scholarly journals Dicyclic and Tricyclic Diaminopyrimidine Derivatives as Potent Inhibitors of Cryptosporidium parvum Dihydrofolate Reductase: Structure-Activity and Structure-Selectivity Correlations

2001 ◽  
Vol 45 (12) ◽  
pp. 3293-3303 ◽  
Author(s):  
Richard G. Nelson ◽  
Andre Rosowsky
Keyword(s):  
Cryptosporidium Parvum ◽  
Dihydrofolate Reductase ◽  
Drug Exposure ◽  
Immunofluorescence Microscopy ◽  
Host Cells ◽  
Indirect Immunofluorescence Microscopy ◽  
Structure Activity ◽  
Dhfr Inhibitors ◽  
Canine Kidney

ABSTRACT A structurally diverse library of 93 lipophilic di- and tricyclic diaminopyrimidine derivatives was tested for the ability to inhibit recombinant dihydrofolate reductase (DHFR) cloned from human and bovine isolates of Cryptosporidium parvum (J. R. Vásquez et al., Mol. Biochem. Parasitol. 79:153–165, 1996). In parallel, the library was also tested against human DHFR and, for comparison, the enzyme from Escherichia coli. Fifty percent inhibitory concentrations (IC50s) were determined by means of a standard spectrophotometric assay of DHFR activity with dihydrofolate and NADPH as the cosubstrates. Of the compounds tested, 25 had IC50s in the 1 to 10 μM range against one or bothC. parvum enzymes and thus were not substantially different from trimethoprim (IC50s, ca. 4 μM). Another 25 compounds had IC50s of <1.0 μM, and 9 of these had IC50s of <0.1 μM and thus were at least 40 times more potent than trimethoprim. The remaining 42 compounds were weak inhibitors (IC50s, >10 μM) and thus were not considered to be of interest as drugs useful against this organism. A good correlation was generally obtained between the results of the spectrophotometric enzyme inhibition assays and those obtained recently in a yeast complementation assay (V. H. Brophy et al., Antimicrob. Agents Chemother. 44:1019–1028, 2000; H. Lau et al., Antimicrob. Agents Chemother. 45:187–195, 2001). Although many of the compounds in the library were more potent than trimethoprim, none had the degree of selectivity of trimethoprim for C. parvum versus human DHFR. Collectively, the results of these assays comprise the largest available database of lipophilic antifolates as potential anticryptosporidial agents. The compounds in the library were also tested as inhibitors of the proliferation of intracellular C. parvum oocysts in canine kidney epithelial cells cultured in folate-free medium containing thymidine (10 μM) and hypoxanthine (100 μM). After 72 h of drug exposure, the number of parasites inside the cells was quantitated by indirect immunofluorescence microscopy. Sixteen compounds had IC50s of <3 μM, and five of these had IC50s of <0.3 μM and thus were comparable in potency to trimetrexate. The finding that submicromolar concentrations of several of the compounds in the library could inhibit in vitro growth of C. parvum in host cells in the presence of thymidine (dThd) and hypoxanthine (Hx) suggests that lipophilic DHFR inhibitors, in combination with leucovorin, may find use in the treatment of intractable C. parvum infections.

Recent Design and Structure-Activity Relationship Studies on Modifications of DHFR Inhibitors as Anticancer Agents

2019 ◽  
Vol 26 ◽  
Author(s):  
Agnieszka Wróbel ◽  
Danuta Drozdowska
Keyword(s):  
Anticancer Activity ◽  
Anticancer Drugs ◽  
Anticancer Agents ◽  
Physicochemical Characterization ◽  
Structure Activity Relationship ◽  
Activity Relationship ◽  
Biological Research ◽  
Structure Activity ◽  
Dhfr Inhibitors

Background: Dihydrofolate reductase (DHFR) has been known for decades as a molecular target for antibacterial, antifungal and anti-malarial treatments. This enzyme is becoming increasingly important in the design of new anticancer drugs, which is confirmed by numerous studies including modelling, synthesis and in vitro biological research. This review aims to present and discuss some remarkable recent advances on the research of new DHFR inhibitors with potential anticancer activity. Methods: The scientific literature of the last decade on the different types of DHFR inhibitors has been searched. The studies on design, synthesis and investigation structure-activity relationship were summarized and divided into several subsections depending on the leading molecule and its structural modification. Various methods of synthesis, potential anticancer activity and possible practical applications as DHFR inhibitors of new chemical compounds were described and discussed. <p> Results: This review presents the current state of knowledge on the modification of known DHFR inhibitors and the structures and searching for over eighty new molecules, designed as potential anticancer drugs. In addition, DHFR inhibitors acting on thymidylate synthase (TS), carbon anhydrase (CA) and even DNA-binding are presented in this paper. <p> Conclusion: Thorough physicochemical characterization and biological investigations it is possible to understand structure-activity relationship of DHFR inhibitors. This will enable even better design and synthesis of active compounds, which would have the expected mechanism of action and the desired activity.

