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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 613 records · Page 34
All sourcesNASA NTRSDOE OSTI
DOE OSTI2014

The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer

In an effort to develop a new therapy for prostate cancer bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β- catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels, and inhibited tumor cell migration. To examine the anti-tumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle to establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum TRACP 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia, and an increase in the animal survival. Finally, based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for prostate cancer bone metastasis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Selective inhibition of human translation termination by a drug-like compound

Methods to directly inhibit gene expression using small molecules hold promise for the development of new therapeutics targeting proteins that have evaded previous attempts at drug discovery. Among these, small molecules including the drug-like compound PF-06446846 (PF846) selectively inhibit the synthesis of specific proteins, by stalling translation elongation. These molecules also inhibit translation termination by an unknown mechanism. Using cryo-electron microscopy (cryo-EM) and biochemical approaches, we show that PF846 inhibits translation termination by arresting the nascent chain (NC) in the ribosome exit tunnel. The arrested NC adopts a compact α-helical conformation that induces 28 S rRNA nucleotide rearrangements that suppress the peptidyl transferase center (PTC) catalytic activity stimulated by eukaryotic release factor 1 (eRF1). These data support a mechanism of action for a small molecule targeting translation that suppresses peptidyl-tRNA hydrolysis promoted by eRF1, revealing principles of eukaryotic translation termination and laying the foundation for new therapeutic strategies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Potent and selective covalent inhibition of the papain-like protease from SARS-CoV-2

Abstract Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we design a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibits PLpro with k inact /K I = 9,600 M −1 s −1 , achieves sub-μM EC 50 values against three SARS-CoV-2 variants in mammalian cell lines, and does not inhibit a panel of human deubiquitinases (DUBs) at >30 μM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validates our design strategy and establishes the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Inhibitor-3 inhibits Protein Phosphatase 1 via a metal binding dynamic protein–protein interaction

To achieve substrate specificity, protein phosphate 1 (PP1) forms holoenzymes with hundreds of regulatory and inhibitory proteins. Inhibitor-3 (I3) is an ancient inhibitor of PP1 with putative roles in PP1 maturation and the regulation of PP1 activity. Here, we show that I3 residues 27–68 are necessary and sufficient for PP1 binding and inhibition. In addition to a canonical RVxF motif, which is shared by nearly all PP1 regulators and inhibitors, and a non-canonical SILK motif, I3 also binds PP1 via multiple basic residues that bind directly in the PP1 acidic substrate binding groove, an interaction that provides a blueprint for how substrates bind this groove for dephosphorylation. Unexpectedly, this interaction positions a CCC (cys-cys-cys) motif to bind directly across the PP1 active site. Using biophysical and inhibition assays, we show that the I3 CCC motif binds and inhibits PP1 in an unexpected dynamic, fuzzy manner, via transient engagement of the PP1 active site metals. Together, these data not only provide fundamental insights into the mechanisms by which IDP protein regulators of PP1 achieve inhibition, but also shows that fuzzy interactions between IDPs and their folded binding partners, in addition to enhancing binding affinity, can also directly regulate enzyme activity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Revealing the atomic and electronic mechanism of human manganese superoxide dismutase product inhibition

Human manganese superoxide dismutase (MnSOD) is a crucial oxidoreductase that maintains the vitality of mitochondria by converting superoxide (O 2 •– ) to molecular oxygen (O 2 ) and hydrogen peroxide (H 2 O 2 ) with proton-coupled electron transfers (PCETs). Human MnSOD has evolved to be highly product inhibited to limit the formation of H 2 O 2 , a freely diffusible oxidant and signaling molecule. The product-inhibited complex is thought to be composed of a peroxide (O 2 2– ) or hydroperoxide (HO 2 – ) species bound to Mn ion and formed from an unknown PCET mechanism. PCET mechanisms of proteins are typically not known due to difficulties in detecting the protonation states of specific residues that coincide with the electronic state of the redox center. To shed light on the mechanism, we combine neutron diffraction and X-ray absorption spectroscopy of the product-bound, trivalent, and divalent states of the enzyme to reveal the positions of all the atoms, including hydrogen, and the electronic configuration of the metal ion. The data identifies the product-inhibited complex, and a PCET mechanism of inhibition is constructed.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Insights into the mechanisms of NH3 inhibition on Cu-CHA SCR catalysts

