Michele C. Connelly

Chemical genetics of Plasmodium falciparum

Malaria caused by Plasmodium falciparum is a disease that is responsible for 880,000 deaths per year worldwide. Vaccine development has proved difficult and resistance has emerged for most antimalarial drugs. To discover new antimalarial chemotypes, we have used a phenotypic forward chemical genetic approach to assay 309,474 chemicals. Here we disclose structures and biological activity of the entire library—many of which showed potent in vitro activity against drug-resistant P. falciparum strains—and detailed profiling of 172 representative candidates. A reverse chemical genetic study identified 19 new inhibitors of 4 validated drug targets and 15 novel binders among 61 malarial proteins. Phylochemogenetic profiling in several organisms revealed similarities between Toxoplasma gondii and mammalian cell lines and dissimilarities between P. falciparum and related protozoans. One exemplar compound displayed efficacy in a murine model. Our findings provide the scientific community with new starting points for malaria drug discovery.

Metabolic pathways for activation of the antiviral agent 9-(2-phosphonylmethoxyethyl)adenine in human lymphoid cells.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA), the acyclic phosphonate analog of adenine monophosphate, is a promising antiviral drug with activity against herpesviruses, Epstein-Barr virus, and retroviruses, including the human immunodeficiency virus. In order to be active, it must be converted to the diphosphate derivative, the putative inhibitor of viral DNA polymerases. The metabolic pathway responsible for activation of PMEA is unclear. The metabolism of PMEA was investigated in human T-lymphoid cells (CEMss) and a PMEA-resistant subline (CEMss(r-1)) with a partial deficiency in adenylate kinase activity. Experiments with [3H]PMEA showed that extracts of CEMss phosphorylated PMEA to its mono- and diphosphate in the presence of ATP as the phosphate donor. No other nucleotides or 5-phosphoribosyl pyrophosphate displayed appreciable activity as a phosphate donor. Subcellular fractionation experiments showed that CEMss cells contained two nucleotide kinase activities, one in mitochondria and one in the cytosol, which phosphorylated PMEA. The PMEA-resistant CEMss mutant proved to have a deficiency in the mitochondrial adenylate kinase activity, indicating that this enzyme was important in the phosphorylation of PMEA. Other effective antiviral purine phosphonate derivatives of PMEA showed a profile of phosphorylating activity similar to that of PMEA. By comparison, phosphorylation of the pyrimidine analog (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine proceeded by an enzyme present in the cytosol. We conclude from these studies that adenylate kinase which has been localized in the intermembrane space of mitochondria is the major route for PMEA phosphorylation in CEMss cells but that another hitherto unidentified enzyme(s) present in the cytosol may contribute to the anabolism of the phosphonates.

Mechanism of uptake of the phosphonate analog (S)-1-(3-hydroxy-2-phosphonylmethoxy-propyl)cytosine (HPMPC) in vero cells

The cellular uptake of phosphonylmethoxypropyl cytosine (HPMPC) was characterized to gain insight into the molecular properties that allow this anticytomegalovirus drug to permeate cell membranes. The time course of uptake of HPMPC into Vero cells was linear between 10 and 75 min and proportional to the concentration in the medium from 10(-6) to 10(-2) M. HPMPC uptake was temperature sensitive and the rate of uptake was considerably lower at 27 degrees than at 37 degrees and almost totally inhibited at 4 degrees. In competition studies with naturally occurring nucleosides, nucleotides or the phosphonylmethoxyethyl derivatives, none affected the uptake of HPMPC at concentrations up to 2000-fold molar excess. The uptake of [3H]HPMPC into Vero cells was compared with that of [14C]sucrose, a probe for fluid-phase endocytosis. Kinetics for both compounds were very similar, as were the effects of the microtubule antagonist colchicine and the tumor promoting agent phorbol myristate acetate. Colchicine and the phorbol ester are known to, respectively, inhibit and stimulate endocytosis. It is concluded from these data that HPMPC enters Vero cells by fluid-phase endocytosis and that once internalized it may accumulate in the lysosome. Protonation of the negative charge on the phosphonyl group in HPMPC may allow its diffusion across the lysosome membrane and eventual activation to its putative active diphosphorylated form in the cell cytoplasm.

