Human African trypanosomiasis (HAT), a major health concern in sub-Saharan Africa, is caused by t... more Human African trypanosomiasis (HAT), a major health concern in sub-Saharan Africa, is caused by the protozoan parasite Trypanosoma brucei. Recent studies have shown that a cathepsin B like protease, TbcatB, is essential to the survival of T. brucei in vitro (Mackey, Z. B.; O'Brien, T. C.; Greenbaum, D. C.; Blank, R. B.; McKerrow, J. H. J. Biol. Chem. 2004, 279, 48426-48433). Herein, we describe the first inhibitors of TbcatB, a series of purine nitriles. The compounds are potent trypanocides, killing the parasite with a high degree of selectivity over a panel of three human cell lines. In addition, a predictive model of trypanocidal activity was developed on the basis of potency against TbcatB and various calculated physical property descriptors.
ABSTRACT Background: Antimicrobial peptides (AMPs) represent an essential component of the innate... more ABSTRACT Background: Antimicrobial peptides (AMPs) represent an essential component of the innate immune system that provides resistance to a variety of pathogenic bacteria. AMPs display very broad spectrum action against bacteria, yeast, fungus, and even viruses. Antiparasitic activities have also been reported for a number of host defense peptides. The peptides appear to kill protozoa by interacting with the cytoplasmic membrane causing excessive permeability, lysis and death; a mechanism that is similar to their mechanism of action against bacteria. Specificity for the parasite versus the host cell is attributed to differences in phospholipid content and the lack of cholesterol in the protozoan membrane. Importantly, because the site of action is at the membrane and not to any specific receptor or intracellular target, the development of resistance to the cytotoxic properties of the antimicrobial peptides is highly unlikely.Recognizing the significant therapeutic limitations of peptides, the Degrado laboratory has developed a series of non-peptidic mimics of these AMPs (SMAMPs) that represent a novel and powerful therapy against many microbes. These SMAMPs include small molecules, sequence-specific oligomers, and polymers that have robust in vivo activity against Staphyococcal aureus in mouse models, suggesting a novel approach to the development of novel therapeutics. Methods: SMAMPs were tested against P.falciparum in culture and a luminescent assay was used to generate IC50s. Results: The Greenbaum laboratory has tested 6 SMAMPs and demonstrate that they kill P.falciparum parasites in culture with a range of IC50s from 50nM-3mM. Phenotoypic analysis has demonstrated that extracellular merozoites appear to be the target of these SMAMPS. Conclusions: We provide preliminary data that non-peptidic SMAMPs represent a novel antimalarial strategy.
A thiocarbazate compound was initially identified as a potent cathepsin L (EC 3.4.22.15) inhibito... more A thiocarbazate compound was initially identified as a potent cathepsin L (EC 3.4.22.15) inhibitor via an HTS effort at Penn Centre for Molecular Discovery. Molecular docking technique was employed to explore the interaction between the inhibitor and the enzyme and identified particular moieties responsible for binding affinity. These efforts have led to the discovery of a tetrahydroquinoline oxocarbazate (PubChem CID 23631927) which was tested to be an nM inhibitor of human cathepsin L and an entry blocker of SARS coronavirus and Ebola pseudotype virus. The compound demonstrated activity in blocking both SARS-CoV (IC50 = 27349 nM) and Ebola virus (IC50 = 19339 nM) entry into HEK 293T cells while the thiocarbazate compound did not show efficacy in the assay. In an attempt to trace the intracellular action of the inhibitors with intracellular cathepsin L, the activity based probe DCG-04 was used to label the active site of cysteine proteases in 293T lysates. The reduction in active c...
Thesis (B.A.)--Williams College, Dept. of Chemistry, 1994. Includes bibliographical references (l... more Thesis (B.A.)--Williams College, Dept. of Chemistry, 1994. Includes bibliographical references (leaves 60-61).