This work elucidates the atomic-scale mechanism behind ammonia (NH3) inhibition during the selective catalytic reduction (SCR) of NO? on Cu-CHA catalysts, a key issue limiting low-temperature emission control. Using SCR kinetic analysis, operando electron paramagnetic resonance (EPR) spectroscopy, and density functional theory (DFT), we demonstrate that NH3 inhibition primarily slows the oxidation half-cycle (OHC), while the reduction half-cycle (RHC) remains unaffected. DFT simulations reveal that excess NH3 substantially increases the diffusion barrier for CuI ions, hindering formation of essential CuII-oxo dimer intermediates and thus suppressing OHC kinetics. Operando EPR studies confirm that this inhibition strongly depends on operating temperature and catalyst Cu loading. Our findings highlight strategies to counteract NH3 inhibition, including optimizing Cu loading, precisely managing NH3:NO feed ratios, and enhancing CuI ion mobility through tailored catalyst design and operational adjustments, thereby advancing the efficiency of emission control technologies in automotive applications.

Deka, Dhruba Jyoti↗
DOE OSTI2018

GlcNAc-1-P-transferase–tunicamycin complex structure reveals basis for inhibition of N-glycosylation

N-linked glycosylation is a predominant post-translational modification of protein in eukaryotes, and its dysregulation is the etiology of several human disorders. The enzyme UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosaminephosphotransferase (GlcNAc-1-P-transferase or GPT) catalyzes the first and committed step of N-linked glycosylation in the endoplasmic reticulum membrane, and it is the target of the natural product tunicamycin. Tunicamycin has potent antibacterial activity, inhibiting the bacterial cell wall synthesis enzyme MraY, but its usefulness as an antibiotic is limited by off-target inhibition of human GPT. Our understanding of how tunicamycin inhibits N-linked glycosylation and efforts to selectively target MraY are hampered by a lack of structural information. Here we present crystal structures of human GPT in complex with tunicamycin. In conclusion, structural and functional analyses reveal the difference between GPT and MraY in their mechanisms of inhibition by tunicamycin. Here, we demonstrate that this difference could be exploited to design MraY-specific inhibitors as potential antibiotics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Pentose sugars inhibit metabolism and increase expression of an AgrD-type cyclic pentapeptide in Clostridium thermocellum

Significant hurdles exist in efforts to domesticate and industrialize a microbial species for biotechnological application as specific metabolic functions found in natural communities disappear in axenic cultures. For the lignocellulose-deconstructing specialist Clostridium thermocellum, the catabolism of hemicellulose-derived pentoses, which the bacterium cannot ferment, is one such function. Here, we report that various xylo-oligomers significantly inhibit C. thermocellum metabolism and growth and that microbe-sugar interactions occur across multiple dimensions. First, stable isotope metabolomics confirmed C. thermocellum s ability to transport and metabolize pentose sugars. This transport occurs, at least in part, through the ATP-dependent transporter, CbpD. Secondly, xylose is an electron sink for C. thermocellum metabolism leading to the production of xylitol. Deletion of Clo1313_0076, annotated as a xylitol dehydrogenase, reduced the total production and molar xylitol yields by 41% and 46%, respectively. However, it also altered the relative end-product distribution patterns confirming that external electron acceptors may influence the bacterium s redox metabolism to a greater extent than previously considered. Finally, xylose-induced inhibition corresponds with the up-regulation and biogenesis of an AgrD-type, lactone cyclized pentapeptide signaling molecule; which is the first report of an AgrD-type signaling peptide in any thermophile. Addition of synthetic versions of the cyclic peptide inhibited cultures grown in the absence of xylose, but had no effect on cultures already inhibited by the pentose sugar. Together, our findings identify that C. thermocellum has evolved previously unrecognized strategies to cope with C5-sugars, but the absence of a native catabolic sink negatively affects strain metabolism and growth.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Biotin attachment domain-containing proteins mediate hydroxy fatty acid-dependent inhibition of acetyl CoA carboxylase