Identification of small molecule proliferating cell nuclear antigen (PCNA) inhibitor that disrupts interactions with PIP-box proteins and inhibits DNA replication

Background: PCNA is a multifunctional component of DNA replication and repair machinery. Results: A novel small molecule inhibitor of the PCNA protein-protein interaction inhibited DNA replication, induced DNA replication stress, and increased cisplatin-mediated DNA damage response in cells. Conclusion: The biochemical PCNA inhibitor can inhibit PCNA functions essential for cells. Significance: Inhibition of the PCNA protein-protein interaction can be a new strategy to sensitize cancer cells to chemotherapy. We have discovered that 3,3′,5-triiodothyronine (T3) inhibits binding of a PIP-box sequence peptide to proliferating cell nuclear antigen (PCNA) protein by competing for the same binding site, as evidenced by the co-crystal structure of the PCNA-T3 complex at 2.1 Å resolution. Based on this observation, we have designed a novel, non-peptide small molecule PCNA inhibitor, T2 amino alcohol (T2AA), a T3 derivative that lacks thyroid hormone activity. T2AA inhibited interaction of PCNA/PIP-box peptide with an IC50 of ∼1 μm and also PCNA and full-length p21 protein, the tightest PCNA ligand protein known to date. T2AA abolished interaction of PCNA and DNA polymerase δ in cellular chromatin. De novo DNA synthesis was inhibited by T2AA, and the cells were arrested in S-phase. T2AA inhibited growth of cancer cells with induction of early apoptosis. Concurrently, Chk1 and RPA32 in the chromatin are phosphorylated, suggesting that T2AA causes DNA replication stress by stalling DNA replication forks. T2AA significantly inhibited translesion DNA synthesis on a cisplatin-cross-linked template in cells. When cells were treated with a combination of cisplatin and T2AA, a significant increase in phospho(Ser139)histone H2AX induction and cell growth inhibition was observed.

Lead optimization of 3-carboxyl-4(1H)-quinolones to deliver orally bioavailable antimalarials.

Malaria is a protozoal parasitic disease that is widespread in tropical and subtropical regions of Africa, Asia, and the Americas and causes more than 800,000 deaths per year. The continuing emergence of multidrug-resistant Plasmodium falciparum drives the ongoing need for the development of new and effective antimalarial drugs. Our previous work has explored the preliminary structural optimization of 4(1H)-quinolone ester derivatives, a new series of antimalarials related to the endochins. Herein, we report the lead optimization of 4(1H)-quinolones with a focus on improving both antimalarial potency and bioavailability. These studies led to the development of orally efficacious antimalarials including quinolone analogue 20g, a promising candidate for further optimization.

Discovery of novel antimalarial compounds enabled by QSAR-based virtual screening

Quantitative structure-activity relationship (QSAR) models have been developed for a data set of 3133 compounds defined as either active or inactive against P. falciparum. Because the data set was strongly biased toward inactive compounds, different sampling approaches were employed to balance the ratio of actives versus inactives, and models were rigorously validated using both internal and external validation approaches. The balanced accuracy for assessing the antimalarial activities of 70 external compounds was between 87% and 100% depending on the approach used to balance the data set. Virtual screening of the ChemBridge database using QSAR models identified 176 putative antimalarial compounds that were submitted for experimental validation, along with 42 putative inactives as negative controls. Twenty five (14.2%) computational hits were found to have antimalarial activities with minimal cytotoxicity to mammalian cells, while all 42 putative inactives were confirmed experimentally. Structural inspection of confirmed active hits revealed novel chemical scaffolds, which could be employed as starting points to discover novel antimalarial agents.

