Title: Preclinical Drug Discovery for New Anti-Pneumocystis compounds Running title: Drug Discovery for Pneumocystis Authors: Melanie T. Cushion, Ph.D.,1,2 Peter D. Walzer, MD., MSc.1,2 Affiliations: 1 Research Service, Veterans Affairs Center, Cincinnati, OH,2

Division of

Infectious Diseases, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH Mailing Addresses: Research Service (151) VA Medical Center 3200 Vine Street Cincinnati, OH 45220 USA MTC Telephone: (513) 861-3100 ext. 4417 FAX: (513) 475-6415 Email: Melanie.cushion@uc.edu or Melanie.cushion@va.gov PDW Telephone: (513) 475-6328 Fax: (513) 475-6415 Email: peter.walzer@va.gov

Financial Support: These studies were supported by the National Institutes of Health contract N01-AI-25467 and R01 grants RO1 AI050450 and RO1 AI44651, and the Department of Veterans Affairs. Conflicts of Interest: None

ABSTRACT Pneumocystis remains an important cause of fatal pneumonia (PCP) in HIV patients and other immunocompromised hosts. Preclinical drug discovery for agents active against PCP has been hindered in large part by the lack of a continuous in vitro growth system. Since approval in 1978, the combination of the folic acid synthesis inhibitor combination trimethoprimsulfamethoxazole has been the primary agent for prophylaxis and therapy. Short term in vitro assays using cell monolayer-based and cell free systems in combination with in vivo studies in rodent models of infection have been the mainstay of candidate screening methods. These systems and their applications are reviewed here. Most strategies have focused on testing compounds already in clinical use, such as dapsone or atovaquone, for activity against Pneumocystis alone or in combination, and as parent compounds for chemical derivation, such as pentamidine and its analogues. Other successes from the bench include primaquine-clindamycin for moderate pneumonia and the family of Beta-glucan synthase inhibitors, which hold promise for clinical use against PCP. Despite the significant obstacles for drug discovery, progress in identifying novel agents has been made with current systems and the promise of future new targets is expected with the annotation of the Pneumocystis genome.

KEY WORDS Pneumocystis Pneumonia (PCP) Drug Development In Vitro Testing Cell-Free Systems In Vivo Testing Mouse and Rat Models Treatment and Prophylaxis Applicability to Humans

1. INTRODUCTION: APPROACHES TO ANTIMICROBIAL DRUG DEVELOPMENT New drugs are typically discovered by identification of a target within the microbe, that if inhibited would lead to the death (ideally) or stasis of the infection. Compounds that are predicted to interact with the target are then synthesized and tested to confirm the interaction and expected outcome. A lead compound or compounds usually emerge from these evaluations that are then further tested directly on the organism in some kind of in vitro system. Those with efficacy are then brought forward into animal testing in models that typically begin in rodent systems such as rats or mice. Efficacious compounds are then tested extensively to ensure that they will be safe to administer to humans. These tests typically last for one to five years and involve toxicity evaluations; pharmacological analysis for organ and blood distribution; and formulation studies to determine delivery vehicles. Should a company, government institution or individual in the United States wish to bring the drug for ultimate use in humans, then the results of all these evaluations must by provided to the FDA to obtain permission for testing in humans. This testing begins with submission of this information and application for permission to administer the drug to healthy volunteers or patients. The vehicle with which to initiate these studies is an Investigational New Drug (IND) request. The compound usually is then evaluated in three successive phases, I to III, with numbers of patients increasing at each phase. In Phase I, the safety and tolerability of the drug in humans is evaluated in healthy volunteers, usually 20100 persons. In Phase II, the efficacy of the drug is evaluated in patients with the condition the drug is intended to treat. Testing can be conducted in several hundred patients to determine safety and effectiveness, and establishes the minimum and maximum effective doses. The drug is often compared to the standard of care. Safety and efficacy testing are further expanded during Phase III trials which typically involve several hundred more patients. In many cases,

after licensing a drug, the parent pharmaceutical company runs an additional Phase IV trial that is designed to learn more about the potential side effects of a drug; determine what the long term risks and benefits may be; and gauge the activity of the drug when it is put into more widespread use. 2. PRECLINICAL DRUG DEVELOPMENT FOR PNEUMOCYSTIS The entire process of drug discovery and development is designed to bring only those products that are both effective and safe to the public. The $900 million price tag is often quoted as the cost to bring a new drug to the public, with an average time line of 10-12 years. In the case of drugs with which to treat AIDS/HIV, the Food and Drug Administration (FDA) has implemented an abbreviated course for testing and approval termed fast tracking. While this approach has been quite successful in bringing new treatments that target HIV to the infected population, efforts to identify new therapies to treat the concomitant co-infections has not been as robust. Thus, effective therapies for fungal pathogens such as Pneumocystis jirovecii or Cryptococcus neoformans; the protozoan agent Cryptosporidium parvum, or the bacterium Mycobacterium avium are either quite limited in number or non-existent. These eukaryotic and prokaryotic agents offer challenges distinct from viral agents in a host with severely compromised immune function. In this review, we focus on the historical aspects of preclinical drug development for the fungal pathogens in the genus Pneumocystis leading to the approaches and strategies used today. The AIDS epidemic in the United States began in the early 1980s. Diagnosis of the condition we now know as HIV/AIDS was often made by the detection of a heretofore rare pneumonia in patients caused by an unusual organism known as Pneumocystis carinii [1,2] . The pneumonia was referred to as PCP (Pneumocystis carinii pneumonia). Thought to be a

zoonotic protozoan, treatment of the pneumonia was limited to an anti-trypanosomal drug, pentamidine isethionate, usually on a compassionate basis, and the combination of trimethoprimsulfamethoxazole (TMP-SMX). Pentamidine was identified in 1958 as an effective treatment for cases of interstitial pneumonia in infants, of which the causal agent was later identified as Pneumocystis. During the succeeding 5 years, mortality was reduced from 50% to about 20%, and in the following 2 years to about 3% [101]. Pentamidine was administered parenterally and the adverse effects were substantial. Nephrotoxicity, neutropenia, hypotension, hypoglycemia, thrombocytopenia and rashes were common side effects [189]. In 1966, Frenkel and colleagues pioneered drug screening for PCP in a corticosteroidimmunosuppressed rat model of infection [71]. Their findings of organism burden reduction and increased survival by administration of the combination of pyrimethamine (a dihydrofolate reductase inhibitor) plus sulfadiazine (dihydropteroate synthase inhibitor) to block folic acid synthesis foreshadowed identification of the most efficacious therapy for PCP at that time and still today, trimethoprim-sulfamethoxazole, which also targets folic acid biosynthesis at the same two targets. In 1974, Hughes et al. showed that the combination of trimethoprimsulfamethoxazole was highly effective against P. carinii pneumonia in the immunosuppressed rat model when administered therapeutically and prophylactically [94]. The combination was approved in 1981 for the treatment of PCP and in 1994 for prophylaxis by Glaxo Wellcome (Approved Drugs for HIV/AIDS or AIDS-related Conditions - Pamphlet by: Food and Drug Administration , July 6, 1999 ). Besides being at least equal in efficacy to pentamidine and with fewer side effects, trimethoprim-sulfamethoxazole was available in oral and parenteral formulations and it soon supplanted pentamidine as the primary drug of choice for PCP.

