Abstract 523: Factor XIIa: New Insights on Chemical Tractability and Target Indications From a Potent and Selective Tool Inhibitor

FXII is an emerging target for thrombosis, yet several questions remain to be addressed. Firstly, from drug discovery perspective, level of enzyme occupancy needed for efficacy (which largely dictates potency and selectivity requirement for small molecule inhibitors) is unclear, as most reported active site inhibitors have some level of off-target activities. Secondly, from disease treatment perspective, it is unclear whether FXIIa inhibition will be a safe strategy for stroke treatment or prevention, as it was recently reported that FXIIa inhibition destabilized the subocclusive thrombi in a plaque rupture model. In this presentation, we set out to address these questions using a previously described molecule, Inf4mut15. We generated the human albumin (HA)-Inf4mut15 fusion protein (Mut-inf) for our studies. In vitro Mut-inf displayed comparable potency as the widely used wild-type HA-Infestin4 (WT-inf) (human FXIIa Ki = 73 and 120 pM, respectively). Both infs acted as competitive reversible active site inhibitors of FXIIa, with no binding to FXII zymogen, hence same mode of action as certain small molecule inhibitors. Mut-inf, however, was much more selective against plasmin compared to WT-inf (20,000- and 75-fold Ki separation, respectively), consistent with results from the functional tPA-induced TEG assay, where Ly60 was reduced dose-dependently by WT- but not Mut-inf. Mut-inf aPTT doubling concentration was 15 uM and FXIIa Ki in 30% plasma was 3.5 nM. Calculated enzyme occupancy for Mut-inf for doubling human aPTT is thus 99.9%. In the rabbit model of cerebral microembolic signals (MES) induced by FeCl 3 injury of the carotid artery, treatment with vehicle (n=7), WT-, and Mut-inf (1mg/kg and n=5 each) produced arterial thrombus of 6.0±0.4, 1.9±0.6, and 0.2±0.1 mg, respectively; incidence of MES detected in the middle cerebral artery was 4.1±1.3, 1.8±0.6, and 0.0±0.0, respectively. In summary, our studies demonstrated that very high enzyme occupancy will be required for achieving a putative aPTT doubling target in human for FXIIa active site inhibitors, highlighting the challenge with the small molecule modality. Our MES studies suggest that targeting FXII may offer a safe strategy for stroke prevention and/or other thromboembolic disorders.

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Faculty Opinions recommendation of Identification of new small molecule inhibitors of cystic fibrosis transmembrane conductance regulator protein: in vitro and in vivo studies.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1069767.522696 ◽

2007 ◽

Author(s):

Kim Barrett

Keyword(s):

Cystic Fibrosis ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

In Vivo Studies ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Regulator Protein ◽

Cystic Fibrosis Transmembrane

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EXTH-31. BRAIN DISTRIBUTION, CLEARANCE AND TOXICITY EVALUATION OF SMALL-MOLECULE INHIBITORS FOLLOWING CONVECTION-ENHANCED DELIVERY TO THE BRAINSTEM

Neuro-Oncology ◽

10.1093/neuonc/noab196.670 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi170-vi170

Author(s):

Erica Power ◽

Juhee Oh ◽

Jonghoon Choi ◽

William Elmquist ◽

David Daniels

Keyword(s):

Small Molecule ◽

Drug Concentration ◽

Small Molecule Inhibitors ◽

Efflux Pump ◽

Sprague Dawley ◽

Convection Enhanced Delivery ◽

Efflux Pump Inhibitor ◽

Delivery Technique ◽

The Brain

Abstract BACKGROUND Diffuse midline gliomas (DMGs) harboring the H3K27M mutation are highly aggressive, fatal brainstem tumors that primarily occur in children. The blood-brain barrier (BBB) prevents numerous drugs from reaching CNS tumors, like DMG, at cytotoxic concentrations. Convection-enhanced delivery (CED) has emerged as a drug delivery technique that bypasses the BBB through a direct interstitial infusion under a pressure gradient. However, drug distribution and clearance from the brain following CED is poorly understood and has been cited as a potential reason for the lack of efficacy observed in prior clinical trials. OBJECTIVE The objective of this study was to understand how two small molecule inhibitors (alisertib, ponatinib) that inhibit cell growth and proliferation in DMG cells in vitro distribute and clear from the brain following CED to the brainstem. METHODS Sprague-dawley rats underwent a single 60mL CED infusion of drug to the brainstem (200mM alisertib, 10mM ponatinib) and were sacrificed 0.083, 1, 2, 4, 8 and 24 hours following the completion of the infusion. Brains were dissected and drug concentration was determined via HPLC analysis. RESULTS No rats showed any clinical or neurological signs of toxicity post-infusion. Both drugs showed significant differences in drug concentration based on anatomical brain region where higher concentrations were observed in the pons and cerebellum compared to the cortex. Drug half-life in the brain was ~0.5 hours for alisertib and ~1 hour for ponatinib, but this was not significantly increased following co-administration of elacridar, a BBB efflux pump inhibitor. CONCLUSIONS These results suggest that elimination of drugs from the brain in a complex, multifactorial mechanism that warrants further preclinical investigation prior to the initiation of a clinical trial.

