (a) erosional systems in which the active agent is confinement one or more container means such as vials, contained in a form within a matrix, found in U.S. Pat. Nos. tubes, and the like, each of the container means comprising 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional one of the separate elements to be used in the method. For systems in which an active component permeates at a example, one of the container means may comprise a controlled rate from a polymer such as described in U.S. Pat. positive control in the assay. Additionally, the kit may Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, include containers for other components, for example, buff pump-based hard wired delivery systems can be used, some ers useful in the assay. of which are adapted for implantation. 0.136 The compounds of the invention may optionally be delivered with other antifungal agents in the form of anti 013.0 Use of a long-term sustained release implant may fungal cocktails, or individually, yet close enough in time to be particularly suitable for treatment of chronic conditions. have a synergistic effect on the treatment of the infection. An 0131 “Long-term' release, as used herein, means that the antifungal cocktail is a mixture of any one of the above implant is constructed and arranged to deliver therapeutic described compounds with another antifungal drug which levels of the active ingredient for at least 7 days, and may or may not be a compound of the invention. The use of preferably 30-60 days. The implant may be positioned at the Such cocktails in pharmaceutical preparations is routine. In site of infection. Long-term Sustained release implants are this embodiment, a common administration vehicle (e.g., well-known to those of ordinary skill in the art and include tablet, implants, injectable Solution, injectable liposome some of the release systems described above. Solution, etc.) could contain both the compound of the 0132) As used herein, “promoted includes all methods invention and the other antifungal agent(s). of doing business including methods of education, hospital 0.137 Anti-fungal agents are useful for the treatment and and other clinical instruction, pharmaceutical industry activ prevention of infective fungi. Some of these treatments are ity including pharmaceutical sales, and any advertising or described above. Anti-fungal agents are sometimes classi other promotional activity including written, oral and elec fied by their mechanism of action. Some anti-fungal agents tronic communication of any form, associated with compo function as cell wall inhibitors by inhibiting glucose Syn sitions of the invention in connection with treatment of thase. These include, but are not limited to, basiungin/ECB. fungal infections. “Instructions' can define a component of Other anti-fungal agents function by destabilizing mem promotion, and typically involve written instructions on or brane integrity. These include, but are not limited to, immi associated with packaging of compositions of the invention. dazoles, such as clotrimazole, Sertaconzole, fluconazole, Instructions also can include any oral or electronic instruc itraconazole, ketoconazole, miconazole, and Voriconacole, tions provided in any manner. The “kit' typically defines a as well as FK 463, amphotericin B, BAY 38-9502, MK991, package including any one or a combination of the compo pradimicin, UK 292, butenafine, and terbinafine. Other sitions of the invention and the instructions, or homologs, anti-fungal agents function by breaking down chitin (e.g. analogs, derivatives, and functionally equivalent composi chitinase) or immunosuppression (501 cream). tions thereof, but can also include a composition of the 0.138 Antifungal agents include Acrisorcin; Ambruticin; invention and instructions of any form that are provided in Amphotericin B; AZaconazole; AZaserine; Basifungin; connection with the composition in a manner Such that a Bifonazole; Biphenamine Hydrochloride; Bispyrithione clinical professional will clearly recognize that the instruc Magsulfex; Butoconazole Nitrate; Calcium Undecylenate: tions are to be associated with the specific composition. Cancidas (Caspofungin Acetate), Candicidin; Carbol-Fuch 0133. The kits described herein may also contain one or sin; Chlordantoin: Ciclopirox: Ciclopirox Olamine: Cilofun more containers, which can contain compounds such as the gin, Cisconazole; Clotrimazole; Cuprimyxin; Denofungin; species, signaling entities, biomolecules and/or particles as Dipyrithione; Doconazole; Econazole; Econazole Nitrate; US 2006/0154991 A1 Jul. 13, 2006