With the overwhelming numbers of patients requiring treatment for PCP during the HIV/AIDS epidemic, disturbing adverse reactions to the sulfa component of the TMP-SMX combination were uncovered and led to increased requests to the CDC for pentamidine as a secondary therapy. Expanded efforts to find new drugs for the treatment of PCP were spearheaded by the National Institutes for Health in the National Institute for Allergy and Infectious Diseases newly created Division of AIDS, leading to contractual agreements with investigators for pre-clinical development of candidate compounds as well as investigator-initiated awards searching for new targets and other strategies. We have held a contract with the NIH for 23 years and these experiences will be discussed in the context of efforts by other investigators in this review. 3. THE GENUS PNEUMOCYSTIS Members of the genus Pneumocystis are fungal organisms that grow within the lungs of mammalian hosts. Recent studies suggest they are harbored in immunologically intact hosts with few consequences. However, in hosts with debilitated immune function, a pneumonia can occur which may result in death if untreated. The Pneumocystis organisms found in different mammalian hosts are morphologically quite similar, which led early investigators to assume that there was just a single genus and species, Pneumocystis carinii, which infected all mammalian species. There is now clear evidence supporting the existence of genetic diversity and host specificity within the genus [149]. The species infecting humans is known as Pneumocystis jirovecii while the most common form in rats is Pneumocystis carinii [70]. The other ratdwelling species, P. wakefieldiae, is found less frequently and most often as a co-infection with P. carinii [52, 60, 96]. Only a single species has been found to infect mice, P. murina [108] and rabbits, P. oryctolagi [64]. More species are anticipated to be formally described as genetic and

host-related evidence grows larger. To the best of the current state of knowledge, drug responses by Pneumocystis infections in animal models closely mimics responses in human beings [184]. 4. PUTATIVE LIFE CYCLE AND DEVELOPMENTAL STAGES Pneumocystis are generally considered to be host-dependent fungi; growth outside the mammalian lung has not been observed for any extended length of time nor has any environmental cycle been identified. Three developmental forms of Pneumocystis are generally recognized: the trophic form (1-5um); the precyst or sporocyte (4-8 um); and the cyst or ascus (5-10 um) (Figure 1). The lifecycle of Pneumocystis is thought to have 2 phases; an asexual phase that is mediated by the process of binary fission rather than budding; and a sexual phase involving mating with progression to ascus formation [47]. Since the lifecycle cannot be verified by in vitro studies, these proposed processes have been based on visual observations at various microscopic levels, kinetic studies in limited in vitro culture systems, and more recently, the presence of gene homologs associated with mating, meiosis and sporulation in the genome of P. carinii that have contributed to a better, but not complete, understanding of its reproductive processes [55]. A proposed lifecycle is shown in Figure 1. 5. IN VITRO METHODS FOR SELECTION OF CANDIDATE ANTI-PNEUMOCYSTIS AGENTS 5.1 General Problems The lack of a continuous in vitro cultivation system for any species of Pneumocystis has put this area of research at a significant disadvantage. All in vitro system have flaws in their ability to predict drug activity in the host, but in the case of Pneumocystis, this problem is exacerbated by the necessity to base drug responses on relatively short term cultures and the inability to take the treated organisms and assess whether the drug effect was static or cidal.

Since these organisms cannot be maintained continuously in culture, animal models have furnished the organism supplies necessary for such studies. Adequate numbers of P. jirovecii from samples such as bronchoalveolar lavage fluid are difficult to obtain. They contain insufficient numbers with which to perform assays and are often contaminated with other human pathogens. Thus, inocula typically have been comprised of Pneumocystis freshly isolated from immunosuppressed animals, usually rats or mice, but more recently, cryopreserved and batched lots have been implemented to create more standardized assays [38]. The latter offers the ability to screen for other microbial contaminants and viability. Quantification of organisms has also been problematic due to the tenacious adherence of the Pneumocystis organisms to one another, and often to host cells. In many studies, a test for viability has not accompanied the evaluation of efficacy by microscopic enumeration. In addition, administration of drugs can adversely affect the monolayer as well as the organisms, leading to false interpretations of efficacy. Likewise the presence of mammalian cells precludes many simple methods of assessment of effect, such as incorporation of radiolabels, which would be taken up by organisms and host cells or multi-well plate assays using chromogenic reactions read on specialized readers, for the same reasons. There have been 2 major approaches to screening of candidate compounds against Pneumocystis in vitro- cell monolayer based and cell free systems. In limited cases, recombinant proteins which are targets of specific drugs have been used to screen families of related compounds [75, 77, 78, 81, 151] or by using the surrogate genes in yeast [121]. 5.2 Cell Monolayer-Based Systems In the 1980s, there was an immediate need for new drugs with which to treat P. jirovecii pneumonia due to the increased numbers of susceptible individuals stemming from the HIV/AIDS epidemic. At that time, most laboratories used a mammalian cell monolayer based

system to which organisms purified from mostly rodent lungs were added in medium containing candidate drugs in varying concentrations. Efficacy was determined by microscopic enumeration of the organisms present in the supernatant over time in untreated vs treated wells or flasks. In these systems, the trophic forms were the major life cycle stage that survived the purification and in vitro culture system process, thus, the actions of the compounds were gauged primarily against this form rather than the cyst stage. This was an obvious shortcoming, since the cyst stage of many microbes provides a measure of resistance to environmental insults as well as drug exposures. The cell lines in the majority of these studies were Chick Embryonic epithelial lung cells (CEL), WI-38 (human embryonic fibroblasts), and A-549 (human lung adenocarcinoma cell line) [46] (Table 1.). Later, human embryonic lung cell lines (HEL 299) were adopted in lieu of the primary line, WI-38 [7, 12] while A549 cells were used in a series of studies using human-derived Pneumocystis (P. jirovecii) as the inocula [33 35] as well as ratderived organisms (45). Other cell lines, such as the Mv 1 Lu mink cell line were used on a limited basis, but followed the basic efficacy assessment using microscopic enumeration of organisms within the supernatant [127]. As these systems evolved, a need for a more efficient assessment became apparent. Enumeration of stained slides was tedious, time consuming and often lacked a viability measurement. A primary consideration in establishing more automated or less biased techniques was the contribution of the mammalian monolayer, necessitating a Pneumocystis-specific approach. In one case, an enzyme-linked immunosorbent assay (ELISA) which used a polyclonal antibody specific to P. carinii antigens to capture organisms and quantify numbers, was applied to drug screening [65]. However, the system was not widely adopted and microscopic enumeration continues as the primary assessment in some laboratories to date [9, 35].