scholarly journals Detection of UV-Induced Thymine Dimers in Individual Cryptosporidium parvum and Cryptosporidium hominis Oocysts by Immunofluorescence Microscopy

2006 ◽  
Vol 73 (3) ◽  
pp. 947-955 ◽  
Author(s):  
B. H. Al-Adhami ◽  
R. A. B. Nichols ◽  
J. R. Kusel ◽  
J. O'Grady ◽  
H. V. Smith
Keyword(s):  
Cryptosporidium Parvum ◽  
Immunofluorescence Microscopy ◽  
Uv Light ◽  
Fluorescein Isothiocyanate ◽  
Immunofluorescence Assay ◽  
Cryptosporidium Hominis ◽  
Specific Fluorescence ◽  
Thymine Dimers ◽  
Texas Red

ABSTRACT To investigate the effect of UV light on Cryptosporidium parvum and Cryptosporidium hominis oocysts in vitro, we exposed intact oocysts to 4-, 10-, 20-, and 40-mJ�cm−2 doses of UV irradiation. Thymine dimers were detected by immunofluorescence microscopy using a monoclonal antibody against cyclobutyl thymine dimers (anti-TDmAb). Dimer-specific fluorescence within sporozoite nuclei was confirmed by colocalization with the nuclear fluorogen 4′,6′-diamidino-2-phenylindole (DAPI). Oocyst walls were visualized using either commercial fluorescein isothiocyanate-labeled anti-Cryptosporidium oocyst antibodies (FITC-CmAb) or Texas Red-labeled anti-Cryptosporidium oocyst antibodies (TR-CmAb). The use of FITC-CmAb interfered with TD detection at doses below 40 mJ�cm−2. With the combination of anti-TDmAb, TR-CmAb, and DAPI, dimer-specific fluorescence was detected in sporozoite nuclei within oocysts exposed to 10 to 40 mJ�cm−2 of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJ�cm−2 of UV light failed to infect neonatal mice, confirming that results of our anti-TD immunofluorescence assay paralleled the outcomes of our neonatal mouse infectivity assay. These results suggest that our immunofluorescence assay is suitable for detecting DNA damage in C. parvum and C. hominis oocysts induced following exposure to UV light.

scholarly journals In SilicoScreening, Synthesis andIn VitroEvaluation of Some Quinazolinone and Pyridine Derivatives as Dihydrofolate Reductase Inhibitors for Anticancer Activity

2009 ◽  
Vol 6 (s1) ◽  
pp. S97-S102 ◽  
Author(s):  
A. G. Nerkar ◽  
A. K. Saxena ◽  
S. A. Ghone ◽  
A. K. Thaker
Keyword(s):  
Anticancer Activity ◽  
Dihydrofolate Reductase ◽  
De Novo ◽  
Human Cancer ◽  
Microwave Assisted Synthesis ◽  
Human Cancer Cell Lines ◽  
Reductase Inhibitors ◽  
Base Derivative ◽  
Dhfr Inhibitors

Dihydrofolate reductase (DHFR) is the important target for anticancer drugs belonging to the class of antimetabolites as the enzyme plays important role in the de novo purine synthesis. We here report thein silicoscreening to obtain best fit molecules as DHFR inhibitors, synthesis of some ʻbest fitʼ quinazolinone from 2-phenyl-3-(substituted-benzilidine-amino) quinazolinones (Quinazolinone Shiff's bases) QSB1-5and pyridine-4-carbohydrazide Shiff's bases (ISB1-5) derivatives and theirin vitroanticancer assay. Synthesis of the molecules was performed using microwave assisted synthesis. The structures of these molecules were elucidated by IR and1H-NMR. These compounds were then subjected forin vitroanticancer evaluation against five human cancer cell-lines for anticancer cyto-toxicity assay. Methotrexate (MTX) was used as standard for this evaluation to give a comparable inhibition of the cell proliferation by DHFR inhibition. Placlitaxel, adriamycin and 5-fluoro-uracil were also used as standard to give a comparable activity of these compounds with other mechanism of anticancer activity. ISB3(4-(N, N-dimethyl-amino)-phenyl) Schiff''s base derivative of pyridine carbohydrazide showed equipotent activity with the standards used inin vitroanticancer assay as per the NCI (National Cancer Institute) guidelines.

