We show that five topoisomerase I inhibitors (two indenoisoquinolines, two camptothecins, and one indolocarbazole) each intercalate
between the base pairs flanking the cleavage site generated during ...the topoisomerase I catalytic cycle and are further stabilized
by a network of hydrogen bonds with topoisomerase I. The interfacial inhibition paradigm described for topoisomerase I inhibitors
can be generalized to a variety of natural products that trap macromolecular complexes as they undergo catalytic conformational
changes that create hotspots for drug binding. Stabilization of such conformational states results in uncompetitive inhibition
and exemplifies the relevance of screening for ligands and drugs that stabilize (“trap”) these macromolecular complexes. Mol
Cancer Ther 2006;5(2):287–95
SARS-CoV-2 is the agent responsible for acute respiratory disease COVID-19 and the global pandemic initiated in early 2020. While the record-breaking development of vaccines has assisted the control ...of COVID-19, there is still a pressing global demand for antiviral drugs to halt the destructive impact of this disease. Repurposing clinically approved drugs provides an opportunity to expediate SARS-CoV-2 treatments into the clinic. In an effort to facilitate drug repurposing, an FDA-approved drug library containing 2400 compounds was screened against the SARS-CoV-2 non-structural protein 7 (nsp7) using a native mass spectrometry-based assay. Nsp7 is one of the components of the SARS-CoV-2 replication/transcription complex essential for optimal viral replication, perhaps serving to off-load RNA from nsp8. From this library, gallic acid was identified as a compound that bound tightly to nsp7, with an estimated K d of 15 μM. NMR chemical shift perturbation experiments were used to map the ligand-binding surface of gallic acid on nsp7, indicating that the compound bound to a surface pocket centered on one of the protein’s four α-helices (α2). The identification of the gallic acid-binding site on nsp7 may allow development of a SARS-CoV-2 therapeutic via artificial-intelligence-based virtual docking and other strategies.
Summary High-resolution three-dimensional structures of essential Mycobacterium tuberculosis (Mtb) proteins provide templates for TB drug design, but are available for only a small fraction of the ...Mtb proteome. Here we evaluate an intra-genus “homolog-rescue” strategy to increase the structural information available for TB drug discovery by using mycobacterial homologs with conserved active sites. Of 179 potential TB drug targets selected for x-ray structure determination, only 16 yielded a crystal structure. By adding 1675 homologs from nine other mycobacterial species to the pipeline, structures representing an additional 52 otherwise intractable targets were solved. To determine whether these homolog structures would be useful surrogates in TB drug design, we compared the active sites of 106 pairs of Mtb and non-TB mycobacterial (NTM) enzyme homologs with experimentally determined structures, using three metrics of active site similarity, including superposition of continuous pharmacophoric property distributions. Pair-wise structural comparisons revealed that 19/22 pairs with >55% overall sequence identity had active site Cα RMSD <1 Å, >85% side chain identity, and ≥80% PSAPF (similarity based on pharmacophoric properties) indicating highly conserved active site shape and chemistry. Applying these results to the 52 NTM structures described above, 41 shared >55% sequence identity with the Mtb target, thus increasing the effective structural coverage of the 179 Mtb targets over three-fold (from 9% to 32%). The utility of these structures in TB drug design can be tested by designing inhibitors using the homolog structure and assaying the cognate Mtb enzyme; a promising test case, Mtb cytidylate kinase, is described. The homolog-rescue strategy evaluated here for TB is also generalizable to drug targets for other diseases.
Pathogenic bacteria adhere to the host cell surface using a family of outer membrane proteins called Trimeric Autotransporter Adhesins (TAAs). Although TAAs are highly divergent in sequence and ...domain structure, they are all conceptually comprised of a C-terminal membrane anchoring domain and an N-terminal passenger domain. Passenger domains consist of a secretion sequence, a head region that facilitates binding to the host cell surface, and a stalk region.
Pathogenic species of Burkholderia contain an overabundance of TAAs, some of which have been shown to elicit an immune response in the host. To understand the structural basis for host cell adhesion, we solved a 1.35 A resolution crystal structure of a BpaA TAA head domain from Burkholderia pseudomallei, the pathogen that causes melioidosis. The structure reveals a novel fold of an intricately intertwined trimer. The BpaA head is composed of structural elements that have been observed in other TAA head structures as well as several elements of previously unknown structure predicted from low sequence homology between TAAs. These elements are typically up to 40 amino acids long and are not domains, but rather modular structural elements that may be duplicated or omitted through evolution, creating molecular diversity among TAAs.
The modular nature of BpaA, as demonstrated by its head domain crystal structure, and of TAAs in general provides insights into evolution of pathogen-host adhesion and may provide an avenue for diagnostics.
Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the ...olfactory nerves, burrowing through the cribriform plate to its destination: the brain's frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogen's high mortality is the lack of sensitivity of N. fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N. fowleri. The genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N. fowleri research. In 2017, 178 N. fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen.