Enilconazole; Ethonam Nitrate; Fenticonazole Nitrate; Fil nutrient broth (DIFCO Labs.), mannitol (Sigma-Aldrich), ipin; Fluconazole; Flucytosine; Fungimycin; Griseofulvin; KHPO, (Sigma-Aldrich) and d-H.O. Hamycin; Isoconazole, Itraconazole; Kalafungin; Ketocona Zole; Lomofungin; Lydimycin; Mepartricin; Miconazole; 0144) Next, small molecules were assayed for their abil Miconazole Nitrate; Monensin; Monensin Sodium; Naftifine ity to inhibit the Spider media-induced hyphal growth. As a Hydrochloride; Neomycin Undecylenate; Nifuratel; Nifur proof-of-concept experiment, latrunculin-A (Lat A) was merone; Nitralamine Hydrochloride: Nystatin; Octanoic added, which reversibly inhibits yeast growth by depoly Acid; Orconazole Nitrate; Oxiconazole Nitrate; Oxifungin merizing actin. Addition of 1.6 uM Lat A inhibited the Hydrochloride; Parconazole Hydrochloride; Partricin; budded-to-hyphal transition and hyphal growth. This inhi Potassium Iodide; Proclonol; Pyrithione Zinc, Pyrrolnitrin; bition was both dose-dependent (effect was lost at 0.1 uM) Rutamycin; Sanguinarium Chloride; Saperconazole; Sco and reversible. This result indicated that the screen was pafungin: Selenium Sulfide: Sinefungin; Sulconazole sensitive to a known cytostatic drug. Nitrate; Terbinafine; Terconazole; Thiram; Ticlatone; Tio 0145 Using this strategy, 70 molecules were screened conazole; Tolciclate; Tolindate; Tolnaftate; Triacetin; Tri from a Chembridge small molecule library (ChemBridge afungin: Undecylenic Acid; Viridofulvin; Zinc Unde Corp., San Diego, Calif.). Of the 70 molecules, most had no cylenate; and Zinoconazole Hydrochloride. effect on hyphal formation, several were either cytostatic or cytotoxic leading to lack of growth of budded or hyphal 0139 Each of the references, patents, and patent appli cells, and some inhibited the budded-to-hyphal transition cations referred to herein is hereby incorporated by refer and hyphal elongation without affecting budded growth. The CCC. inhibitory molecules include the following compounds: EXAMPLES Example 1 1)N-1Ns1 New High-Throughput Screen for Inhibitors and C Enhancers of the Budded-to-Hyphal Transition 0140 C. albicans is a constitutively diploid organism and, thus, standard sexual genetic approaches for the isola tion of recessive, loss-of-function mutants are not currently feasible. Therefore, alternative strategies for the identifica tion of components participating in the budded-to-hyphal transition are employed. s--O 0141 One such approach is a high-throughput small molecule screen, in which a large diverse collection of individual small organic molecules (<1500 Da) are tested for their ability to inhibit or enhance a biological process, in this C case the budded-to-hyphal transition. The active molecules OH and their structural derivatives are used to identify the component or components involved in the process. This N-- “phenotype-based' approach has been used successfully to identify novel components involved in a variety of different biological processes (reviewed in Ward, G. E., et al., 2002, Cell. Microbiol. 4:471-482). C 0142. A small molecule screen was developed to identify O inhibitors and enhancers of the budded-to-hyphal transition. A proof-of-concept study verified that the assay is robust and reproducible. In addition, several known and new molecules inI NH2 that inhibit the budded-to-hyphal transition were identified. leN 0143 For the screen, C. albicans cells were grown in YNB media that inhibits hyphal growth and then transferred N to 384-well optical plates containing Spider media to induce the budded-to-hyphal transition and hyphal elongation. N Microscopic examination indicated that C. albicans cells reproducibly undergo the budded-to-hyphal transition and show significant hyphal elongation within 4 hours (h) at 37° C. in Spider media shown FIG. 3. In FIG. 3, the indicated Small molecules were added at the indicated concentrations at time 0 and growth was allowed to continue for 4 h at 37° C. before the cells were fixed and observed microscopically. YNB media is well known in the art and contains yeast nitrogen base (DIFCO Labs.), glucose (US Biological) and d-HO. Spyder media is well known in the art and contains US 2006/0154991 A1 Jul. 13, 2006 15