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Synthesis and in vitro evaluation of novel small molecule inhibitors of bacterial arylamine N-acetyltransferases (NATs)

Bioorganic & Medicinal Chemistry Letters ◽

10.1016/s0960-894x(03)00484-0 ◽

2003 ◽

Vol 13 (15) ◽

pp. 2527-2530 ◽

Cited By ~ 49

Author(s):

Edward W. Brooke ◽

Stephen G. Davies ◽

Andrew W. Mulvaney ◽

Minoru Okada ◽

Frédérique Pompeo ◽

...

Keyword(s):

Small Molecule ◽

Small Molecule Inhibitors ◽

In Vitro Evaluation

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High Throughput in Silico Identification of Novel Phytochemical Inhibitors for the Master Regulator of Inflammation (TNFα)

10.26434/chemrxiv.14178212.v2 ◽

2021 ◽

Author(s):

Pratap Kumar Parida ◽

Dipak Paul ◽

Debamitra Chakravorty

Keyword(s):

Small Molecule ◽

High Throughput ◽

In Silico ◽

Small Molecule Inhibitors ◽

Binding Free Energy ◽

Natural Compounds ◽

Free Energy Calculations ◽

Compound Libraries ◽

Relative Binding

<a>The over expression of Tumor necrosis factor-α (TNFα) has been implicated in a variety of disease and is classified as a therapeutic target for inflammatory diseases (Crohn disease, psoriasis, psoriatic arthritis, rheumatoid arthritis).Commercially available therapeutics are biologics which are associated with several risks and limitations. Small molecule inhibitors and natural compounds (saponins) were identified by researchers as lead molecules against TNFα, however, </a>they were often associated with high IC50 values which can lead to their failure in clinical trials. This warrants research related to identification of better small molecule inhibitors by screening of large compound libraries. Recent developments have demonstrated power of natural compounds as safe therapeutics, hence, in this work, we have identified TNFα phytochemical inhibitors using high throughput in silico screening approaches of 6000 phytochemicals followed by 200 ns molecular dynamics simulations and relative binding free energy calculations. The work yielded potent hits that bind to TNFα at its dimer interface. The mechanism targeted was inhibition of oligomerization of TNFα upon phytochemical binding to restrict its interaction with TNF-R1 receptor. MD simulation analysis resulted in identification of two phytochemicals that showed stable protein-ligand conformations over time. The two compounds were triterpenoids: Momordicilin and Nimbolin A with relative binding energy- calculated by MM/PBSA to be -190.5 kJ/Mol and -188.03 kJ/Mol respectively. Therefore, through this work it is being suggested that these phytochemicals can be used for further in vitro analysis to confirm their inhibitory action against TNFα or can be used as scaffolds to arrive at better drug candidates.

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Novel Small-Molecule Inhibitors of RNA Polymerase III

Eukaryotic Cell ◽

10.1128/ec.2.2.256-264.2003 ◽

2003 ◽

Vol 2 (2) ◽

pp. 256-264 ◽

Cited By ~ 47

Author(s):

Liping Wu ◽

Jing Pan ◽

Vala Thoroddsen ◽

Deborah R. Wysong ◽

Ronald K. Blackman ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

Rna Polymerase Iii ◽

Wild Type ◽

Biochemical Assays ◽

Transcription In Vitro ◽

Pol Iii

ABSTRACT A genetic approach utilizing the yeast Saccharomyces cerevisiae was used to identify the target of antifungal compounds. This analysis led to the identification of small molecule inhibitors of RNA polymerase (Pol) III from Saccharomyces cerevisiae. Three lines of evidence show that UK-118005 inhibits cell growth by targeting RNA Pol III in yeast. First, a dominant mutation in the g domain of Rpo31p, the largest subunit of RNA Pol III, confers resistance to the compound. Second, UK-118005 rapidly inhibits tRNA synthesis in wild-type cells but not in UK-118005 resistant mutants. Third, in biochemical assays, UK-118005 inhibits tRNA gene transcription in vitro by the wild-type but not the mutant Pol III enzyme. By testing analogs of UK-118005 in a template-specific RNA Pol III transcription assay, an inhibitor with significantly higher potency, ML-60218, was identified. Further examination showed that both compounds are broad-spectrum inhibitors, displaying activity against RNA Pol III transcription systems derived from Candida albicans and human cells. The identification of these inhibitors demonstrates that RNA Pol III can be targeted by small synthetic molecules.

