Diversity-Oriented Synthesis Yields a Novel Lead for the Treatment of Malaria

Here, we describe the discovery of a novel antimalarial agent using phenotypic screening of Plasmodium falciparum asexual blood-stage parasites. Screening a novel compound collection created using diversity-oriented synthesis (DOS) led to the initial hit. Structure–activity relationships guided the synthesis of compounds having improved potency and water solubility, yielding a subnanomolar inhibitor of parasite asexual blood-stage growth. Optimized compound 27 has an excellent off-target activity profile in erythrocyte lysis and HepG2 assays and is stable in human plasma. This compound is available via the molecular libraries probe production centers network (MLPCN) and is designated ML238.Chemistry and Chemical Biolog

Effect of Stalling after Mismatches on the Error Catastrophe in Nonenzymatic Nucleic Acid Replication

The frequency of errors during genome replication limits the amount of functionally important information that can be passed on from generation to generation. During the origin of life, mutation rates are thought to have been quite high, raising a classic chicken-and-egg paradox: could nonenzymatic replication propagate sequences accurately enough to allow for the emergence of heritable function? Here we show that the theoretical limit on genomic information content may increase substantially as a consequence of dramatically slowed polymerization after mismatches. As a result of postmismatch stalling, accurate copies of a template tend to be completed more rapidly than mutant copies and the accurate copies can therefore begin a second round of replication more quickly. To quantify this effect, we characterized an experimental model of nonenzymatic, template-directed nucleic acid polymerization. We found that most mismatches decrease the rate of primer extension by more than 2 orders of magnitude relative to a matched (Watson-Crick) control. A chemical replication system with this property would be able to propagate sequences long enough to have function. Our study suggests that the emergence of functional sequences during the origin of life would be possible even in the face of the high intrinsic error rates of chemical replication

Can an Infectious Disease Genomics Project Predict and Prevent the Next Pandemic?

Infectious diseases need a globally coordinated genomic-based movement linking sequencing efforts to development of response tools to mitigate the impact of existing and emerging threats

Functional Characterization of the Plasmodium falciparum Chloroquine-Resistance Transporter (PfCRT) in Transformed Dictyostelium discoideum Vesicles

Chloroquine (CQ)-resistant Plasmodium falciparum malaria has been a global health catastrophe, yet much about the CQ resistance (CQR) mechanism remains unclear. Hallmarks of the CQR phenotype include reduced accumulation of protonated CQ as a weak base in the digestive vacuole of the erythrocyte-stage parasite, and chemosensitization of CQ-resistant (but not CQ-sensitive) P. falciparum by agents such as verapamil. Mutations in the P. falciparum CQR transporter (PfCRT) confer CQR; particularly important among these mutations is the charge-loss substitution K→T at position 76. Dictyostelium discoideum transformed with mutant PfCRT expresses key features of CQR including reduced drug accumulation and verapamil chemosensitization.We describe the isolation and characterization of PfCRT-transformed, hematin-free vesicles from D. discoideum cells. These vesicles permit assessments of drug accumulation, pH, and membrane potential that are difficult or impossible with hematin-containing digestive vacuoles from P. falciparum-infected erythrocytes. Mutant PfCRT-transformed D. discoideum vesicles show features of the CQR phenotype, and manipulations of vesicle membrane potential by agents including ionophores produce large changes of CQ accumulation that are dissociated from vesicular pH. PfCRT in its native or mutant form blunts the ability of valinomycin to reduce CQ accumulation in transformed vesicles and decreases the ability of K(+) to reverse membrane potential hyperpolarization caused by valinomycin treatment.Isolated vesicles from mutant-PfCRT-transformed D. discoideum exhibit features of the CQR phenotype, consistent with evidence that the drug resistance mechanism operates at the P. falciparum digestive vacuole membrane in malaria. Membrane potential apart from pH has a major effect on the PfCRT-mediated CQR phenotype of D. discoideum vesicles. These results support a model of PfCRT as an electrochemical potential-driven transporter in the drug/metabolite superfamily that (appropriately mutated) acts as a saturable simple carrier for the facilitated diffusion of protonated CQ