shown recently to mate under laboratory conditions at a -continued reasonable frequency to form tetraploids (Miller, M. G., et al., 2002, Cell 110:293-302). However, these tetraploids do not undergo a demonstrated meiosis or meiotic recombina tion, making classical sexual genetic approaches difficult. In order to circumvent this limitation, a “forward chemical genetic' approach was developed to identify Small mol ecules that either inhibit or stimulate the budded-to-hyphal HO Or transition, described below. C 0150. Several molecules that inhibit the budded-to-hy phal transition have been identified, including:

s1 OESEO s1N1 N C

OH O

N OH C N-- )

0146 Interestingly, a known actin-stabilizing drug jas plakinolide (Jas) was identified as a dose-dependent cyto C toxic molecule in the screen, leading to an apparent cell OH cycle arrest as small budded cells (shown in FIG. 3). N-N- Jasplakinolide was previously shown to have anti-fungal O N activity (Scott, V. R., et al., 1988, Antimicrob. Agents Chemother. 32:1154-1157), and its isolation from a random selection of small molecules further supports the efficacy of the screen. The effects of the active molecules that inhibited the budded-to-hyphal transition were also shown to be C dose-dependent and reversible, indicating that the inability O tocytotoxic undergo effect. the budded-to-hyphal transition was not due to a in 0147 Molecules from the Chembridge library that are leN structurally related to the active molecules were also assayed for their efficacy. N 0148. These results indicate that the screen is robust, reproducible, amenable to high-throughput protocols, and N able to identify known and novel molecules that can inhibit the budded-to-hyphal transition. This approach significantly augments the genetic and biochemical strategies currently N O used to identify and characterize the components that regu late the budded-to-hyphal transition and enhance the iden- N-N-1N1 tification of potential therapeutic molecules. Example 2 Identification of New Components of the Budded-to-Hyphal Transition Pathway Using a New High-Throughput Small Molecule Screen 0149 While typical genetic approaches are relatively NF facile with modern techniques, the use of genetic screens to identify new components of the budded-to-hyphal transition pathway is complicated by the fact that C. albicans is a HO constitutive diploid. Certain C. albicans isolates have been US 2006/0154991 A1 Jul. 13, 2006 16

molecule and adding fresh Spider media; cells that resume -continued hyphal growth indicate that the molecule is reversible in C action and that the molecule's initial effects were not due to cytotoxicity.

What is claimed is: 1. A method for treating a fungal infection in a Subject in need of Such treatment comprising administering to the OFSFO Subject a compound that modulates a phenotypic transition of yeast cells. 2. The method of claim 1 wherein the compound is of the OH O formula:

N H RI C A 1 (CRR)-N S R2 R5 N N 0151. These molecules were further characterized in sec X, it --Y ondary screens for their ability to inhibit hyphal induction 2 2 under the variety of hyphal-inducing signals described pre viously and for effects on specific cell biological processes or structures (described below). In addition, these molecules or a pharmaceutically acceptable salt thereof, are used as reagents to identify their cellular targets and their wherein R' and R are independently selected from the cognate pathways. Additional molecules are also identified group consisting of hydrogen, C-C alkyl, C-C alk using similar techniques and in a high-throughput version of enyl, C-C alkynyl, aryl, and heteroaryl; the screen. Not only does this approach allow the dissection of the budded-to-hyphal transition pathway, but may also R. R', and Rare independently selected from the group lead to new therapeutic molecules and targets. consisting of hydrogen, hydroxy, halo C-C alkoxy, 0152 The basic microscopic screen was described in C-C alkyl, C-C alkenyl, and C-C alkynyl: detail above. To identify inhibitors, C. albicans cells are P is an integer varying from 1 to 5: initially grown in YNB media that inhibits hyphal growth each X and each Y are independently selected from the and then transferred to 384-well optical assay plates con group consisting of halo, hydroxy, C-C alkoxy, nitro, taining Spider media to induce the transition to hyphae. C-C alkyl, C-C alkenyl, C-C alkynyl, amido, aryl, Individual molecules from the Chembridge small molecule heteroaryl, and acyl: or two Xs or two Ys together form library (Kao, R. Y. T., et al., 2002, Proc. Natl. Acad. Sci. a 5, 6, or 7-membered ring: USA99:10066-10071), or other library, are transferred from a 384-well plate containing stock solutions to individual m is an integer varying from 0 to 5; and wells of the assay plates. For convenience, positive and negative controls may be located at each corner of the plate. n is an integer varying from 0 to 5: These plates are incubated at 37° C. for 4 h to allow for in an amount effective to treat the fungal infection. hyphal elongation before the cells are fixed and screened. 3-6. (canceled) Screening occurs on an inverted Nikon microscope with a 7. The method of claim 1, wherein the fungal infection is computer driven XY stage with DIC/Hoffman optics, digital caused by a yeast of the Candida genus. SPOTR camera and automatic shutter. Pictures are auto 8. (canceled) matically taken of each well with the plate number and well 9. The method of claim 1 wherein the compound is position embedded in the picture through a script written for administered in combination with a second antifungal agent. OpenLab(R) image analysis software from Improvision 10. (canceled) (INVIVO, from QED Imaging, Inc.). Inhibitory molecules 11. The method of claim 1 wherein the first phenotype is are identified by rapidly screening a picture of each well for a budded form and the second phenotype is a hyphal form. the presence of budded cells and absence of hyphal cells. Each inhibitor is retested at both 4 h and 16 h to eliminate 12-13. (canceled) those compounds that are cytotoxic, delay hyphal formation, 14. A method for treating a fungal infection in a subject or delay elongation through a growth defect. Molecules that in need of Such treatment comprising administering to the give consistent inhibition of the budded-to-hyphal transition Subject a compound of the formula: with continued budded growth with no hyphae out to 16 are further characterized as described below. A dose-response for each molecule is established through serial dilutions in order to determine the threshold concentration of each X X \ Sy molecule. Typical threshold concentrations range from about 1 to about 100 micromolar (LM). In addition, the reversibility of each molecule is tested by washing out the US 2006/0154991 A1 Jul. 13, 2006 17

or a pharmaceutically acceptable salt thereof, 29-35. (canceled) wherein R' and R are independently selected from the 36. The method of claim 28, wherein the fungal infection group consisting of hydrogen, C-C alkyl, C-C, is caused by a yeast of the Candida genus. alkoxy, hydroxy, and halo; 37. (canceled) 38. The method of claim 28 wherein the compound is p is an integer ranging from 1 to 5: administered in combination with a second antifungal agent. each X and each Y are independently selected from the 39. A method for treating a fungal infection in a subject group consisting of halo, hydroxy, nitro, C-C alkoxy, in need of Such treatment comprising administering to the C-C alkyl, C-C alkenyl, C-C alkynyl, amido, aryl, Subject a compound of the formula: heteroaryl, and acyl: or two Xs or two Ys together form a 5, 6, or 7-membered ring: m is an integer varying from 0 to 11; and n is an integer varying from 0 to 11: N O in an amount effective to treat the fungal infection. Nu-N-1N1 nu 15-24. (canceled) 25. The method of claim 14, wherein the fungal infection is caused by a yeast of the Candida genus. 26. (canceled) 27. The method of claim 14 wherein the compound is administered in combination with a second antifungal agent. 28. A method for treating a fungal infection in a subject in need of Such treatment comprising administering to the or a pharmaceutically acceptable salt thereof, Subject a compound of the formula: wherein R is selected from the group consisting of hydro gen, hydroxy, C-C alkoxy, halo, C-C alkyl, C-C, OH alkenyl, and C-C alkynyl: each X and each Y are independently selected from the N-- group consisting of halo, hydroxy, C-C alkoxy, nitro, C-C alkynyl, amido, aryl, heteroaryl, and acyl: or two Xs or two Ys together form a 5, 6, or 7-membered ring: m is an integer ranging from 0 to 5; and n is an integer ranging from 0 to 5: or a pharmaceutically acceptable salt thereof, in an amount effective to treat the fungal infection. wherein R' and R are independently selected from the 40-45. (canceled) group consisting of hydrogen, C-C alkyl, C-C alk 46. The method of claim 39, wherein the fungal infection enyl, C-C alkynyl, aryl, and heteroaryl; is caused by a yeast of the Candida genus. 47. (canceled) wherein R. R. R. R. and R7 are independently selected 48. The method of claim 39 wherein the compound is from the group consisting of hydrogen, hydroxy, C-C, administered in combination with a second antifungal agent. alkoxy, halo, C-C alkyl, C-C alkenyl, and C-C, 49. A method for treating a fungal infection in a subject alkynyl; or R and R together are a carbonyl oxygen; in need of Such treatment comprising administering to the n is an integer ranging from 1 to 5: Subject a compound of the formula:

O s1 CRR3 ". CRR3 ); s1 CRR2),ha -(CR'R)CRR2 N Xin 3 Y, i. Z, it 2 R 2 O 2

each X and each Y are independently selected from the or a pharmaceutically acceptable salt thereof, group consisting of halo, hydroxy, C-C alkoxy, nitro, wherein each R', each R, and each Rare independently C-C alkyl, C-C alkenyl, C-C alkynyl, amido, aryl, Selected from the group consisting of hydrogen, heteroaryl, and acyl: or two Xs or two Ys together form hydroxy, C-C alkoxy, halo, C-C alkyl, C-C alk a 5, 6, or 7-membered ring: enyl, and C-C alkynyl: or R' and R together are a m is an integer varying from 0 to 5; and carbonyl oxygen; p is an integer varying from 0 to 5: each q is an integer ranging from 0 to 6: in an amount effective to treat the fungal infection. each r is an integer ranging from 0 to 6; US 2006/0154991 A1 Jul. 13, 2006 18

each S is an integer ranging from 0 to 6; observing whether or not there is a measurable change in the cells from the first phenotypic form to a second each t is an integer ranging from 0 to 6: phenotypic form; and each X, each Y, and each Z are independently selected classifying the test compound as a modulator of the from the group consisting of halo, hydroxy, C-C, phenotypic transition if there is a change or classifying alkoxy, nitro, C-C alkynyl, amido, aryl, heteroaryl, the test compound as not being a modulator of the and acyl: or two Xs or two Ys, or two Zs, together form phenotypic transition if there is no change. a 5, 6, or 7-membered ring: 64-65. (canceled) m is an integer ranging from 0 to 5: 66. The method of claim 63 wherein the first phenotype is a budded form and the second phenotype is a hyphal form. n is an integer ranging from 0 to 4; and 67-70. (canceled) p is an integer ranging from 0 to 5: 71. A method for identifying an inhibitor of a budded-to hyphal transition of Candida albicans cells comprising: in an amount effective to treat the fungal infection. 50-59. (canceled) providing a sample of the cells characterized by the 60. The method of claim 49, wherein the fungal infection budded form; is caused by a yeast of the Candida genus. adding a test compound to the sample of the cells; 61. (canceled) observing whether or not there is a measurable change in 62. The method of claim 49 wherein the compound is the Candida albicans cells from the budded form to the administered in combination with a second antifungal agent. hyphal form; and 63. A method for identifying a modulator of a phenotypic classifying the test compound as an inhibitor of the transition of Candida cells comprising: phenotypic transition if there is no increase in the providing a sample of the cells characterized by a first budded form or classifying the test compound as not phenotypic form; being an inhibitor if there is no change. adding a test compound to the sample of the cells; k k k k k