About 30% of human tumours carry ras gene mutations. Of the three genes in this family (composed ... more About 30% of human tumours carry ras gene mutations. Of the three genes in this family (composed of K-ras, N-ras and H-ras), K-ras is the most frequently mutated member in human tumours, including adenocarcinomas of the pancreas ( approximately 70-90% incidence), colon ( approximately 50%) and lung ( approximately 25-50%). To construct mouse tumour models involving K-ras, we used a new gene targeting procedure to create mouse strains carrying oncogenic alleles of K-ras that can be activated only on a spontaneous recombination event in the whole animal. Here we show that mice carrying these mutations were highly predisposed to a range of tumour types, predominantly early onset lung cancer. This model was further characterized by examining the effects of germline mutations in the tumour suppressor gene p53, which is known to be mutated along with K-ras in human tumours. This approach has several advantages over traditional transgenic strategies, including that it more closely recapitu...
Peptidases are essential for the establishment and survival of the medically important parasite, ... more Peptidases are essential for the establishment and survival of the medically important parasite, Schistosoma mansoni. This helminth expresses a number of gut-associated peptidases that degrade host blood proteins, including hemoglobin, as a means of nutrition. Using irreversible affinity probes, we demonstrate that S. mansoni cathepsin B-like endopeptidase 1 (SmCB1) is the most abundant papain family cysteine peptidase in both the parasite gut and somatic extracts. SmCB1 zymogen (SmCB1pm) was functionally expressed in Pichia pastoris (4-11mgl(-1)). Monospecific and immunoselected antibodies raised against SmCB1pm localized the enzyme exclusively to the gut lumen and surrounding gastrodermis of adult worms. Recombinant SmCB1pm was unable to catalyze its activation, even at low pH. However, recombinant S. mansoni asparaginyl endopeptidase (SmAE), another gut-associated cysteine peptidase, processed and activated SmCB1pm in trans. Consistent with the known specificity of AEs, processin...
Human African trypanosomiasis (HAT), a major health concern in sub-Saharan Africa, is caused by t... more Human African trypanosomiasis (HAT), a major health concern in sub-Saharan Africa, is caused by the protozoan parasite Trypanosoma brucei. Recent studies have shown that a cathepsin B like protease, TbcatB, is essential to the survival of T. brucei in vitro (Mackey, Z. B.; O'Brien, T. C.; Greenbaum, D. C.; Blank, R. B.; McKerrow, J. H. J. Biol. Chem. 2004, 279, 48426-48433). Herein, we describe the first inhibitors of TbcatB, a series of purine nitriles. The compounds are potent trypanocides, killing the parasite with a high degree of selectivity over a panel of three human cell lines. In addition, a predictive model of trypanocidal activity was developed on the basis of potency against TbcatB and various calculated physical property descriptors.
ABSTRACT Background: Antimicrobial peptides (AMPs) represent an essential component of the innate... more ABSTRACT Background: Antimicrobial peptides (AMPs) represent an essential component of the innate immune system that provides resistance to a variety of pathogenic bacteria. AMPs display very broad spectrum action against bacteria, yeast, fungus, and even viruses. Antiparasitic activities have also been reported for a number of host defense peptides. The peptides appear to kill protozoa by interacting with the cytoplasmic membrane causing excessive permeability, lysis and death; a mechanism that is similar to their mechanism of action against bacteria. Specificity for the parasite versus the host cell is attributed to differences in phospholipid content and the lack of cholesterol in the protozoan membrane. Importantly, because the site of action is at the membrane and not to any specific receptor or intracellular target, the development of resistance to the cytotoxic properties of the antimicrobial peptides is highly unlikely.Recognizing the significant therapeutic limitations of peptides, the Degrado laboratory has developed a series of non-peptidic mimics of these AMPs (SMAMPs) that represent a novel and powerful therapy against many microbes. These SMAMPs include small molecules, sequence-specific oligomers, and polymers that have robust in vivo activity against Staphyococcal aureus in mouse models, suggesting a novel approach to the development of novel therapeutics. Methods: SMAMPs were tested against P.falciparum in culture and a luminescent assay was used to generate IC50s. Results: The Greenbaum laboratory has tested 6 SMAMPs and demonstrate that they kill P.falciparum parasites in culture with a range of IC50s from 50nM-3mM. Phenotoypic analysis has demonstrated that extracellular merozoites appear to be the target of these SMAMPS. Conclusions: We provide preliminary data that non-peptidic SMAMPs represent a novel antimalarial strategy.