We conducted extensive Pneumocystis culture, life cycle, and drug screening studies using monolayers of A549 cell line and a transformed lung fibroblast line, WI-38 VA13 [53, 54, 56, 57]. A standard method of Pneumocystis enumeration was established by counting of Giemsa/Diff-Quik stained organism nuclei rather than a selected life cycle stage (e.g. cyst, trophic form) and use of the rapid variant of the Giemsa stain, Diff-Quik [53, 56]. The rapid Giemsa stain has been adopted by almost every laboratory employing microscopic enumeration of Pneumocystis nuclei (sometimes referred to as trophozoites) as well as in the clinical laboratory for rapid diagnosis of Pneumocystis pneumonia [48]. Pneumocystis was counted in preparations prior to inoculation to avoid a false impression of growth due to release of the organisms from the monolayer over time. Our observations and experience with these tissue culture systems made it apparent that use of monolayers for in vitro drug screening was problematic. Unless a viability stain was included in the counts over the culture period, dead Pneumocystis could stain with Diff-Quik or a cyst stain and lead to misinterpretation of a drug's effects (leading to a false negative). Our later studies using quantification of ATP as a viability assessment of the Pneumocystis verified the lack of viability in cultures where the Pneumocystis stained with Diff-Quik [30, 49]. Certain drugs could influence the viability and metabolic processes of the monolayer cells, leading to an incorrect conclusion of efficacy for Pneumocystis. Not all inocula produced the same level of replication in vitro, leading us to exclude all experiments where untreated Pneumocystis failed to increase 3-fold. Besides these concerns, we found it difficult to evaluate uptake of radiolabeled precursors without interference by the monolayer, although attempts were made to block metabolism radiometrically or by chemical means (e.g. gamma radiation, muconomycin A).

5.3 Cell-Free Systems (Intact Organisms) Alternative approaches to screening of anti-Pneumocystis compounds have taken advantage of a cell-free environment by using incorporation of radiolabeled precursors or a global assessment of viability such as ATP. Initially, cell extracts were used to evaluate the activities of inhibitors on specific enzymes such as dihydrofolate reductase (DHFR) [4, 24], dihydropteroate synthase (DHPS) [129] and topoisomerase [66]; later most of these enzymes were expressed as recombinant proteins and used for inhibitor screenings [88, 176, 193] (Table 2). 5.3.1 DHFR DHFR inhibitors act by blocking the enzyme dihydrofolate reductase which reduces dihydrofolic acid to tetrahydrofolic acid , using NADPH as the electron donor . This reaction product is an essential cofactor in the conversion of deoxyuridylate (dUMP) to deoxythymidylate (dTMP) by thymidylate synthetase and is therefore, a critical enzyme in DNA synthesis. DHFR is also essential for the production of folic acid, which Pneumocystis cannot salvage, and thus is dependent upon its de novo synthesis. Early enzymatic assays for the assessment of DHFR inhibitors employed sonication of purified P. carinii organisms with subsequent concentration of the supernatants, usually with the addition of protease inhibitors, as the standard method for preparation of the whole cell extract assays [4, 17, 24]. Host cell contamination was reduced by various methods including low speed centrifugations to separate out the heavier host cells or by separation on density gradients. Even with these precautions there was always a question regarding purity. A spectrophotometric assay was used to measure the reaction catalyzed by DHFR. In this assay, NADPH was added to the P. carinii supernatants and the reaction was initiated by the addition of dihydrofolic acid. The reaction velocity was measured by

disappearance of the NADPH at 340 nm. Inhibitors were directly added to the reaction and the rate of NADPH depletion relative to the untreated controls formed the basis of the assay. IC50s were generated by assay with several inhibitor concentrations, often after the percentages were converted to probits [17]. Recombinant P. carinii DHFR has supplanted the use of cell free extracts for evaluation of DHFR inhibitors [76, 79, 80, 151]. The enzyme is routinely expressed in E. coli using the pET8C expression system and released from the bacteria by sonication with subsequent centrifugation. The supernatant is used directly in the spectrophotometric enzyme assay and the concentration yielding 50% inhibition vs the untreated extracts (IC50) is calculated. 5.3.2 DHPS Sulfa drugs inhibit the enzyme dihydropteroate synthetase, which catalyzes the linkage of 7,8-dihydro-6-hydroxymethylpterin pyrophosphate with para- aminobenzoic acid to form dihydropteroate, a necessary step in the synthesis of folic acid. Like DHFR, early studies to assess the effect of specific inhibitors on P. carinii DHPS activity used extracts of proteins from organisms that were disrupted by sonication and subsequently concentrated [129]. Initially, DHPS production was assessed by an HPLC method which was based on incorporation of (14C) para-aminobenzoic acid into the product, dihydropteroate [129]. Efficacy of inhibitors was assessed by calculation of the Ki and IC50s calculated with varying doses of the inhibitors as compared to untreated sonicates. In another system, dihydropteroate synthase activity of P. carinii lysates was determined by using (14C)-labeled para-aminobenzoic acid as a substrate and ascending chromatography on strips Whatman 3MM paper in a phosphate buffer to separate the labeled from unlabeled products [32]. Scintillation counting was then used to quantify the reaction and IC50s were calculated from the scintillation counts of the treated vs untreated lysates.

Ki values were determined by solving in the Michaelis-Menton equation for competitive inhibitors; solving for the point of intersection of double-reciprocal plots, and solving for the slope of the Dixon plot. The P. carinii DHPS is located in tandem on a multi-functional protein with 2 other folate biosynthetic enzymes, dihyroneopterin aldolase and hydroxymethyldihydropterin pyrophosphokinase. The P. carinii folic acid synthesis, Fas, gene was first cloned in a baculovirus system [178] and this system was used to provide recombinant DHPS in crude cell extracts for enzymatic and inhibitory studies [88]. DHPS activity was assessed by tracking the incorporation of (3H) para-aminobenzoic acid into the product, dihydropteroate, and the reduction of (3H) para-aminobenzoic acid in the reaction mixture after separation of the 2 compounds by ether extraction. Inhibition was evaluated in the presence of 4 concentrations of the inhibitors and their absence. The logarithm of drug concentration was plotted against percent inhibition to determine the IC50 of each compound. 5.3.3 Topoisomerase Topoisomerase inhibitors have been evaluated in cell free extract systems [66] in standard monolayer based systems [68] and in our ATP cytotoxicity assay [49]. Topoisomerase I and II were partially purified from P. carinii organisms using a FPLC method, fractionated according to ATP-dependent relaxing activities and further purified on hydroxyapatite columns and subsequently assayed for relaxing activities with additional column purifications [66]. One unit of P. carinii topoisomerase I was defined as the amount of enzyme required to relax 50% of the form I supercoiled plasmid DNA into form II relaxed molecules at 250nM at 37oC in 30 minutes. The early eluting ATP-dependent fractions contained the topoisomerase II activity and one unit was defined as the amount of enzyme required to completely decatenate 125 ng of kDNA

