The
development of new antimalarial compounds remains a pivotal part of
the strategy for malaria elimination. Recent large-scale phenotypic
screens have provided a wealth of potential starting points for hit-to-lead
campaigns. One such public set is explored, employing an open source
research mechanism in which all data and ideas were shared in real
time, anyone was able to participate, and patents were not sought.
One chemical subseries was found to exhibit oral activity but contained
a labile ester that could not be replaced without loss of activity,
and the original hit exhibited remarkable sensitivity to minor structural
change. A second subseries displayed high potency, including activity
within gametocyte and liver stage assays, but at the cost of low solubility.
As an open source research project, unexplored avenues are clearly
identified and may be explored further by the community; new findings
may be cumulatively added to the present work.
Discerning false positives from true actives in high-throughput screening (HTS) output is fraught with difficulty as the reason of anomalous activity seen for compounds is often not clear-cut. In this study, we introduce a novel medium-throughput NMR assay for the identification of redox-cycling compounds (RCCs), which is based on detection of oxidation of a reducing agent. We compare its outcomes to those from horseradish peroxidase (HRP)/phenol red and resazurin (RZ)-based assays that are more commonly used for triaging HTS outputs. Data from NMR, RZ, and HRP redox assay are shown to correlate, with the NMR assay showing the greatest accuracy. In addition, historical data analysis was used to identify compounds frequently active in assays for redox-susceptible targets. We provide examples of compound classes found and conclude that the NMR redox assay offers a novel and reliable way of identifying RCCs at a medium throughput. The HRP and RZ assays are reasonable higher-throughput alternatives, with both showing similar sensitivity to redox-cycling and false-positive compounds. The RZ assay has a higher hit rate, reflecting its ability to pick up multiple modes of action.
Electronic computers have revolutionized virtually all aspects of our lives. However, long before these existed, cells have relied on computations implemented using biochemistry to make decisions about how to improve their chance of survival. The emerging field of synthetic biology offers a new perspective on life, attempting to apply engineering principles to modify and repurpose biological systems or even create new ones from scratch. This is opening up exciting opportunities to reprogram cellular functions, enabling us to better understand how biological computations are implemented, as well as providing a window into the inner workings of the living computers that surround us.
Transcriptional terminators signal where transcribing RNA polymerases (RNAPs) should halt and disassociate from DNA. However, because termination is stochastic, two different forms of transcript could be produced: one ending at the terminator and the other reading through. An ability to control the abundance of these transcript isoforms would offer bioengineers a mechanism to regulate multi-gene constructs at the level of transcription. Here, we explore this possibility by repurposing terminators as ‘transcriptional valves’ that can tune the proportion of RNAP read-through. Using one-pot combinatorial DNA assembly, we iteratively construct 1780 transcriptional valves for T7 RNAP and show how nanopore-based direct RNA sequencing (dRNA-seq) can be used to characterize entire libraries of valves simultaneously at a nucleotide resolution in vitro and unravel genetic design principles to tune and insulate termination. Finally, we engineer valves for multiplexed regulation of CRISPR guide RNAs. This work provides new avenues for controlling transcription and demonstrates the benefits of long-read sequencing for exploring complex sequence-function landscapes.
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