Hundreds of naturally occurring specialized fatty acids (FAs) have potential as desirable chemical feedstocks if they could be produced at large scale by crop plants; however, transgenic expression of their biosynthetic genes has generally been accompanied by dramatic reductions in oil yield. For example, expression of castor (Ricinus communis) FA hydroxylase (FAH) in the Arabidopsis thaliana FA elongation mutant fae1 resulted in a 50% reduction of FA synthesis rate that was attributed to inhibition of acetyl-CoA carboxylase (ACCase) by an undefined mechanism. Here, we tested the hypothesis that the ricinoleic acid-dependent decrease in ACCase activity is mediated by biotin attachment domain-containing (BADC) proteins. BADCs are inactive homologs of biotin carboxy carrier protein that lack a biotin cofactor and can inhibit ACCase. Arabidopsis contains three BADC genes. To reduce expression levels of BADC1 and BADC3 in fae1/FAH plants, a homozygous badc1,3/fae1/FAH line was created. The rate of FA synthesis in badc1,3/fae1/FAH seeds doubled relative to fae1/FAH, restoring it to fae1 levels, increasing both native FA and HFA accumulation. Total FA per seed, seed oil content, and seed yield per plant all increased in badc1,3/fae1/FAH, to 5.8 µg, 37%, and 162 mg, respectively, relative to 4.9 µg, 33%, and 126 mg, respectively, for fae1/FAH. Transcript levels of FA synthesis-related genes, including those encoding ACCase subunits, did not significantly differ between badc1,3/fae1/FAH and fae1/FAH. These results demonstrate that BADC1 and BADC3 mediate ricinoleic acid-dependent inhibition of FA synthesis. We propose that BADC-mediated FAS inhibition as a general mechanism that limits FA accumulation in specialized FA-accumulating seeds.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Patulin Inhibition of Specific Apple Microbiome Members Uncovers Hanseniaspora uvarum as a Potential Biocontrol Agent

Penicillium expansum is a major postharvest pathogen of apples, causing loss in fruits through tissue damage, as well as in apple products due to contamination with the mycotoxin patulin. During infections, patulin is a cultivar-dependent virulence factor that facilitates apple lesion development. Patulin also has characterized antimicrobial activity and is important for inhibiting other competitive phytopathogens, but the role of this inhibitory activity has not been investigated in the context of the apple microbiome. Here, in our current study, we isolated 68 apple microbiota and characterized their susceptibility to P. expansum extracts. We found gram-negative bacteria and basidiomycete yeast to demonstrate largely patulin-specific growth inhibition compared with gram-positive and ascomycete isolates. From co-cultures, we identified a Hanseniaspora and Gluconobacter pairing that reduced P. expansum biomass and found that H. uvarum alone is sufficient to reduce apple disease progression in vivo. We investigated possible mechanisms of H. uvarum biocontrol activity and found modest inhibition on apple puree plates, as well as a trend toward lower patulin levels at the wound site. Active biocontrol activity required live yeast, which was also effective in controlling Botrytis cinerea apple infections. Lastly, we explored the breadth of H. uvarum biocontrol activity with over 30 H. uvarum isolates and found consistent inhibition of P. expansum apple disease.

Hanseniaspora uvarum↗
DOE OSTI2018

Structural Insight into Allosteric Inhibition of Mycobacterium tuberculosis Tryptophan Synthase