The rise in antimicrobial resistance is a global health crisis and necessitates the development of novel strategies to treat infections. For example, in 2022 tuberculosis (TB) was the second leading ...infectious killer after COVID-19, with multi-drug-resistant strains of TB having an ∼40% fatality rate. Targeting essential biosynthetic pathways in pathogens has proven to be successful for the development of novel antimicrobial treatments. Fatty-acid synthesis (FAS) in bacteria proceeds via the type II pathway, which is substantially different from the type I pathway utilized in animals. This makes bacterial fatty-acid biosynthesis (Fab) enzymes appealing as drug targets. FabG is an essential FASII enzyme, and some bacteria, such as Mycobacterium tuberculosis, the causative agent of TB, harbor multiple homologs. FabG4 is a conserved, high-molecular-weight FabG (HMwFabG) that was first identified in M. tuberculosis and is distinct from the canonical low-molecular-weight FabG. Here, structural and functional analyses of Mycolicibacterium smegmatis FabG4, the third HMwFabG studied to date, are reported. Crystal structures of NAD
and apo MsFabG4, along with kinetic analyses, show that MsFabG4 preferentially binds and uses NADH when reducing CoA substrates. As M. smegmatis is often used as a model organism for M. tuberculosis, these studies may aid the development of drugs to treat TB and add to the growing body of research that distinguish HMwFabGs from the archetypal low-molecular-weight FabG.
Paraburkholderia xenovorans degrades organic wastes, including polychlorinated biphenyls. The atomic structure of a putative dehydrogenase/reductase (SDR) from P. xenovorans (PxSDR) was determined in ...space group P21 at a resolution of 1.45 Å. PxSDR shares less than 37% sequence identity with any known structure and assembles as a prototypical SDR tetramer. As expected, there is some conformational flexibility and difference in the substrate‐binding cavity, which explains the substrate specificity. Uniquely, the cofactor‐binding cavity of PxSDR is not well conserved and differs from those of other SDRs. PxSDR has an additional seven amino acids that form an additional unique loop within the cofactor‐binding cavity. Further studies are required to determine how these differences affect the enzymatic functions of the SDR.
The high‐resolution structure of a putative short‐chain reductase from the commercially important bacterium Paraburkholderia xenovorans is reported. P. xenovorans degrades organic wastes such as polychlorinated biphenyls.
Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections globally and is one of the most commonly reported infections in the United States. There is a need to develop ...new therapeutics due to drug resistance and the failure of current treatments to clear persistent infections. Structures of potential C. trachomatis rational drug‐discovery targets, including C. trachomatis inorganic pyrophosphatase (CtPPase), have been determined by the Seattle Structural Genomics Center for Infectious Disease. Inorganic pyrophosphatase hydrolyzes inorganic pyrophosphate during metabolism. Furthermore, bacterial inorganic pyrophosphatases have shown promise for therapeutic discovery. Here, a 2.2 Å resolution X‐ray structure of CtPPase is reported. The crystal structure of CtPPase reveals shared structural features that may facilitate the repurposing of inhibitors identified for bacterial inorganic pyrophosphatases as starting points for new therapeutics for C. trachomatis.
Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections. C. trachomatis inorganic pyrophosphatase (CtPPase) hydrolyzes inorganic pyrophosphate during metabolism. A 2.2 Å resolution X‐ray structure of CtPPase reveals shared structural features that may facilitate the repurposing of inhibitors identified for bacterial inorganic pyrophosphatases as starting points for new therapeutics.
Inorganic pyrophosphate (PP
) is generated as an intermediate or byproduct of many fundamental metabolic pathways, including DNA/RNA synthesis. The intracellular concentration of PP
must be regulated ...as buildup can inhibit many critical cellular processes. Inorganic pyrophosphatases (PPases) hydrolyze PP
into two orthophosphates (P
), preventing the toxic accumulation of the PP
byproduct in cells and making P
available for use in biosynthetic pathways. Here, the crystal structure of a family I inorganic pyrophosphatase from Legionella pneumophila is reported at 2.0 Å resolution. L. pneumophila PPase (LpPPase) adopts a homohexameric assembly and shares the oligonucleotide/oligosaccharide-binding (OB) β-barrel core fold common to many other bacterial family I PPases. LpPPase demonstrated hydrolytic activity against a general substrate, with Mg
being the preferred metal cofactor for catalysis. Legionnaires' disease is a severe respiratory infection caused primarily by L. pneumophila, and thus increased characterization of the L. pneumophila proteome is of interest.
is the causative agent of Buruli ulcer, a debilitating chronic disease that mainly affects the skin. Current treatments for Buruli ulcer are efficacious, but rely on the use of antibiotics with ...severe side effects. The enzyme dihydrofolate reductase (DHFR) plays a critical role in the
biosynthesis of folate species and is a validated target for several antimicrobials. Here we describe the biochemical and structural characterization of
DHFR and identified P218, a safe antifolate compound in clinical evaluation for malaria, as a potent inhibitor of this enzyme. We expect our results to advance
DHFR as a target for future structure-based drug discovery campaigns.
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