(kinetoplast DNA) in 30 min at 37oC. Inhibitors were added to the topoisomerase assays and reported as the concentration of compound required to inhibit 50% of the relaxation of the supercoiled pBluescriptII KS+ plasmid DNA in the presence of 0.5U of P. carinii topoisomerase I activity. The supercoiled and relaxed DNA were quantified by scanning of photographic negatives of the DNAs electrophoresed through agarose gels. The topoisomerase II drug inhibition assays were based on the concentration of compound that decreased the decatenation activity by 50%. This assay is specific for topoisomerase II because only these enzymes can cause the double strand break and resealing of the substrate DNA leading to decatenation. One unit of the P. carinii topoisomerase II was mixed with the drugs and DNA substrate and incubated, stopped, and the results visualized by agarose gel electrophoresis. A 50% inhibition of decatenation was calculated by densitometric scanning of photographic negatives. 5.3.4 Heterologous studies In P. jirovecii, mutations have arisen in the Fas gene which confer decreased susceptibility to sulfa drugs in other human pathogens [5, 106, 107, 167]. These mutations include amino acid changes at positions 55 (Trp to Ala) and 57 (Pro to Ser) of the DHPS domain, either as a single or double mutation. The double mutations are significantly associated with failure of sulfa prophylaxis in patients with P. jirovecii infection [5, 106, 117]. Heterologous expression of the P. jirovecii Fas proteins has not yet been possible, likely due to the differences in codon usage and AT content between P. jirovecii and other model systems, such as S. cerevisiae. To study the consequences of these and other mutations and to determine susceptibilities to sulfa drugs, investigators have had to genetically engineer the same mutations into the S. cerevisiae FOL1gene, the putative ortholog of the P. jirovecii Fas gene [98, 121]. Both groups of investigators found that the double mutation in the wild type S. cerevisiae

conferred a requirement for p-aminobenzoic acid supplementation, suggesting reduced affinity for this substrate. The heterologous system and the enzymatic inhibition studies described above are useful for drug discovery targeting a single known enzymatic step, but a more global assessment is needed for discovery of new targets. 5.4 Development of a Cell-Free Screening System in Our Laboratory Because of the inherent problems with tissue culture, we explored the use of cell-free systems for growth and drug screening of Pneumocystis. Our first system used a peptone-based medium using incorporation of (35S)-methionine into Pneumocystis-specific proteins over time as a marker of viability [51]. The Pneumocystis were responsive to the standard anti-Pneumocystis compounds, TMP-SMX and pentamidine, in that they did not increase over inocula levels, yet viability assay using erythrosin B dye exclusion showed many of the organisms that stained with Diff-Quik were dead. Subsequently, another laboratory showed the specific uptake of radiolabeled methionine into P. carinii antigens which was decreased upon treatment with pentamidine, atovaquone, amphotericin B, piritrexim, sulfamethoxazole, TMP-SMX, and an experimental compound, RO11-8958 (epiroprim, a DHFR inhibitor) [119]. Inhibition was not observed when the organisms were exposed to dapsone, trimethoprim, clarithromycin or TNF and gamma interferon in the same study. The problems of host cell interference and viability led us to then explore the use of ATP content, a global assessment of viability, to screen drugs [30, 38, 49, 50, 105]. ATP is the universal energy unit in almost all living cells. When a cell dies, ATP is rapidly degraded by ATPases; the decline in ATP levels permits an immediate assessment of cell viability. ATP reflects the viability of an entire cell and does not target a single enzyme or pathway. The luciferin/luciferase driven bioluminescent ATP cytotoxicity assay has been shown to be a simple,

rapid and sensitive test to measure ATP levels in a variety of eukaryotic and prokaryotic cell systems [112, 113, 142, 159]. The ATP-driven reaction evolves light in a linear manner. We found that the response of Pneumocystis to test compounds in the ATP assay could be assessed within hours rather than over a period of days by microscopic enumeration [49]. The assay also enabled us to discern any discrepancies between microscopic counts and viability of the Pneumocystis populations. The system was adapted to a higher throughput platform by use of 96-well plates and a PolarStar Optima Microplate reader (BMG LabTech). The use of multi-well plates reduced reaction volumes, the need for large numbers of Pneumocystis, and amounts of drugs, while decreasing the time needed for set-up, sampling, and analysis. Significantly, the measurement of evolved light by the luminometer provided an unbiased quantitation of a drug response (ATP levels) in contrast to enumeration of organism numbers which could be influenced by technician bias or error. Our experience indicates that the ATP assay is a highly efficient system of screening candidate drugs. The ATP levels also correlated well with data obtained in our rat model: the ATP assay had a true positivity rate of 73%, a true negative rate of 89%, a specificity of 84%, and a sensitivity of 80% [49]. These values were comparable with other in vitro assays of drug screening (e.g., the Ames test for mutagenic potential) and animal studies. The ATP assay also correlates well with results obtained in our mouse model of Pneumocystis pneumonia. We also adapted the ATP assay system to assess the potential toxicity of promising candidate compounds identified in the Pneumocystis in vitro system to mammalian cell monolayers (i.e. A549 lung cells, Hep2 liver cells) as an intermediate screening step prior to testing in the animal model of Pneumocystis pneumonia [58, 181]. We feel this step is an important one that has helped to screen out compounds that may produce toxicity in the animals

and for guidance in determining the dosage in the animal model. Potentially toxic compounds were routinely started at lower dose finding ranges than apparently non-toxic ones, for example. 5.4.1Assessment of Efficacy Each test compound is initially evaluated at 100g/ml to screen out inactive compounds. If a 50% decrease in ATP compared to untreated organisms is observed for the compound, a 3 concentration series is then run to determine the IC 50 (10-, 1and 0.1g/ml). Compounds with a high degree of activity (reduction to less than 50% of control at all concentrations) are tested again at nanogram levels. The effects of drugs on the ATP of Pneumocystis pools are calculated by the following formula: Av. Control RLU Av. Drug RLU/ Av. Control RLU x100 = % decrease in ATP where Av. is the average; Control represents the vehicle control (untreated); RLU is the relative light units evolved; and Drug is the compound being tested. 5.4.1.1 IC50 Rank Scale The percent decrease in ATP content of a compound is used in a linear regression formula with the log drug concentrations to determine the 50% inhibitory concentration (IC50) (GraphPad Software v2 for Science, San Diego, CA ). Based on their IC 50 values, each agent is classified using an activity scale. The original scale had 4 categories: very marked, marked, moderate, and none. As our experience with drug testing has increased, we have added a fifth category of activity: slight. This was done in order to have as many categories as our in vivo activity scale and to accommodate drugs that attain serum levels higher than 10g/ml. The categories in the current drug activity scale are: very

marked, 50g/ml.