scholarly journals In Vitro Activities of Several Diaminomethylpyridopyrimidines against Mycobacterium avium Complex

1998 ◽  
Vol 42 (12) ◽  
pp. 3315-3316 ◽  
Author(s):  
Carolyn M. Shoen ◽  
Olga Choromanska ◽  
Robert C. Reynolds ◽  
James R. Piper ◽  
Cheryl A. Johnson ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Mycobacterium Avium ◽  
Mycobacterium Avium Complex ◽  
Dhfr Inhibitors ◽  
Better Than

ABSTRACT Three recently synthesized dihydrofolate reductase (DHFR) inhibitors designated SoRI 8890, 8895, and 8897 were evaluated for their in vitro activities against 25 isolates of Mycobacterium avium complex. The MICs at which 50 and 90% of isolates were inhibited were 1 and 2, 4 and 8, and 4 and 8 μg/ml for SoRI 8890, 8895, and 8897, respectively. Although the addition of dapsone at 0.5 μg/ml did not significantly enhance the in vitro activities of these compounds, their activities alone were comparable to, if not better than, results seen with other DHFR inhibitors, such as pyrimethamine or WR99210.

scholarly journals Diaminoquinazoline MMV675968 from Pathogen Box inhibits Acinetobacter baumannii growth through targeting of dihydrofolate reductase

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Warangkhana Songsungthong ◽  
Suganya Yongkiettrakul ◽  
Louise E. Bohan ◽  
Eric S. Nicholson ◽  
Sunisa Prasopporn ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Dihydrofolate Reductase ◽  
Selective Inhibition ◽  
Resistance Mechanisms ◽  
New Drugs ◽  
Activity Assay ◽  
Antibiotic Development ◽  
Chemical Structures ◽  
Dhfr Inhibitors

Abstract Antibiotic resistance in Acinetobacter baumannii is a major global health threat. New drugs with novel chemical structures are needed to overcome a myriad of resistance mechanisms in A. baumannii. In this study, we screened an open-source Pathogen Box library for anti-A. baumannii compounds. Compound MMV675968 (a diaminoquinazoline analog) was the only non-reference compound found to inhibit the growth of all four A. baumannii test strains with IC50 of 0.6–2.7 μM, IC90 of 0.7–3.9 μM, and MIC of 1.6–10 μM. We showed that MMV675968 targeted A. baumannii dihydrofolate reductase (AbDHFR) as determined by an E. coli surrogate whose growth was dependent on AbDHFR function and by an in vitro DHFR activity assay. Additionally, chemical scaffolds of DHFR inhibitors that are effective as antibiotics against A. baumannii were identified using an in vitro DHFR activity assay and A. baumannii growth inhibition. MMV675968 was the most potent among DHFR inhibitors tested in inhibiting A. baumannii growth. This study shows for the first time that MMV675968 inhibits A. baumannii growth via selective inhibition of AbDHFR and is therefore a promising scaffold for further antibiotic development against A. baumannii.

scholarly journals Spontaneous dormancy protects Trypanosoma cruzi during extended drug exposure

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Fernando J Sánchez-Valdéz ◽  
Angel Padilla ◽  
Wei Wang ◽  
Dylan Orr ◽  
Rick L Tarleton
Keyword(s):  
Trypanosoma Cruzi ◽  
Drug Treatment ◽  
Drug Exposure ◽  
Host Cells ◽  
New Host ◽  
Cytotoxic Compounds ◽  
Drug Treatments ◽  
Disease Agent

The ability of the Chagas disease agent Trypanosoma cruzi to resist extended in vivo exposure to highly effective trypanocidal compounds prompted us to explore the potential for dormancy and its contribution to failed drug treatments in this infection. We document the development of non-proliferating intracellular amastigotes in vivo and in vitro in the absence of drug treatment. Non-proliferative amastigotes ultimately converted to trypomastigotes and established infections in new host cells. Most significantly, dormant amastigotes were uniquely resistant to extended drug treatment in vivo and in vitro and could re-establish a flourishing infection after as many as 30 days of drug exposure. These results demonstrate a dormancy state in T. cruzi that accounts for the failure of highly cytotoxic compounds to completely resolve the infection. The ability of T. cruzi to establish dormancy throws into question current methods for identifying curative drugs but also suggests alternative therapeutic approaches.

scholarly journals Identification of Cryptosporidium parvum Dihydrofolate Reductase Inhibitors by Complementation in Saccharomyces cerevisiae