The rise of antibiotic‐resistant pathogens requires redirecting drug discovery effort toward new cellular targets to provide new treatment options. Such alternative, yet unexplored opportunities exist in central metabolism, where many enzymes are only conditionally essential and thus their successful exploitation requires understanding of metabolic fluxes and pathway regulations under certain environmental conditions. In Mycobacterium tuberculosis , tryptophan biosynthesis represents one such promising pathway that could be targeted. In Mtb, the multistep L‐Trp synthesis involves several enzymes: TrpEG, TrpD, TrpC and TrpAB, which is validated as conditionally essential in vivo . The whole‐cell screening of a diversity‐oriented synthetic library identified a novel bactericidal azetidine derivative, BRD4592, that specifically inhibits TrpAB (tryptophan synthase). TrpAB is a heterotetrameric, PLP‐dependent complex catalyzing two last reactions in L‐Trp synthesis: the conversion of indole‐3‐glycerol phosphate to indole and glyceraldehyde 3‐phosphate (TrpA) followed by indole and L‐Ser condensation to yield L‐Trp (TrpB). During the catalytic cycle, indole is transferred from the TrpA to TrpB active site through 25 Å hydrophobic channel to react with the L‐Ser‐PLP adduct. The enzymatic activities and substrate channeling are coordinated by complex allosteric communication between the subunits. This regulation involves cycling between various conformational states, with both subunits adopting either an open (inactive) or closed (active) state. Detailed kinetic, biophysical and structural evidence reveals that BRD4592 is an allosteric, mixed‐type inhibitor, which binds specifically to a novel site within the intersubunit channel, at the TrpA‐TrpB interface. The molecule promotes closed, active state of the TrpB subunit and increases enzyme affinity for the L‐Ser substrate. BRD4592 acts on three levels: it limits flexibility of the interface potentially disrupting mutual activation of the subunits, prevents indole translocation, and increases product inhibition providing means to successfully inhibit both, TrpA and TrpB reactions. Our results indicate that allosteric binders may represent an effective tool to deactivate central metabolism enzymes, which often are allosterically regulated and might be predisposed for this type of inhibition. Support or Funding Information This work was supported by the Broad Institute Tuberculosis donor group, the Pershing Square Foundation, the Bill & Melinda Gates Foundation under grant OPP1032518, the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contracts HHSN272200700058C and HHSN272201200026C and the U. S. Department of Energy, Office of Biological and Environmental Research, under contract DE‐AC02‐06CH11357. For this work, University of Chicago is a subgrantee of Structural Genomics Consortium under a Bill & Melinda Gates Foundation grant for the Structure‐Guided Drug Discovery Coalition.

Michalska, Karolina↗
DOE OSTI2012

Structural basis of the inhibition of class C acid phosphatases by adenosine 5;#8242;-phosphorothioate

The inhibition of phosphatases by adenosine 5'-phosphorothioate (AMPS) was first reported in the late 1960s; however, the structural basis for the inhibition has remained unknown. Here, it is shown that AMPS is a submicromolar inhibitor of class C acid phosphatases, a group of bacterial outer membrane enzymes belonging to the haloacid dehalogenase structural superfamily. Furthermore, the 1.35-{angstrom} resolution crystal structure of the inhibited recombinant Haemophilus influenzae class C acid phosphatase was determined; this is the first structure of a phosphatase complexed with AMPS. The conformation of AMPS is identical to that of the substrate 5'-AMP, except that steric factors force a rotation of the thiophosphoryl out of the normal phosphoryl-binding pocket. This conformation is catalytically nonproductive, because the P atom is not positioned optimally for nucleophilic attack by Asp64, and the O atom of the scissile O-P bond is too far from the Asp (Asp66) that protonates the leaving group. The structure of 5'-AMP complexed with the Asp64 {yields} Asn mutant enzyme was also determined at 1.35-{angstrom} resolution. This mutation induces the substrate to adopt the same nonproductive binding mode that is observed in the AMPS complex. In this case, electrostatic considerations, rather than steric factors, underlie the movement of the phosphoryl. The structures not only provide an explanation for the inhibition by AMPS, but also highlight the precise steric and electrostatic requirements of phosphoryl recognition by class C acid phosphatases. Moreover, the structure of the Asp64 {yields} Asn mutant illustrates how a seemingly innocuous mutation can cause an unexpected structural change.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Horizontal transfer of a pathway for coumarate catabolism unexpectedly inhibits purine nucleotide biosynthesis