6. UPDATE ON RECENT DRUG DEVELOPMENT STUDIES 6.1 Assessment of Sterol Biosynthetic Pathway Inhibitors Treatment of Pneumocystis pneumonia in humans or animal models with standard antifungal compounds in the azole family or with amphotericin B has largely been unsuccessful. But, P. carinii synthesizes sterols with a double bond at C-7 of the sterol nucleus and an alkyl group with 1-2 carbons at C-24 of the side chain, raising the possibility that sterol biosynthesis would be a reasonable drug target. A comprehensive study using inhibitors targeted to enzymatic reactions involved in sterol biosynthesis was conducted using the ATP assay method which showed efficacy among several inhibitors [105]. The most effective drugs were those targeted to squalene synthase, squalene epoxide-lanosterol cyclase, and 8 to 7 isomerase, which showed moderate levels of activities with some compounds. A striking result of this study was the susceptibility of P. carinii in this system to some lanosterol demethylase inhibitors, the target of the azoles. Although resistant to Fluconazole, moderate reductions in ATP levels were observed after 48- 72 hours of treatment with 2 proprietary imidazoles, suggesting the lanosterol demethylase of P. carinii could have different binding affinities than those in other fungi, and may still be a viable target in these fungi. Likewise, amphotericin B also had moderate effects on the ATP pools of these organisms. Polyene antibiotics exert their effects by binding to sterols within biomembranes, causing the formation of pores and leading to the collapse of chemical gradients. Although most have a higher affinity to ergosterol, cholesterol will also be affected at sufficient concentrations. The direct administration to organisms in the cell free milieu could have facilitated this mechanism of action since the membranes of P. carinii are comprised primarily of cholesterol. Subsequent to these studies, sequencing of the P. carinii genome revealed the presence of genes necessary for an operational sterol biosynthetic pathway.

Organism transcripts isolated from organisms during fulminate infection showed that these genes were actively transcribed [55]. Reduction of organism ATP levels after exposure to inhibitors targeted to the sterol pathway together with evidence of the transcription of sterol pathway genes suggest that this pathway is operational and could provide new therapeutic targets, perhaps in combination with other drugs targeting different pathways or within the same pathway. 6.2 Pentamidine analogues Pentamidine was once the primary treatment for PCP prior to the advent of TMP-SMX [91]. Toxicity and the need for parenteral administration reduced the attractiveness of this compound, ceding the standard of care to TMP-SMX since the late 1970s. However, pentamidine was commonly used for prophylaxis, especially when delivered via an aerosol route, during the height of the AIDS epidemic and became the secondary treatment after adverse reactions to TMP-SMX or therapeutic failures. This aromatic cationic diamidine still remains a popular parent compound which has been derivatized over the decades in an effort to reduce toxicity and to permit oral delivery. The drug DB75, [2,5-bis(4-amidinophenyl)furan was found to be active against Trypanosoma spp and subsequently derivatized to the amidoxime prodrug, DB289 [2,5-bis(4-amidinophenyl)furan bis-O-methyl-amidoxime [114] . This prodrug is orally active and metabolically converted to DB75. DB289 has undergone phase IIb human clinical trials for the treatment of PCP, malaria, and early stage human African trypanosomiasis; however, these trials were discontinued because of concerns about drug therapy. In our laboratory, we identified a series of highly active piperazine- and alkanediamidelinked bisbenzamidines in our ATP in vitro cytotoxicity system [59, 177]. To test the relationship of the in vitro results with in vivo studies in the mouse model of pneumocystosis, 9 bisbenzamidines that exhibited a range of in vitro activity from highly active to inactive were

evaluated at 3-5 doses [58]. All 6 of the compounds that exhibited high in vitro activity significantly decreased the infection in vivo; 2 compounds with marked to moderate in vitro activities had slight or no activity in vivo, while the compound that was inactive in vitro was also ineffective in vivo. It was our conclusion that an in vitro selection strategy should first focus on the highly active compounds for subsequent in vivo studies. 6.3 Combination drug studies A recent application of the ATP assay system was to use it as a means to identify synergistic drug combinations. In this set of studies we chose to target the sterol biosynthetic pathway and in combination with other metabolic targets such as mitochondrial function. In these studies, the concentration of one compound was held constant while a series of concentrations of another compound were added. The results of the combination treatment were analyzed with normalized isobolograms (CalcuSynv2, Biosoft, UK). Six sterol biosynthesis and related enzymes were targeted with inhibitory compounds: HMG CoA synthase (Erg13) and reductase (Hmg1); DOXP synthase (dxs); squalene epoxidase (Erg1); sterol 14 demethylase (Erg11), and sterol C24 methyltransferase (Erg6). One of the more efficacioius combinations was the Erg6 inhibitor, berberine, with cerulenin, an Erg 13 inhibitor. As shown in Figure 3A, the isobologram illustrates the super additive combinations of these 2 compounds as represented by the points underneath the line. Synergism was also attained with certain combinations of simvastatin and berberine; palmitine and terbinafine; cerulenin and terbinafine; simvastatin and tebuconazole; tolnaftate and tebuconazole. Equivocal or antagonistic results were achieved with combinations of simvastatin and tolnaftate; tebuconazole and berberine; berberine and terbinafine; palmitate and tebuconazole; cerulenin and tebuconazole. Surprisingly, fosmidomycin, though it had no effect

as a monotherapy, showed synergism with simvastatin in this system (Figure 3B). Taken together, these results demonstrate the value of this assay in studying drug combinations. 6.4 Protease inhibitors The effects of protease inhibitors on P. carinii in vitro is a controversial topic. Reports of the efficacy on P. carinii in vitro by protease inhibitors administered to patients with HIV/AIDS as anti-viral therapies [8] prompted our group to evaluate the efficacy of this class of compounds in our ATP assay and in the mouse model of PCP [180]. The authors of the first study found indinavir, ritonavir, nelfinavir and saquinavir to inhibit the proliferation of P. carinii on confluent monolayers of HEL299 cells by 40- to almost 50% at the highest concentrations of each compound (93.7 to 66.4 ng/ml). A later study by the same group reported a significant reduction of P. carinii on day 8 of exposure by amprenavir concentrations of 320 nM to 11.58 uM [9]. In contrast, results from the ATP assay showed an IC50 of 35.4 ug/ml for indinavir and 23.3 ug/ml for saquinovir; both drug levels exceed those that can be clinically achieved in serum. Amprenavir and ritonavir showed no activity [180]. The protease inhibitors were evaluated at 50- and 200 mg/kg/day in the mouse model and no activity was observed for indinavir, nelfinavir, saquinavir, amprenavir or combinations of lopinavir/ritonavir or lopinavir/ritonavir at these levels [180]. While the method of in vitro evaluation may have influenced the results between the 2 laboratories, the lack of efficacy in the mouse model of Pneumocystis pneumonia supports the lack of efficacy of this class of compounds. 6.5 Sordarins Sordarins represent a new class of antifungal agents that target protein synthesis by interfering with the elongation step by binding to fungal EF-2 [87]. A series of these derivatives were shown to exhibit in vitro activity against P. carinii using a DMEM based cell free system