2000 ◽  
Vol 44 (4) ◽  
pp. 1019-1028 ◽  
Author(s):  
Victoria Hertle Brophy ◽  
John Vasquez ◽  
Richard G. Nelson ◽  
John R. Forney ◽  
Andre Rosowsky ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cryptosporidium Parvum ◽  
Dihydrofolate Reductase ◽  
Pneumocystis Carinii ◽  
Lead Compounds ◽  
Reductase Inhibitors ◽  
Protozoan Pathogen ◽  
Dhfr Inhibitors ◽  
Potential Inhibitors ◽  
Dihydrofolate Reductase Inhibitors

ABSTRACT There is a pressing need for drugs effective against the opportunistic protozoan pathogen Cryptosporidium parvum. Folate metabolic enzymes and enzymes of the thymidylate cycle, particularly dihydrofolate reductase (DHFR), have been widely exploited as chemotherapeutic targets. Although many DHFR inhibitors have been synthesized, only a few have been tested against C. parvum. To expedite and facilitate the discovery of effective anti-Cryptosporidium antifolates, we have developed a rapid and facile method to screen potential inhibitors of C. parvum DHFR using the model eukaryote, Saccharomyces cerevisiae. We expressed the DHFR genes of C. parvum, Plasmodium falciparum, Toxoplasma gondii, Pneumocystis carinii, and humans in the same DHFR-deficient yeast strain and observed that each heterologous enzyme complemented the yeast DHFR deficiency. In this work we describe our use of the complementation system to screen known DHFR inhibitors and our discovery of several compounds that inhibited the growth of yeast reliant on the C. parvum enzyme. These same compounds were also potent or selective inhibitors of the purified recombinantC. parvum DHFR enzyme. Six novel lipophilic DHFR inhibitors potently inhibited the growth of yeast expressing C. parvumDHFR. However, the inhibition was nonselective, as these compounds also strongly inhibited the growth of yeast dependent on the human enzyme. Conversely, the antibacterial DHFR inhibitor trimethoprim and two close structural analogs were highly selective, but weak, inhibitors of yeast complemented by the C. parvum enzyme. Future chemical refinement of the potent and selective lead compounds identified in this study may allow the design of an efficacious antifolate drug for the treatment of cryptosporidiosis.

scholarly journals Structure-Activity Relationships of Bacillus cereus and Bacillus anthracis Dihydrofolate Reductase: toward the Identification of New Potent Drug Leads

2006 ◽  
Vol 50 (10) ◽  
pp. 3435-3443 ◽  
Author(s):  
Tammy M. Joska ◽  
Amy C. Anderson
Keyword(s):  
Bacillus Cereus ◽  
Bacillus Anthracis ◽  
Dihydrofolate Reductase ◽  
Antimicrobial Agents ◽  
Structure Activity Relationships ◽  
Structure Activity ◽  
Phenyl Rings ◽  
Potent Drug

ABSTRACT New and improved therapeutics are needed for Bacillus anthracis, the etiological agent of anthrax. To date, antimicrobial agents have not been developed against the well-validated target dihydrofolate reductase (DHFR). In order to address whether DHFR inhibitors could have potential use as clinical agents against Bacillus, 27 compounds were screened against this enzyme from Bacillus cereus, which is identical to the enzyme from B. anthracis at the active site. Several 2,4-diamino-5-deazapteridine compounds exhibit submicromolar 50% inhibitory concentrations (IC50s). Four of the inhibitors displaying potency in vitro were tested in vivo and showed a marked growth inhibition of B. cereus; the most potent of these has MIC50 and minimum bactericidal concentrations at which 50% are killed of 1.6 μg/ml and 0.09 μg/ml, respectively. In order to illustrate structure-activity relationships for the classes of inhibitors tested, each of the 27 inhibitors was docked into homology models of the B. cereus and B. anthracis DHFR proteins, allowing the development of a rationale for the inhibition profiles. A combination of favorable interactions with the diaminopyrimidine and substituted phenyl rings explains the low IC50 values of potent inhibitors; steric interactions explain higher IC50 values. These experiments show that DHFR is a reasonable antimicrobial target for Bacillus anthracis and that there is a class of inhibitors that possess sufficient potency and antibacterial activity to suggest further development.

scholarly journals Role of CpSUB1, a Subtilisin-Like Protease, in Cryptosporidium parvum Infection In Vitro

