Discovery and Evaluation of Anti-Fibrinolytic Plasmin Inhibitors Derived from 5‑(4-Piperidyl)isoxazol-3-ol (4-PIOL)

Inhibition of plasmin has been found to effectively reduce fibrinolysis and to avoid hemorrhage. This can be achieved by addressing its kringle 1 domain with the known drug and lysine analogue tranexamic acid. Guided by shape similarities toward a previously discovered lead compound, 5-(4-piperidyl)­isoxazol-3-ol, a set of 16 structurally similar compounds was assembled and investigated. Successfully, <i>in vitro</i> measurements revealed one compound, 5-(4-piperidyl)­isothiazol-3-ol, superior in potency compared to the initial lead. Furthermore, a strikingly high correlation (R² = 0.93) between anti-fibrinolytic activity and kringle 1 binding affinity provided strong support for the hypothesized inhibition mechanism, as well as revealing opportunities to fine-tune biological effects through minor structural modifications. Several different ligand-based (Freeform, shape, and electrostatic-based similarities) and structure-based methods (e.g., Posit, MM/GBSA, FEP+) were used to retrospectively predict the binding affinities. A combined method, molecular alignment using Posit and scoring with <i>T</i>_combo, lead to the highest coefficient of determination (R² = 0.6)

Discovery and Evaluation of Anti-Fibrinolytic Plasmin Inhibitors Derived from 5‑(4-Piperidyl)isoxazol-3-ol (4-PIOL)

Inhibition of plasmin has been found to effectively reduce fibrinolysis and to avoid hemorrhage. This can be achieved by addressing its kringle 1 domain with the known drug and lysine analogue tranexamic acid. Guided by shape similarities toward a previously discovered lead compound, 5-(4-piperidyl)­isoxazol-3-ol, a set of 16 structurally similar compounds was assembled and investigated. Successfully, <i>in vitro</i> measurements revealed one compound, 5-(4-piperidyl)­isothiazol-3-ol, superior in potency compared to the initial lead. Furthermore, a strikingly high correlation (R² = 0.93) between anti-fibrinolytic activity and kringle 1 binding affinity provided strong support for the hypothesized inhibition mechanism, as well as revealing opportunities to fine-tune biological effects through minor structural modifications. Several different ligand-based (Freeform, shape, and electrostatic-based similarities) and structure-based methods (e.g., Posit, MM/GBSA, FEP+) were used to retrospectively predict the binding affinities. A combined method, molecular alignment using Posit and scoring with <i>T</i>_combo, lead to the highest coefficient of determination (R² = 0.6)

Changes in cortical network activity induced by 7–8 cumulatively increasing concentrations of THIP in the absence and in the presence of 300 nM, 1 μM or 3 μM DS2.

<p>The heat maps to the left of the vertical hatched line present statistically significant changes in 40 activity parameters relative to native activity (no drug, 100%) (Student’s paired t-test, p≤0.05). The concentration-response relationships for the drug combinations at 10 selected activity parameters are given as mean ± S.E.M. relative to native activity (no drug, 100%). The heat maps to the right of the vertical hatched line present statistically significant changes in the 40 activity parameters between the activity induced by a specific THIP concentration co-applied with 300 nM, 1 μM or 3 μM DS2 relative to the activity induced by that specific THIP concentration on its own (100%) (Student’s paired t-test, p≤0.05).</p

Changes in cortical network activity induced by 7–9 cumulatively increasing concentrations of the functionally selective δ-GABA_AR agonists THIP and Thio-THIP.

<p>The heat maps present statistically significant changes in 40 activity parameters relative to native activity (no drug, 100%) (Student’s paired t-test, p≤0.05). The concentration-response relationships for the drugs at 10 selected activity parameters are given as mean ± S.E.M. relative to native activity (no drug, 100%).</p

Functional characterization of GABA_A receptor-mediated modulation of cortical neuron network activity in microelectrode array recordings