and in the immunosuppressed rat model of PCP [10]. Sordarins are of particular interest for clinical development since some new derivatives, the azasordarins, were found to be active against a wide variety of fungi including the human pathogenic Candida species (with the exception of C. krusei), some emerging pathogens such as Rhizopus arrhizus, and dermatophytes, although no activity was observed against Cryptococcus neoformans [87]. Such broad spectrum activity holds promise for future therapeutic options, especially for the immunocompromised host. 6.6 Beta-glucan synthase inhibitors The efficacy of the Beta-glucan synthase inhibitors against experimental Pneumocystis pneumonia was evident early in their development as pharmaceutical agents [158]. Merck subsequently developed an echinocandin, caspofungin acetate (Cancidas), which was licensed for the treatment of Candida infections. We recently conducted in vitro and in vivo efficacy assays (6)of three 1-3, Beta glucan inhibitors in clinical use: anidulafungin (Eraxis, Pfizer,Inc.), caspofungin (Cancidas, Merck) and micafungin (Mycamine Astellas Pharma US, Inc) using the ATP cytotoxicity system and the mouse model of PCP. In our standard ATP assay system which uses a supplement of 20% calf serum, no effect on ATP levels was detected at concentrations up to 100 ug/ml. However, a recent article reported on the increased MICs of echinocandins in medium containing sera for Candida and Aspergillus [134]. When we reduced the serum to 5% or less in our ATP assay, dramatic changes in IC50s resulted for each of the 3 glucan synthase inhibitors which resulted in a definite hierarcy of efficacy among these compounds. For example, after 48 hr of exposure in 5% calf serum, the IC50s for anidulafungin, caspofungin, and micafungin were 0.41-, 7.6- and 9.9 ug/ml, respectively. Studies in the mouse model followed this general pattern. Anidulafungin

and caspofungin significantly reduced lung burdens by 2 logs, from 7.13 to ~ 5 log10 after 3 weeks of treatment at all doses ranging from 0.1 mg/kg to 10 mg/kg. Micafungin was less effective at the lower drug doses and required 2.5- to 10 mg/kg to reduce lung burdens by 2 logs [6]. Thus, in the case of the echinocandins, using a reduced serum medium, the in vitro assay reflected the in vivo therapeutic results. Despite the concerted efforts of many laboratories, and despite the identification of many promising compounds, in vitro assays have not yet directly led to the clinical application of any anti-PCP therapy. Rather, the therapies that are in current clinical use were a result of studies in rodent animal models or in the case of pentamidine, empiric usage. In the case of the echinocandins, preclinical evaluation using an in vitro ATP assay reflected the hierarchy of efficacy in the mouse model of Pneumocystis infection. 7. IN VIVO APPROACHES FOR DRUG DEVELOPMENT 7.1 Anti-Pneumocystis Drugs in Clinical Use Trimethoprim (TMP)-sulfamethoxazole (SMX) is the drug of choice for mild, moderate and severe PCP [111]. This drug combination, which has been used for almost three decades, works by inhibiting the synthesis of folic acid. TMP-SMX, which can be given orally or intravenously, causes frequent adverse effects in HIV patients, but is well-tolerated by other immunocompromised hosts. Several alternative oral regimens are available for the treatment of mild to moderate cases of PCP. The combinations of TMP with dapsone (a sulfone) and clindamycin (a lincosamide) with primaquine (an 8-aminoquinolone) work well and may cause fewer side effects in HIV patients. The mechanism of action of clindamycin and primaquine is unknown. Atovaquone, (a hydroxynaphthoquinone) which appears to inhibit mitochondrial electron transport, is less effective but well tolerated. Pentamidine isethionate (a diamidine) is

usually reserved as an alternative to the treatment of severe PCP. Pentamidine is an old drug that is quite effective but highly toxic; its mechanism of action is unknown. Trimetrexate is another alternative that has been mainly used as salvage therapy for patients who have limited treatment options. Trimotrexate is a lipophilic analogue of methotrexate which acts by inhibiting Pneumocystis dihydrofolate reductase. Corticosteroids are used as adjunctive therapy in severe PCP to decrease the lung inflammatory response in HIV patients. Chemoprophylaxis of PCP is indicated for both first episode (primary) and recurrent episodes (secondary) of the disease, and should be continued as long as the risk factors for PCP exist [168]. Among adult HIV patients, chemoprophylaxis is usually begun when CD4 cell count is below 200/L, with TMP-SMX being the drug of choice. Alternative oral regimens include dapsone alone, dapsone with pyrimethamine, or atovaquone. Another option is pentamidine administered as an aerosol in a special nebulizer once a month. Recommendations for Pneumocystis prophylaxis in nonHIV patients are less well defined. Primary chemoprophylaxis may be considered for patients with primary immune deficiency diseases or severe malnutrition; patients receiving immunosuppressive or cytotoxic agents for conditions such as organ transplantation or cancer; persons on prolonged corticosteroids at a dose equivalent to 20 mg prednisone per day; and patients with persistent CD4+ cell counts < 200/l [85]. 7.2 Studies in Animal Models 7.2.1 Rats Animal models have been extensively used to analyze new compounds for in vivo activity against Pneumocystis. The immunosuppressed rat model of PCP developed by Frenkel et

al [71] in the 1960s served as the basis for most of this work. Rats administered corticosteroids for about 8 weeks spontaneously developed PCP, which had histopathologic features very similar to the disease in humans; the mechanism was considered to be reactivation of latent infection. Systematic use of this model to test new drugs began in the 1970s [94] and has continued since then. With improved microbial containment procedures in commercial breeding colonies, latent P. carinii infection was reduced but not entirely eliminated [97, 195]. In order to ensure adequate infection in the rats used in drug testing studies, investigators began to intratracheally inoculate the rats with Pneumocystis [22] or systematically house them with rats that had an established infection (co-housing or seeding) [141]. Overall, the rat model has proven to be a reliable predictor of activity in humans; candidate drugs usually have to demonstrate antiPneumocystis activity in this system before the FDA will permit studies in humans. 7.2.2 Mice The use of immunosuppressed normal mice and congenitally immunodeficient mice as models of PCP began in the 1970s [174, 190, 192]. Since that time, the use of mice as models for chemotherapy and immunotherapy of PCP gradually increased [23, 86, 120, 152, 153, 161, 162] so that by today, these animals have become the first animal species in which to test new agents. Mice offer several advantages over rats: they are only about 10% of the size and thus consume smaller quantities of drugs, which may be in short supply; mice have lower housing costs; and mice provide a broader variety of strains and reagents for investigational use. P. murina infection is produced in mice by intranasal or intratracheal inoculation or by seeding/co-housing[141]. PCP can be induced in normal mice by the administration of corticosteroids in a manner similar to that in rats [190, 192]. Immunodeficient mouse models of PCP have included athymic (nude) mice, which lack T cells; uMT mice, which lack B cells;