2009 ◽  
Vol 8 (4) ◽  
pp. 470-477 ◽  
Author(s):  
Jane W. Wanyiri ◽  
Patsharaporn Techasintana ◽  
Roberta M. O'Connor ◽  
Michael J. Blackman ◽  
Kami Kim ◽  
...  
Keyword(s):  
Serine Protease ◽  
Cryptosporidium Parvum ◽  
Protease Activity ◽  
Diarrheal Disease ◽  
Genomic Sequence ◽  
Host Cells ◽  
Pcr Analysis ◽  
Apical Region ◽  
Gene Encoding

ABSTRACTThe apicomplexan parasiteCryptosporidiumis a significant cause of diarrheal disease worldwide. Previously, we reported that aCryptosporidium parvumsubtilisin-like serine protease activity with furin-type specificity cleaves gp40/15, a glycoprotein that is proteolytically processed into gp40 and gp15, which are implicated in mediating infection of host cells. Neither the enzyme(s) responsible for the protease activity inC. parvumlysates nor those that process gp40/15 are known. There are no furin or other proprotein convertase genes in theC. parvumgenome. However, a gene encoding CpSUB1, a subtilisin-like serine protease, is present. In this study, we cloned the CpSUB1 genomic sequence and expressed and purified the recombinant prodomain. Reverse transcriptase PCR analysis of RNA fromC. parvum-infected HCT-8 cells revealed that CpSUB1 is expressed throughout infection in vitro. In immunoblots, antiserum to the recombinant CpSUB1 prodomain revealed two major bands, of ∼64 kDa and ∼48 kDa, forC. parvumlysates and proteins “shed” during excystation. In immunofluorescence assays, the antiserum reacted with the apical region of sporozoites and merozoites. The recombinant prodomain inhibited protease activity and processing of recombinant gp40/15 byC. parvumlysates but not by furin. Since prodomains are often selective inhibitors of their cognate enzymes, these results suggest that CpSUB1 may be a likely candidate for the protease activity inC. parvumand for processing of gp40/15. Importantly, the recombinant prodomain inhibitedC. parvuminfection of HCT-8 cells. These studies indicate that CpSUB1 plays a significant role in infection of host cells by the parasite and suggest that this enzyme may serve as a target for intervention.

scholarly journals Cryptosporidium parvum Pyruvate Kinase Inhibitors With in vivo Anti-cryptosporidial Efficacy

2022 ◽  
Vol 12 ◽  
Author(s):  
Shahbaz M. Khan ◽  
Xuejin Zhang ◽  
William H. Witola
Keyword(s):  
Pyruvate Kinase ◽  
Cryptosporidium Parvum ◽  
Diarrheal Disease ◽  
Severe Disease ◽  
The United States ◽  
Metabolic Energy ◽  
Host Cells ◽  
Infection Model

Cryptosporidium parvum is a highly prevalent protozoan parasite that causes a diarrheal disease in humans and animals worldwide. Thus far, the moderately effective nitazoxanide is the only drug approved by the United States Food and Drug Administration for treating cryptosporidiosis in immunocompetent humans. However, no effective drug exists for the severe disease seen in young children, immunocompromised individuals and neonatal livestock. C. parvum lacks the Krebs cycle and the oxidative phosphorylation steps, making it dependent solely on glycolysis for metabolic energy production. Within its glycolytic pathway, C. parvum possesses two unique enzymes, the bacterial-type lactate dehydrogenase (CpLDH) and the plant-like pyruvate kinase (CpPyK), that catalyze two sequential steps for generation of essential metabolic energy. We have previously reported that inhibitors of CpLDH are effective against C. parvum, both in vitro and in vivo. Herein, we developed an in vitro assay for the enzymatic activity of recombinant CpPyK protein and used it to screen a chemical compound library for inhibitors of CpPyK’s activity. The identified inhibitors were tested (at non-toxic concentrations) for efficacy against C. parvum using in vitro assays, and an in vivo mouse infection model. We identified six CpPyK inhibitors that blocked in vitro growth and proliferation of C. parvum at low micromolar concentrations (EC50 values ranging from 10.29 to 86.01 μM) that were non-toxic to host cells. Among those six compounds, two (NSC252172 and NSC234945) were found to be highly efficacious against cryptosporidiosis in immunocompromised mice at a dose of 10 mg/kg body weight, with very significant reduction in parasite load and amelioration of intestinal pathologies. Together, these findings have unveiled inhibitors for an essential molecular target in C. parvum and demonstrated their efficacy against the parasite in vitro and in vivo. These inhibitors are, therefore, potential lead-compounds for developing efficacious treatments for cryptosporidiosis.

Sign in / Sign up

Export Citation Format

Share Document