A thiocarbazate compound was initially identified as a potent cathepsin L (EC 3.4.22.15) inhibito... more A thiocarbazate compound was initially identified as a potent cathepsin L (EC 3.4.22.15) inhibitor via an HTS effort at Penn Centre for Molecular Discovery. Molecular docking technique was employed to explore the interaction between the inhibitor and the enzyme and identified particular moieties responsible for binding affinity. These efforts have led to the discovery of a tetrahydroquinoline oxocarbazate (PubChem CID 23631927) which was tested to be an nM inhibitor of human cathepsin L and an entry blocker of SARS coronavirus and Ebola pseudotype virus. The compound demonstrated activity in blocking both SARS-CoV (IC50 = 27349 nM) and Ebola virus (IC50 = 19339 nM) entry into HEK 293T cells while the thiocarbazate compound did not show efficacy in the assay. In an attempt to trace the intracellular action of the inhibitors with intracellular cathepsin L, the activity based probe DCG-04 was used to label the active site of cysteine proteases in 293T lysates. The reduction in active c...
Thesis (B.A.)--Williams College, Dept. of Chemistry, 1994. Includes bibliographical references (l... more Thesis (B.A.)--Williams College, Dept. of Chemistry, 1994. Includes bibliographical references (leaves 60-61).
About 30% of human tumours carry ras gene mutations. Of the three genes in this family (composed ... more About 30% of human tumours carry ras gene mutations. Of the three genes in this family (composed of K-ras, N-ras and H-ras), K-ras is the most frequently mutated member in human tumours, including adenocarcinomas of the pancreas ( approximately 70-90% incidence), colon ( approximately 50%) and lung ( approximately 25-50%). To construct mouse tumour models involving K-ras, we used a new gene targeting procedure to create mouse strains carrying oncogenic alleles of K-ras that can be activated only on a spontaneous recombination event in the whole animal. Here we show that mice carrying these mutations were highly predisposed to a range of tumour types, predominantly early onset lung cancer. This model was further characterized by examining the effects of germline mutations in the tumour suppressor gene p53, which is known to be mutated along with K-ras in human tumours. This approach has several advantages over traditional transgenic strategies, including that it more closely recapitu...
Peptidases are essential for the establishment and survival of the medically important parasite, ... more Peptidases are essential for the establishment and survival of the medically important parasite, Schistosoma mansoni. This helminth expresses a number of gut-associated peptidases that degrade host blood proteins, including hemoglobin, as a means of nutrition. Using irreversible affinity probes, we demonstrate that S. mansoni cathepsin B-like endopeptidase 1 (SmCB1) is the most abundant papain family cysteine peptidase in both the parasite gut and somatic extracts. SmCB1 zymogen (SmCB1pm) was functionally expressed in Pichia pastoris (4-11mgl(-1)). Monospecific and immunoselected antibodies raised against SmCB1pm localized the enzyme exclusively to the gut lumen and surrounding gastrodermis of adult worms. Recombinant SmCB1pm was unable to catalyze its activation, even at low pH. However, recombinant S. mansoni asparaginyl endopeptidase (SmAE), another gut-associated cysteine peptidase, processed and activated SmCB1pm in trans. Consistent with the known specificity of AEs, processin...
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Papers by Doron Greenbaum