A microbe’s ecological niche and biotechnological utility are confirmed by its specific set of co-evolved metabolic pathways. The acquisition of new pathways, through horizontal gene transfer or genetic engineering, can have unpredictable consequences. In this work, we show that two different pathways for coumarate catabolism failed to function when initially transferred into Escherichia coli . Using laboratory evolution, we elucidated the factors limiting activity of the newly acquired pathways and the modifications required to overcome these limitations. Both pathways required host mutations to enable effective growth with coumarate, but the necessary mutations differed. In one case, a pathway intermediate inhibited purine nucleotide biosynthesis, and this inhibition was relieved by single amino acid replacements in IMP dehydrogenase. A strain that natively contains this coumarate catabolism pathway, Acinetobacter baumannii , is resistant to inhibition by the relevant intermediate, indicating that natural pathway transfers have faced and overcome similar challenges. Molecular dynamics simulation of the wild type and a representative single-residue mutant provide insight into the structural and dynamic changes that relieve inhibition. These results reflect how deleterious interactions can limit pathway transfer, that these interactions can be traced to specific molecular interactions between host and pathway, and how evolution or engineering can alleviate these limitations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Inhibition of the eukaryotic initiation factor-2α kinase PERK decreases risk of autoimmune diabetes in mice

Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress–responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eukaryotic translation initiation factor-2α (eIF2α). In T1D, maladaptive unfolded protein response (UPR) in insulin-producing β cells renders these cells susceptible to autoimmunity. We found that inhibition of the eIF2α kinase PKR-like ER kinase (PERK), a common component of the UPR and ISR, reversed the mRNA translation block in stressed human islets and delayed the onset of diabetes, reduced islet inflammation, and preserved β cell mass in T1D-susceptible mice. Single-cell RNA-Seq of islets from PERK-inhibited mice showed reductions in the UPR and PERK signaling pathways and alterations in antigen-processing and presentation pathways in β cells. Spatial proteomics of islets from these mice showed an increase in the immune checkpoint protein programmed death-ligand 1 (PD-L1) in β cells. Golgi membrane protein 1, whose levels increased following PERK inhibition in human islets and EndoC-βH1 human β cells, interacted with and stabilized PD-L1. Collectively, our studies show that PERK activity enhances β cell immunogenicity and that inhibition of PERK may offer a strategy for preventing or delaying the development of T1D.

Research & Experimental Medicine↗
DOE OSTI2018

The orphan nuclear receptor EAR-2 (NR2F6) inhibits hematopoietic cell differentiation and induces myeloid dysplasia in vivo

Background: In patients with myelodysplastic syndrome (MDS), bone marrow cells have an increased predisposition to apoptosis, yet MDS cells outcompete normal bone marrow (BM)–suggesting that factors regulating growth potential may be important in MDS. We previously identified v-Erb A related-2 (EAR-2, NR2F6) as a gene involved in control of growth ability. Methods: Bone marrow obtained from C57BL/6 mice was transfected with a retrovirus containing EAR-2-IRES-GFP. Ex vivo transduced cells were flow sorted. In some experiments cells were cultured in vitro, in other experiments cells were injected into lethally irradiated recipients, along with non-transduced bone marrow cells. Short-hairpin RNA silencing EAR-2 was also introduced into bone marrow cells cultured ex vivo. Results: Here, we show that EAR-2 inhibits maturation of normal BM in vitro and in vivo and that EAR-2 transplant chimeras demonstrate key features of MDS. Competitive repopulation of lethally irradiated murine hosts with EAR2-transduced BM cells resulted in increased engraftment and increased colony formation in serial replating experiments. Recipients of EAR-2-transduced grafts had hypercellular BM, erythroid dysplasia, abnormal localization of immature precursors and increased blasts; secondary transplantation resulted in acute leukemia. Animals were cytopenic, having reduced numbers of erythrocytes, monocytes and granulocytes. Suspension culture confirmed that EAR-2 inhibits granulocytic and monocytic differentiation, while knockdown induced granulocytic differentiation. We observed a reduction in the number of BFU-E and CFU-GM colonies and the size of erythroid and myeloid colonies. Serial replating of transduced hematopoietic colonies revealed extended replating potential in EAR-2-overexpressing BM, while knockdown reduced re-plating ability. EAR-2 functions by recruitment of histone deacetylases, and inhibition of differentiation in 32D cells is dependent on the DNA binding domain. Conclusions: This data suggest that NR2F6 inhibits maturation of normal BM in vitro and in vivo and that the NR2F6 transplant chimera system demonstrates key features of MDS, and could provide a mouse model for MDS.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Revisiting the Effects of Xenon on Urate Oxidase and Tissue Plasminogen Activator: No Evidence for Inhibition by Noble Gases