Discovery of trypanocidal thiosemicarbazone inhibitors of rhodesain and TbcatB

Human African trypanosomiasis (HAT) is caused by the protozoan parasite Trypanosoma brucei. The cysteine proteases of T. brucei have been shown to be crucial for parasite replication and represent an attractive point for therapeutic intervention. Herein we describe the synthesis of a series of thiosemicarbazones and their activity against the trypanosomal cathepsins TbcatB and rhodesain, as well as human cathepsins L and B. The activity of these compounds was determined against cultured T. brucei, and specificity was assessed with a panel of four mammalian cell lines.

Synthesis and Structure-Activity Relationships of Antimalarial 4-oxo-3-carboxyl quinolones

Malaria is endemic in tropical and subtropical regions of Africa, Asia, and the Americas. The increasing prevalence of multi-drug-resistant Plasmodium falciparum drives the ongoing need for the development of new antimalarial drugs. In this light, novel scaffolds to which the parasite has not been exposed are of particular interest. Recently, workers at the Swiss Tropical Institute discovered two novel 4-oxo-3-carboxyl quinolones active against the intra-erythrocytic stages of P. falciparum while carrying out rationally directed low-throughput screening of potential antimalarial agents as part of an effort directed by the World Health Organization. Here we report the design, synthesis, and preliminary pharmacologic characterization of a series of analogues of 4-oxo-3-carboxyl quinolones. These studies indicate that the series has good potential for preclinical development.

Synthesis and evaluation of 7-substituted 4-aminoquinoline analogues for antimalarial activity.

We previously reported that substituted 4-aminoquinolines with a phenyl ether substituent at the 7-position of the quinoline ring and the capability of intramolecular hydrogen bonding between the protonated amine on the side chain and a hydrogen bond acceptor on the amines alkyl substituents exhibited potent antimalarial activity against the multidrug resistant strain P. falciparum W2. We employed a parallel synthetic method to generate diaryl ether, biaryl, and alkylaryl 4-aminoquinoline analogues in the background of a limited number of side chain variations that had previously afforded potent 4-aminoquinolines. All subsets were evaluated for their antimalarial activity against the chloroquine-sensitive strain 3D7 and the chloroquine-resistant K1 strain as well as for cytotoxicity against mammalian cell lines. While all three arrays showed good antimalarial activity, only the biaryl-containing subset showed consistently good potency against the drug-resistant K1 strain and good selectivity with regard to mammalian cytotoxicity. Overall, our data indicate that the biaryl-containing series contains promising candidates for further study.

Methylsulfonylnitrobenzoates, a New Class of Irreversible Inhibitors of the Interaction of the Thyroid Hormone Receptor and Its Obligate Coactivators That Functionally Antagonizes Thyroid Hormone

Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor (NR) superfamily and regulate development, growth, and metabolism. Upon binding thyroid hormone, TR undergoes a conformational change that allows the release of corepressors and the recruitment of coactivators, which in turn regulate target gene transcription. Although a number of TR antagonists have been developed, most are analogs of the endogenous hormone that inhibit ligand binding. In a screen for inhibitors that block the association of TRβ with steroid receptor coactivator 2 (SRC2), we identified a novel methylsulfonylnitrobenzoate (MSNB)-containing series that blocks this interaction at micromolar concentrations. Here we have studied a series of MSNB analogs and characterized their structure activity relationships. MSNB members do not displace thyroid hormone T3 but instead act by direct displacement of SRC2. MSNB series members are selective for the TR over the androgen, vitamin D, and PPARγ NR members, and they antagonize thyroid hormone-activated transcription action in cells. The methylsulfonylnitro group is essential for TRβ antagonism. Side-chain alkylamine substituents showed better inhibitory activity than arylamine substituents. Mass spectrum analysis suggested that MSNB inhibitors bind irreversibly to Cys-298 within the AF-2 cleft of TRβ to disrupt SRC2 association.