<div><p>The numerous γ-aminobutyric acid type A receptor (GABA_AR) subtypes are differentially expressed and mediate distinct functions at neuronal level. In this study we have investigated GABA_AR-mediated modulation of the spontaneous activity patterns of primary neuronal networks from murine frontal cortex by characterizing the effects induced by a wide selection of pharmacological tools at a plethora of activity parameters in microelectrode array (MEA) recordings. The basic characteristics of the primary cortical neurons used in the recordings were studied in some detail, and the expression levels of various GABA_AR subunits were investigated by western blotting and RT-qPCR. In the MEA recordings, the pan-GABA_AR agonist muscimol and the GABA_BR agonist baclofen were observed to mediate phenotypically distinct changes in cortical network activity. Selective augmentation of αβγ GABA_AR signaling by diazepam and of δ-containing GABA_AR (δ-GABA_AR) signaling by DS1 produced pronounced changes in the majority of the activity parameters, both drugs mediating similar patterns of activity changes as muscimol. The apparent importance of δ-GABA_AR signaling for network activity was largely corroborated by the effects induced by the functionally selective δ-GABA_AR agonists THIP and Thio-THIP, whereas the δ-GABA_AR selective potentiator DS2 only mediated modest effects on network activity, even when co-applied with low THIP concentrations. Interestingly, diazepam exhibited dramatically right-shifted concentration-response relationships at many of the activity parameters when co-applied with a trace concentration of DS1 compared to when applied alone. In contrast, the potencies and efficacies displayed by DS1 at the networks were not substantially altered by the concomitant presence of diazepam. In conclusion, the holistic nature of the information extractable from the MEA recordings offers interesting insights into the contributions of various GABA_AR subtypes/subgroups to cortical network activity and the putative functional interplay between these receptors in these neurons.</p></div

Changes in cortical network activity induced by 8–9 cumulatively increasing concentrations of the δ-GABA_AR-selective modulators DS1 and DS2.

<p>The heat maps present statistically significant changes in 40 activity parameters relative to native activity (no drug, 100%) (Student’s paired t-test, p≤0.05). The concentration-response relationships for the drugs at 10 selected activity parameters are given as mean ± S.E.M. relative to native activity (no drug, 100%).</p

Biochemical characterization of cortical network characteristics and GABA_AR subunit expression in primary cortical neurons and in mouse frontal cortex tissue.

<p><b>A.</b> Immunocytochemistry characterization of the primary cortical neurons (28 DIV). <i>i</i>. Staining with antibodies for synapsin-1 (<i>green</i>), β3-tubulin (<i>red</i>) and Hoechst-Bisbenzimid (<i>blue</i>). <i>ii</i>. Neurons. <i>iii</i>. Isolated synaptic punctae. <i>iv</i>. Nuclei. <b>B.</b> Immunocytochemistry characterization of α₁ GABA_AR protein expression in the primary cortical neurons (28 DIV). Staining for α₁ (<i>red</i>), β3-tubulin (<i>green</i>) and Hoechst-Bisbenzimid (<i>blue</i>). <b>C.</b> Western blot analysis of α₁, α₃ and α₅ GABA_AR protein expression levels in the primary neurons (7, 14, 21, 28 and 35 DIV). <b>D.</b> Western blot analysis of α₁ and α₃ protein expression levels in the primary neurons (7, 14, 21 and 28 DIV) and in postnatal mouse frontal cortex (7, 14, 21 and 28 days postnatally). <b>E.</b> RT-qPCR analysis of α₄, β₁, β₂, β₃ and δ mRNA expression levels (± S.D.) in the primary neurons and in postnatal mouse cortex tissue (relative to the expression of the reference gene RPL13a).</p

Changes in cortical network activity induced by 8–9 cumulatively increasing concentrations of DS1 in the absence and in the presence of 50 nM diazepam.

<p>The heat maps to the left of the vertical hatched line present statistically significant changes in 40 activity parameters relative to native activity (no drug, 100%) (Student’s paired t-test, p≤0.05). The concentration-response relationships for the drug combinations at 10 selected activity parameters are given as mean ± S.E.M. relative to native activity (no drug, 100%). The heat maps to the right of the vertical hatched line present statistically significant changes in the 60 activity parameters between the activity induced by a specific DS1 concentration co-applied with 50 nM diazepam relative to the activity induced by 50 nM diazepam on its own (100%) or relative to the activity induced by that specific DS1 concentration on its own (100%) (Student’s paired t-test, p≤0.05).</p

Sequences of primers used in the RT-qPCR.

<p>Sequences of primers used in the RT-qPCR.</p

Changes in cortical network activity induced by 8–9 cumulatively increasing concentrations of diazepam in the absence and in the presence of 30 nM DS1.

<p>The heat maps to the left of the vertical hatched line present statistically significant changes in 40 activity parameters relative to native activity (no drug, 100%) (Student’s paired t-test, p≤0.05). The concentration-response relationships for the drug combinations at 10 selected activity parameters are given as mean ± S.E.M. relative to native activity (no drug, 100%). The heat maps to the right of the vertical hatched line present statistically significant changes in the 40 activity parameters between the activity induced by a specific diazepam concentration co-applied with 30 nM DS1 relative to the activity induced by 30 nM DS1 on its own (100%) or relative to the activity induced by that specific diazepam concentration on its own (100%) (Student’s paired t-test, p≤0.05).</p