severe combined immunodeficiency (scid) mice, which lack B and T cells; and a variety of knockout mice [163]. Some investigators have also administered steroids to speed the development of PCP in immunodeficient mice [40]. Another approach in normal mice has been to deplete the animals of CD4 cells either by administration of a monoclonal antibody (MAb) or the hybridoma itself [120, 161]. CD4 cell depletion results in less immunosuppression than corticosteroids; this is probably the reason why some drugs exhibit greater anti-Pneumocystis activity in mice given anti-CD4 antibody [13]. There is also some question about which immunosuppressive regimen more closely mimics HIV infection. Depletion of CD4 cells resembles one of the prominent clinical manifestations in HIV patients. On the other hand, HIV infects a variety of cell types and its effects on the immune system are essentially global; this situation is more closely approximated by steroids. 7.3 Experimental Protocols Similar methods to develop PCP and evaluate the efficacy of candidate drugs have been used in rats and mice. Corticosteroids have been the most commonly used type of immunosuppressive agent, although a number of other compounds (e.g. cyclophosphamide, cyclosporine) have also been used [179, 185, 195]. Administration of a low (8%) protein diet also enhances the effects of the steroids, but it is quite expensive and used less frequently than in the past. Since any type of immunocompromised state increases the risks of other microbial infections, barrier facilities with autoclaved food, water, and bedding have been increasingly employed as a means to help control the occurrence of these infections. The use of hyperchlorinated drinking water or the addition of an antibiotic to the water is also common. Drug studies last 6-12 weeks and are mainly of two types: treatment, in which the candidate

anti-Pneumocystis agents are given over the last 2-3 weeks of immunosuppression; prophylaxis, in which the agents are administered throughout the period of immunosuppression. Studies of relapse of PCP have been infrequent. The rationale for 2-3 week time for administration in treatment studies is that most anti-Pneumocystis drugs are slow acting agents; echinocandins and papulocandins, which can eliminate Pneumocystis cysts a few days, are notable exceptions [132, 140, 154, 156, 158]. Drugs have been given by a variety of oral, parenteral, and aerosol routes that can materially affect pharmacokinetics, metabolism, and efficacy. For example, the results achieved by administering a drug by oral gavage may differ from results achieved by mixing the drug in the food or water and allowing the rats to ingest it ad libitum. Drug efficacy had previously been assessed by analyzing the extent or severity of PCP rather than by survival because most of the animals live until the end of an experiment; of those that do not survive, it is often not possible to determine whether they died from PCP, another microbial infection, or drug toxicity. Recently, survival curves using Kaplan-Meier plots have been introduced into the analysis, and have provided important information [58]. The extent or severity of PCP has been analyzed using a variety of different scoring systems, based either on organism quantitation in lung homogenates, imprint smears, or histopathologic staining [204]. Stains to identify Pneumocystis have usually been of one of two types: stains such as methenamine silver, toluidine blue, or cresyl echt violet which selectively stain Pneumocystis cysts, but not its other developmental forms; stains such as Wright-Giemsa or one of its more rapid variants (Diff-Quik) which stain the nuclei of all Pneumocystis life cycle stages. In general, nuclei counts are about 10-fold higher than cyst counts. Immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and molecular (PCR) techniques have also been used [65, 115, 133]. The lack of standardization among these

techniques has deterred direct comparison of the results from different labs. 7.4 Active drugs Drugs that have been tested in experimental models of Pneumocystis pneumonia have ranged from agents in clinical use to newly synthesized compounds. Agents that have been effective against Pneumocystis in rats have usually been effective in mice, and vice versa. One possible exception was diacetyldapsone, a prodrug that can be metabolized to dapsone in mice but not in rats (Smith, TJ, Queener, SF, Bartlett, MS, et. al. Diacetyldapsone inhibits Pneumocystis carinii in inoculated mouse models. Presented at the American Society of Tropical Medicine Hygiene, Cincinnati, OH, November, 1994). This compound was also found to be inactive in our rat model of Pneumocystis infection. The following classes of drugs have shown anti-Pneumocystis activity in rats and/or mice: antifolate compounds, including inhibitors of dihydropteroate synthase (sulfonamides, sulfones, and sulfonylureas) alone or in combination with inhibitors of dihydrofolate reductase (DHFR) [61, 71, 84, 94, 95, 109, 110, 143, 183, 184, 188, 192]; sulfonamides combined with macrolides [3, 90, 93]; diamidines and related cationionic compounds [20, 21, 63, 66, 69, 83, 89, 103, 130, 131, 150, 171, 173, 182, 186, 198, 199]; 8-aminoquinolones alone or combined with clindamycin [16, 144 146]; purine nucleoside analogues [164, 183]; polyamine inhibitors [31, 37, 122, 124, 126, 128]; nitrofurans [187]; papulocandins and echinocandins [11, 72, 74, 99, 139, 154, 156, 157]; sodarins [10, 102]; allylamines [43, 45]; streptogrammins [181]; fluoroquinolones [28]; hydroxynaphthoquinones alone or combined with DHFR inhibitors, macrolides, or rifamycins [40, 42, 92, 191]; ionophores [135]; benzonaphthacenes [200]; iron chelators [36, 123, 125]; immunological agents (e.g. cytokines, lymphocytes, antibodies, vaccines) [18, 67, 82, 116, 118, 148, 160, 166, 169, 170, 196, 197]; and compounds with novel mechanisms of action [62]. Several reports have

examined the efficacy of drug combinations in animals infected with Pneumocystis and other microbial pathogens [25, 27, 29, 136, 137]. 7.5 Bench to Bedside Of all the anti-Pneumocystis drugs in clinical use, only pentamidine did not undergo prior testing in animal models. Pentamidine was first used clinically many years ago for the treatment African trypanosomiasis, and then for PCP in infants who were debilitated or malnourished. However, pentamidine was later used in many animal models as the standard agent with which to compare new diamidines. One of these compounds, DB 285, which can be taken orally, is currently undergoing clinical trials [114], but recently these studies have been halted because of concerns about toxicity. The development of new anti-Pneumocystis drugs for use in humans has presented major challenges. Faced with the lack of a reliable culture system, limited commercial market and high costs, pharmaceutical companies have focused on testing drugs already in clinical use for antiPneumocystis activity. Examples of this approach included TMP-SMX, dapsone, clindamycinprimaquine, and atovaquone. In some cases, members of a new drug class (e.g., echinocandins) displayed good activity against Pneumocystis in animals, but were developed for clinical use against organisms for which there was a larger market (e.g., Candida, Aspergillus). Another approach to drug development has been conducted by the National Institute of Health (NIH). In the 1980s, NIH established contracts with investigators to develop and test new or existing drugs for activity against Pneumocystis in vitro and in animal models. The ultimate goal was to bring a few of these agents to clinical trials. This program has produced new methods to test candidate drugs in vitro and in vivo; identified a number of promising agents that could be developed for clinical trials; and provided a wealth of scientific info about