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Small-Molecule Inhibitors of Staphylococcus aureus RnpA-Mediated RNA Turnover and tRNA Processing

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04352-14 ◽

2015 ◽

Vol 59 (4) ◽

pp. 2016-2028 ◽

Cited By ~ 9

Author(s):

Tess M. Eidem ◽

Nicole Lounsbury ◽

John F. Emery ◽

Jeffrey Bulger ◽

Andrew Smith ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

Antimicrobial Activities ◽

Rna Degradation ◽

Dual Function ◽

Content Type ◽

Trna Processing ◽

Cellular Assays

ABSTRACTNew agents are urgently needed for the therapeutic treatment ofStaphylococcus aureusinfections. In that regard,S. aureusRNase RnpA may represent a promising novel dual-function antimicrobial target that participates in two essential cellular processes, RNA degradation and tRNA maturation. Accordingly, we previously used a high-throughput screen to identify small-molecule inhibitors of the RNA-degrading activity of the enzyme and showed that the RnpA inhibitor RNPA1000 is an attractive antimicrobial development candidate. In this study, we used a series ofin vitroand cellular assays to characterize a second RnpA inhibitor, RNPA2000, which was identified in our initial screening campaign and is structurally distinct from RNPA1000. In doing so, it was found thatS. aureusRnpA does indeed participate in 5′-precursor tRNA processing, as was previously hypothesized. Further, we show that RNPA2000 is a bactericidal agent that inhibits both RnpA-associated RNA degradation and tRNA maturation activities bothin vitroand withinS. aureus. The compound appears to display specificity for RnpA, as it did not significantly affect thein vitroactivities of unrelated bacterial or eukaryotic ribonucleases and did not display measurable human cytotoxicity. Finally, we show that RNPA2000 exhibits antimicrobial activity and inhibits tRNA processing in efflux-deficient Gram-negative pathogens. Taken together, these data support the targeting of RnpA for antimicrobial development purposes, establish that small-molecule inhibitors of both of the functions of the enzyme can be identified, and lend evidence that RnpA inhibitors may have broad-spectrum antimicrobial activities.

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The Design and Synthesis of Small Molecule Inhibitors of Collagen Binding to Integrin α2β1 as Antithrombotic Agents.

Blood ◽

10.1182/blood.v110.11.306.306 ◽

2007 ◽

Vol 110 (11) ◽

pp. 306-306

Author(s):

Meredith W. Miller ◽

Soni Basra ◽

Paul C. Billings ◽

Jamie Gewirtz ◽

William F. DeGrado ◽

...

Keyword(s):

Small Molecule ◽

Platelet Adhesion ◽

Small Molecule Inhibitors ◽

Critical Event ◽

Platelet Activity ◽

Allosteric Site ◽

Collagen Binding ◽

Glycoprotein Vi

Abstract Vascular damage due to trauma or disease exposes circulating platelets to collagen in the subendothelial matrix. This is a critical event in the formation of a hemostatic plug or an occluding thrombus because collagen is not only a substrate for platelet adhesion but is also a strong platelet agonist. Platelets possess two physiologic collagen receptors: glycoprotein VI, a member of the immunoglobin superfamily, and the integrin α2β1. To design small molecule inhibitors of the interaction of platelets with collagen, we focused on α2β1 as a target because murine models of α2β1 deficiency display normal bleeding times and only a slight decrease in platelet activation by collagen and because the small number of reported patients with congenital α2β1 deficiency demonstrated only a mild bleeding diathesis. Thus, α2β1 antagonists could be effective anti-thrombotic agents with minimal toxicity, especially when combined with other anti-platelet drugs. We have developed a class of compounds that target the I-like domain of the β1 subunit, an allosteric site that regulates collagen binding to α2β1 by preventing the conversion of α2β1 from an inactive (low affinity) to an active (high affinity) conformation. This class of compounds is based on a proline-substituted 2,3-diaminopropionic acid scaffold. Structure-activity relationship studies of the scaffold have focused on optimization of the proline moiety, the urea functionality, and the sulfonyl group and have resulted in the development of potent inhibitors of α2β1-mediated platelet adhesion to collagen with IC50’s in the high picomolar to low nanomolar range. In particular, optimization of the proline moiety lead to compounds with high potency: transitioning from proline (DB496, IC50 of 29–62 nM) to a thiazolidine (SB68A) improved the IC50 to 2–8 nM; adding a methyl group at the 2 position of the thiazolidine (SB68B) slightly improved the IC50 to 1–12 nM; adding two methyl groups at the 5 position of the thiazolidine (SW4-161) resulted in a lead compound with an IC50 of 0.33–8 nM. As expected, the compounds had no effect on the binding of isolated α2 I-domains to collagen, consistent with their I-like domain mode of activity. Further, they were specific for α2β1-mediated platelet adhesion to collagen because they had no impact on ADP-stimulated platelet aggregation when added at 2 μM, a concentration more than 100-fold greater than the IC50 for inhibition of platelet adhesion to collagen. The compounds were also strong inhibitors of murine platelet adhesion to collagen and when tested in the ferric chloride-initiated murine carotid artery injury model, displayed activity similar to aspirin. Thus, 71% of untreated mice in this thrombosis model developed occlusive thrombi that remained stable for the 30 min duration of the assay, whereas stable thrombi developed in only 32% of mice treated with 1g/kg aspirin orally and in 41% of mice receiving 60 mg/kg CSW4-161intravenously. In summary, we have developed a class of potent inhibitors of the integrin α2β1 that demonstrate both in vitro and in vivo anti-platelet activity. Further development of this class of compounds may result in novel and relatively non-toxic anti-thrombotic agents.

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