Although chemically inert, Xe and other noble gases have been shown to have functional effects on biological systems. For example, Xe is a powerful anesthetic with neuroprotective properties. Recent reports have claimed that Xe inhibits the activity of tissue plasminogen activator (tPA) and urate oxidase (UOX), indicating that the use of Xe as an anesthetic may have undesirable side effects. Here, we revisited the methods used to demonstrate Xe inhibition of UOX and tPA, testing both indirect and direct gas delivery methods with variable bubble sizes and gas flowrates. Our results indicate that Xe or Kr do not affect the activity of UOX or tPA and that the previously reported inhibition is due to protein damage attendant to directly bubbling gases into protein solutions. The lack of evidence to support Xe inhibition of UOX or tPA alleviates concerns regarding possible side effects for the clinical application of Xe as an anesthetic. Furthermore, this study illustrates the importance of using indirect methods of gas dissolution for studying gas-protein interactions in aqueous solution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Inhibition of the C1s Protease and the Classical Complement Pathway by 6-(4-Phenylpiperazin-1-yl)Pyridine-3-Carboximidamide and Chemical Analogs

Abstract The classical pathway (CP) is a potent mechanism for initiating complement activity and is a driver of pathology in many complement-mediated diseases. The CP is initiated via activation of complement component C1, which consists of the pattern recognition molecule C1q bound to a tetrameric assembly of proteases C1r and C1s. Enzymatically active C1s provides the catalytic basis for cleavage of the downstream CP components, C4 and C2, and is therefore an attractive target for therapeutic intervention in CP-driven diseases. Although an anti-C1s mAb has been Food and Drug Administration approved, identifying small-molecule C1s inhibitors remains a priority. In this study, we describe 6-(4-phenylpiperazin-1-yl)pyridine-3-carboximidamide (A1) as a selective, competitive inhibitor of C1s. A1 was identified through a virtual screen for small molecules that interact with the C1s substrate recognition site. Subsequent functional studies revealed that A1 dose-dependently inhibits CP activation by heparin-induced immune complexes, CP-driven lysis of Ab-sensitized sheep erythrocytes, CP activation in a pathway-specific ELISA, and cleavage of C2 by C1s. Biochemical experiments demonstrated that A1 binds directly to C1s with a K d of ∼9.8 μM and competitively inhibits its activity with an inhibition constant (K i) of ∼5.8 μM. A 1.8-Å-resolution crystal structure revealed the physical basis for C1s inhibition by A1 and provided information on the structure–activity relationship of the A1 scaffold, which was supported by evaluating a panel of A1 analogs. Taken together, our work identifies A1 as a new class of small-molecule C1s inhibitor and lays the foundation for development of increasingly potent and selective A1 analogs for both research and therapeutic purposes.

Immunology↗
NASA NTRS1982

Quantitation of chlorpromazine-bound calmodulin during chlorpromazine inhibition of gravitropism

The regulatory protein, calmodulin (CaM), controls the activity of a plasma membrane localized ATPase in plants which serves to pump calcium out of cells. Recent data are consistent with the hypothesis that activation of this pump is one of the early steps necessary for gravitropism. Chlorpromazine (CPZ), a CaM antagonist, reversibly inhibits gravitropism in oat coleoptiles at concentrations which permit normal growth rates. C-14-labeled CPZ was used to photo-affinity label endogenous CaM in vivo to learn whether the drug is actually binding to some portion of endogenous CaM when it inhibits gravitropism. Under conditions in which CPZ inhibits gravitropism for over an hour, at least 11% of the CaM in gravitropically stimulated coleoptiles is bound to CPZ. In a given CPZ experiment the degree of inhibition of gravitropism correlates well with the amount of CaM bound to CPZ.

Roux, S. J.↗