pharmacology, structure-activity relationships, mechanisms of action, and drug resistance. However, the issues that dissuaded pharmaceutical companies from undertaking clinical trials also came into play here. The one successful clinical application from this basic research was the development of the combination of clindamycin and primaquine alternative therapy in for PCP in patients with failed or could not tolerate TMP-SMX (145). Since these drugs had already been in clinical use, the cost of conducting clinical trials for PCP was manageable. Recent attention has focused on echinocandins, particularly caspofungin. There have been several publications describing the use of caspofungin as adjunctive therapy of PCP in small numbers of patients [19, 104, 175, 194]. Identification of new drugs and targets for the treatment of PCP has been an arduous task. Unresponsive to standard anti-fungal therapies such as the azoles and polyenes, results from various laboratories have also found these fungal organisms to be largely refractory to a variety of other families of compounds. Faced with a lack of long term in vitro culture method, investigators have been forced to conduct screening studies in short term cultures or by assessing affects directly on known target enzymes in in vitro assays. Scale up of experimental compounds for testing in animal models has also challenged the development process. Despite these problems, animal testing studies identified the primary therapy used for the treatment of PCP in humans, trimethoprim-sulfamethoxazole. This combination remains the treatment of choice today. Likewise, alternative treatments such as atovaquone, clindamycin-primaquine, and the Beta-glucan synthase inhibitors were products of screening in models. Although in vitro approaches have not yet led to a clinical product, many promising agents and combinations have been identified that could be used for the development of future therapeutic options.

ACKNOWLEDGEMENTS The authors sincerely acknowledge the LONG-TERM funding support they have received from the National Institutes of Health and the Department of Veterans Affairs where a large portion of this work was performed. In addition, we would like to recognize our Program Officer, Dr. Chris Lambros, for his direction and guidance over the past 20 plus years and to Margaret S. Collins, the senior technician who perfected the ATP assay and worked tirelessly to evaluate the compounds referenced here.

FIGURE LEGENDS Figure 1. Proposed life cycle of Pneumocystis. (A) Asexual binary fission. A trophic form replicates its nuclear and cytoplasmic contents, separates by binary fission and produces two genetically identical progeny. (B) Sexual replication. There are two proposed mating types of P. carinii that are morphologically indistinguishable, although on at least one of the types is a Ste3like pheromone receptor [165]. The two mating types fuse and undergo karyogamy to produce a diploid zygote or precyst. Meiosis proceeds with a reduction division followed by a mitotic replication resulting in 4 nuclei. An additional mitotic replication of the haploid nuclei results in eight nuclei, which are then sequestered into eight individual spores or daughter forms. It is assumed that the spores are released from the mature cyst (ascus) and become new vegetative trophic forms. Figure 2. Pipeline of in vitro and in vivo preclinical drug discovery for anti-Pneumocystis compounds. The steps from in vitro screening to selection based on activity to analysis in the rodent models of Pneumocystis infection are showed as a decision schematic. Figure 3. Isobolograms illustrating synergistic and efficacious combinations of drugs in vitro. 3A. Cerulenin is combined with four concentrations of berberine. The two lowest ratios appearsuperadditive while the higher ratios were near additive in their interaction.

3B. Fosmidomycin has very limited anti-Pneumocystis activity when used as a monotherapy (7.8% ATP reduction at 100g/ml). However, the possibility of blocking the non-mevalonate pathway, if one exists in Pneumocystis, was of interest. In this trial, both compounds were added at varying concentrations, and some synergistic combinations were observed.

Table 1. Cell-based systems used for in vitro drug screening Cells/Cell Line WI38 (fetal lung fibroblast primary cells) HEL (human embryonic lung) cells Inocula P. carinii P. murina P. carinii Method of Quantification Microscopic enumeration of Giemsa-stained trophozoites Microscopic enumeration of Giemsa-stained trophozoites ELISA to surface antigens Efficacy Assessment % reduction in treated vs untreated organisms; IC50 % reduction in treated vs untreated organisms; IC50 % reduction of absorbance vs untreated A549 cell line (human lung carcinoma) P. carinii Microscopic enumeration of Diff-Quik stained organism nuclei organisms % reduction in treated vs untreated organisms; IC50 [56] Reference (s) [14, 15, 68, 145, 147]

[8, 9, 11, 100]

[65]

P. jirovecii

Microscopic enumeration of Giemsa-stained trophozoites

% reduction in treated vs untreated organisms; IC50

[33, 35] [41, 43, 44]

CEL (chick embryonic lung) Mv 1 Lu (mink lung cell line)

P. carinii

Microscopic enumeration of toluidine stained

% reduction in treated vs untreated organisms % reduction in treated vs untreated organisms

[138]

P. carinii

cysts Microscopic enumeration of Giemsa-stained trophozoites

[127]

Table 2. Cell free systems used for in vitro drug screening. Inocula Intact P. carinii organisms Method of Quantification Incorporation of radiolabeled paminobenzoic acid Efficacy Reference (s)

Assessment 50% inhibition [39] vs untreated control; IC50 50% inhibition [51, 87, 119] vs untreated control; IC50 50% inhibition vs untreated control; IC50

Intact P. carinii organisms

Incorporation of radiolabeled methionine

Intact P. carinii organisms

ATP bioluminescent assay

[23, 38, 48, 65, 67, 79, 80, 83, 135, 137,

Cell-free sonicates of P. carinii

DHFR activity by radiolabel incorporation into ..

202, 207] 50% inhibition [17] of activity; IC50 50% inhibition [17, 76, 80, of activity; 81]

Recombinant DHFR

DHFR activity by radiolabel incorporation into ..

Cell free sonicates of P. carinii

DHPS activity by radiolabel incorporation of radiolabeled paminobenzoic acid into dihydropteroate

IC50 50% inhibition [32, 129] of activity; IC50

Recombinant DHPS

DHPS activity by radiolabel incorporation of radiolabeled paminobenzoic acid into dihydropteroate and reduction of p-aminobenzoic acid in the reaction mixture

50% inhibition [88] of activity; IC50

Partial purification of enzyme activity from cell-free sonicates of P. carinii

Topoisomerase activities (I and II) by relaxation of coiled DNA; decatenation

50% inhibition [66] of activity; IC50

Figure 1. Putative lifecycle of Pneumocystis.

Figure 2. Pipeline for preclinical drug discovery.

Figure 3. Experimental in vitro drug combinations. A

B

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