Aspartate transcarbamoylase (ATC) is the first committed step in de novo pyrimidine biosynthesis in eukaryotes and plants. A potent transition state analog of human ATCase (PALA) has previously been assessed in clinical trials for the treatment of cancer, but was ultimately unsuccessful. Additionally, inhibition of this pathway has been proposed to be a target to suppress cell proliferation in E. coli, the malarial parasite and tuberculosis. In this manuscript we screened a 70-member library of ATC inhibitors developed against the malarial and tubercular ATCases for inhibitors of the human ATC. Four compounds showed low nanomolar inhibition (IC50 30-120 nM) in an in vitro activity assay. These compounds significantly outperform PALA, which has a triphasic inhibition response under identical conditions, in which significant activity remains at PALA concentrations above 10 μM. Evidence for a druggable allosteric pocket in human ATC is provided by both in vitro enzyme kinetic, homology modeling and in silico docking. These compounds also suppress the proliferation of U2OS osteoblastoma cells by promoting cell cycle arrest in G0/G1 phase. This report provides the first evidence for an allosteric pocket in human ATC, which greatly enhances its druggability and demonstrates the potential of this series in cancer therapy.

Fragment-based approaches offer rapid screening of chemical space and have become a mainstay in drug discovery. This manuscript provides a recent example that highlights the initial and intermediate stages involved in the fragment-based discovery of an allosteric inhibitor of the malarial aspartate transcarbamoylase (ATCase), subsequently shown to be a potential novel anti-malarial. The initial availability of high-resolution diffracting crystals allowed the collection of a number of protein fragment complexes, which were then assessed for inhibitory activity in an in vitro assay, and binding was assessed using biophysical techniques. Elaboration of these compounds in cycles of structure-based drug design improved activity and selectivity between the malarial and human ATCases. A key element in this process was the use of multicomponent reaction chemistry as a multicomponent compatible fragment library, which allowed the rapid generation of elaborated compounds, the rapid construction of a large (70 member) chemical library, and thereby efficient exploration of chemical space around the fragment hits. This review article details the steps along the pathway of the development of this library, highlighting potential limitations of the approach and serving as an example of the power of combining multicomponent reaction chemistry with fragment-based approaches.

Aspartate transcarbamoylase (ATCase) plays a key role in the second step of de novo pyrimidine biosynthesis in eukaryotes and has been proposed to be a target to suppress cell proliferation in E. coli, human cells and the malarial parasite. We hypothesized that a library of ATCase inhibitors developed for malarial ATCase (PfATCase) may also contain inhibitors of the tubercular ATCase and provide a similar inhibition of cellular proliferation. Of the 70 compounds screened, 10 showed single-digit micromolar inhibition in an in vitro activity assay and were tested for their effect on M. tuberculosis cell growth in culture. The most promising compound demonstrated a MIC90 of 4 & mu;M. A model of MtbATCase was generated using the experimental coordinates of PfATCase. In silico docking experiments showed this compound can occupy a similar allosteric pocket on MtbATCase to that seen on PfATCase, explaining the observed species selectivity seen for this compound series.

Malaria remains one of the most prominent and dangerous tropical diseases. While artemisinin and analogs have been used as first-line drugs for the past decades, due to the high mutational rate and rapid adaptation to the environment of the parasite, it remains urgent to develop new antimalarials. The pyrimidine biosynthesis pathway plays an important role in cell growth and proliferation. Unlike human host cells, the malarial parasite lacks a functional pyrimidine salvage pathway, meaning that RNA and DNA synthesis is highly dependent on the de novo synthesis pathway. Thus, direct or indirect blockage of the pyrimidine biosynthesis pathway can be lethal to the parasite. Aspartate transcarbamoylase (ATCase), catalyzes the second step of the pyrimidine biosynthesis pathway, the condensation of L-aspartate and carbamoyl phosphate to form N-carbamoyl aspartate and inorganic phosphate, and has been demonstrated to be a promising target both for anti-malaria and anti-cancer drug development. This is highlighted by the discovery that at least one of the targets of Torin2 - a potent, yet unselective, antimalarial - is the activity of the parasite transcarbamoylase. Additionally, the recent discovery of an allosteric pocket of the human homology raises the intriguing possibility of species selective ATCase inhibitors. We recently exploited the available crystal structures of the malarial aspartate transcarbamoylase to perform a fragment-based screening to identify hits. In this review, we summarize studies on the structure of Plasmodium falciparum ATCase by focusing on an allosteric pocket that supports the catalytic mechanisms.

The discovery and development of new drugs against malaria remain urgent. Aspartate transcarbamoylase (ATC) has been suggested to be a promising target for antimalarial drug development. Here, we describe a series of small-molecule inhibitors of P. falciparum ATC with low nanomolar binding affinities that selectively bind to a previously unreported allosteric pocket, thereby inhibiting ATC activation. We demonstrate that the buried allosteric pocket is located close to the traditional ATC active site and that reported compounds maintain the active site of PfATC in its low substrate affinity/low activity conformation. These compounds inhibit parasite growth in blood stage cultures at single digit micromolar concentrations, whereas limited effects were seen against human normal lymphocytes. To our knowledge, this series represent the first PfATC-specific allosteric inhibitors.

IL-17a is a major inflammation target, with several approved antibodies in clinical use. Small-molecule IL-17a antagonists are an emerging hot topic, with the recent advancement of three compounds into clinical trials. Here, we describe the design, discovery, synthesis, and screening of macrocyclic compounds that bind to IL-17a. We found that all currently described IL-17a modifiers belong to the same pharmacophore model, likely resulting in a similar receptor binding mode on IL-17a. A pipeline of pharmacophore analysis, virtual screening, resynthesis, and protein biophysics resulted in a potent IL-17a macrocyclic modifier.

Lipoic acid (LA) is an organic compound that plays a key role in cellular metabolism. It participates in a posttranslational modification (PTM) named lipoylation, an event that is highly conserved and that occurs in multimeric metabolic enzymes of very distinct microorganisms such as Plasmodium sp. and Staphylococcus aureus, including pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KDH). In this mini review, we revisit the recent literature regarding LA metabolism in Plasmodium sp. and Staphylococcus aureus, by covering the lipoate ligase proteins in both microorganisms, the role of lipoate ligase proteins and insights for possible inhibitors of lipoate ligases.