Normalization of the Lymph Node T Cell Stromal Microenvironment in lpr/lpr Mice Is Associated with SU5416-Induced Reduction in Autoantibodies

The vascular-stromal elements of lymph nodes can play important roles in regulating the activities of the lymphocytes within. During model immune responses, the vascular-stromal compartment has been shown to undergo proliferative expansion and functional alterations. The state of the vascular-stromal compartment and the potential importance of this compartment in a spontaneous, chronic model of autoimmunity have not been well studied. Here, we characterize the vascular expansion in MRL-lpr/lpr lymph nodes and attempt to ask whether inhibiting this expansion can interfere with autoantibody generation. We show that characteristics of vascular expansion in enlarging MRL-lpr/lpr lymph nodes resemble that of the VEGF-dependent expansion that occurs in wild-type mice after model immunization. Surprisingly, treatment with SU5416, an inhibitor of VEGF and other receptor tyrosine kinases, did not have sustained effects in inhibiting vascular growth, but attenuated the anti-dsDNA response and altered the phenotype of the double negative T cells that are expanded in these mice. In examining for anatomic correlates of these immunologic changes, we found that the double negative T cells are localized within ectopic follicles around a central B cell patch and that these T cell-rich areas lack the T zone stromal protein ER-TR7 as well as other elements of a normal T zone microenvironment. SU5416 treatment disrupted these follicles and normalized the association between T zone microenvironmental elements and T cell-rich areas. Recent studies have shown a regulatory role for T zone stromal elements. Thus, our findings of the association of anti-dsDNA responses, double negative T cell phenotype, and altered lymphocyte microenvironment suggest the possibility that lymphocyte localization in ectopic follicles protects them from regulation by T zone stromal elements and functions to maintain autoimmune responses. Potentially, altering the lymphocyte microenvironment that is set up by the vascular-stromal compartment can be a means by which to control undesired autoimmune responses

The secondary structure and sequence optimization of an RNA ligase ribozyme.

In vitro selection can generate functional sequence variants of an RNA structural motif that are useful for comparative analysis. The technique is particularly valuable in cases where natural variation is unavailable or non-existent. We report the extension of this approach to a new extreme--the identification of a 112 nt ribozyme secondary structure imbedded within a 186 nt RNA. A pool of 10(14) variants of an RNA ligase ribozyme was generated using combinatorial chemical synthesis coupled with combinatorial enzymatic ligation such that 172 of the 186 relevant positions were partially mutagenized. Active variants of this pool were enriched using an in vitro selection scheme that retains the sequence variability at positions very close to the ligation junction. Ligases isolated after four rounds of selection catalyzed self-ligation up to 700 times faster than the starting sequence. Comparative analysis of the isolates indicated that when complexed with substrate RNAs the ligase forms a nested, double pseudo-knot secondary structure with seven stems and several important joining segments. Comparative analysis also suggested the identity of mutations that account for the increased activity of the selected ligase variants; designed constructs incorporating combinations of these changes were more active than any of the individual ligase isolates

Follicular stromal cells and lymphocyte homing to follicles

Follicular dendritic cells (FDCs), the best defined stromal cell subset within lymphoid follicles, play a critical role in presenting intact antigen to B lymphocytes. The discovery that many follicular stromal cells make B-lymphocyte chemoattractant (BLC), a CXC chemokine that attracts CXCR5+ cells, provides a basis for understanding how motile B cells come into contact with stationary FDCs. Here we review our work on BLC and discuss properties of BLC-expressing follicular stromal cells. We also review the properties of primary follicle and germinal center FDCs and suggest a model of FDC development that incorporates information about BLC expression. Finally, we consider how antigen recognition causes T and B lymphocytes to undergo changes in chemokine responsiveness that may help direct their movements into, or out of, lymphoid follicles