Module 2 Lecture 3: Pain Research

Question 1

Front: What are the challenges of using Gabapentinoids and Opioids in treating chronic neuropathic pain?

Answer 1

Gabapentinoids: Partially effective in only a small proportion of patients.
Opioids: Often ineffective; can lead to drug addiction and potential overdoses.

Question 2

Front: What is the role of descending noradrenergic and serotonergic inhibitory fibers in pain modulation?

Answer 2

Back: These fibers inhibit pain by modulating signals at the dorsal horn of the spinal cord, reducing the transmission of pain signals to the brain. Pharmacological agents like opioids, α2 agonists, TCAs, and SSRIs can enhance this inhibition.

Question 3

Front: Explain the Gate Control Theory and the role of glycinergic neurons.

Answer 3

Gate Control Theory: Pain perception is modulated at the spinal cord level by the relative activity of different types of nerve fibers.

Glycinergic Neurons: Inhibitory neurons that help close the “gate” by inhibiting pain signals (especially from C-fibers). When large fiber input (Aβ fibers) is high, the gate closes, reducing pain perception.

Question 4

Front: How does glycine neurotransmission change in chronic pain conditions?

Answer 4

Chronic Pain: Increased excitation and loss of glycinergic inhibition lead to enhanced pain transmission.

Restored Normal Sensations: Drugs that enhance glycinergic signaling can restore inhibition and reduce chronic pain.

Question 5

Front: How does glycine function in normal sensation versus neuropathic pain in the dorsal horn?

Answer 5

Normal Sensation: Glycine inhibits signals from entering the pain pathway, maintaining normal sensation.
Neuropathic Pain: There’s a reduction in glycinergic neurotransmission, allowing more pain signals to pass to the brain, contributing to chronic pain.

Question 6

Front: Describe the dual role of glycine as a neurotransmitter.

Answer 6

Inhibitory Role: Glycine inhibits excitatory signals at inhibitory synapses.
Excitatory Role: Glycine can also act as an excitatory neurotransmitter in certain synapses, balancing excitation and inhibition.

Question 7

Front: What is GlyT2, and how can its inhibition help in neuropathic pain?

Answer 7

GlyT2: A transporter that reabsorbs glycine from the synaptic cleft, reducing its availability.
Inhibition of GlyT2: Leads to increased glycine levels in inhibitory synapses, enhancing glycinergic neurotransmission, which can reduce neuropathic pain.

Question 8

Front: How is the activity of glycine transport inhibitors measured experimentally?

Answer 8

Procedure:
Oocytes from Xenopus laevis are extracted and prepared.
cRNA is injected to express the protein of interest.
Electrophysiological measurements are taken using a two-electrode voltage clamp (TEVC) to assess the impact of glycine transport inhibitors.

Question 9

Front: What is the role of N-arachidonoyl-glycine in neuropathic pain, and how does it exert its effects?

Answer 9

N-arachidonoyl-glycine: An endogenous compound derived from arachidonic acid that provides analgesia in neuropathic pain.
Mechanism: Inhibits GlyT2, stimulates GlyRs, and modulates various ion channels, though the exact mechanisms are not fully understood.

Question 10

Front: How is glycine transport measured, and how do inhibitors affect this process?

Answer 10

Measurement: Glycine transport generates an inward current.
Effect of Inhibitors: Adding increasing concentrations of inhibitors reduces the amount of glycine transport, as seen by a reduction in inward current. This is represented by a decrease in I/Imax in concentration-response curves.

Question 11

Front: What are the key structural features of N-arachidonoyl-glycine, and how were they optimized in drug design?

Answer 11

N-arachidonoyl-glycine: A non-selective, unstable compound with an IC50 of ~10 µM.
Optimization: Modifying the structure led to the development of Oleoyl-D-lysine, which is selective for GlyT2, stable, crosses the blood-brain barrier (BBB), and has an IC50 of ~25 nM.

Question 12

Front: What were the findings from testing Oleoyl-D-Lysine in a rat model of neuropathic pain?

Answer 12

Findings:
The maximal dose of Oleoyl-D-Lysine reduced allodynia for up to 6 hours.
It caused marginal side effects compared to ORG25543, another tested compound.

Question 13

Front: What are the limitations of Oleoyl-D-Lysine in drug design?

Answer 13

Limitations:
Oleoyl-D-Lysine readily crosses the BBB and remains stable for at least 24 hours.
However, it remains in the CNS at high concentrations well after analgesia wears off.
It has very high brain binding and very low bioavailability.

Question 14

Front: How was the drug binding site on GlyT2 identified, and what does it entail?

Answer 14

Identification: Through a combination of site-directed mutagenesis and molecular dynamics simulations.
Binding Site: The identified site is critical for the binding of inhibitory drugs to GlyT2, allowing for targeted drug design.

Question 15

Front: What advances have been made in developing second-generation GlyT2 inhibitors?

Answer 15

Advances:
Discovery of an extracellular allosteric lipid binding site on GlyT2.
This site is used to identify new drugs that can bind and inhibit GlyT2.
Machine learning models screened 20 million compounds, identifying the top 100 hits, with 4 new drugs being identified as effective inhibitors.

Question 16

Front: How does N-arachidonoyl-glycine modulate glycine receptors, and what was discovered through analog screening?

Answer 16

N-arachidonoyl-glycine: Stimulates the activity of glycine receptors.
Screening: Around 70 lipid analogs were screened for their ability to stimulate glycine receptors, leading to the identification of OPOG as a potent positive allosteric modulator.

Question 17

Front: Where do allosteric modulators bind on glycine receptors?

Answer 17

Binding Sites: Multiple drug binding sites have been identified on the glycine receptor, including the Ivermectin site, which is crucial for modulating receptor activity.

Question 18

Front: What is the effect of OPOG on glycine receptors, and how does it influence glycine potency?

Answer 18

Effect of OPOG: OPOG enhances glycine receptor activity, shifting the glycine dose-response curve to the left, making glycine more potent.
Experiment: Increasing concentrations of OPOG with 5 µM glycine showed a dose-dependent enhancement of receptor activation.

Question 19

Front: How does OPOG interact with glycine receptors, and what is its binding site?

Answer 19

Binding Interaction: OPOG binds at the Ivermectin site on glycine receptors.
Mechanism: It binds at the interface between subunits to stimulate the opening of the ion channel.

Question 20

Front: What are the challenges with OPOG, and what are the strategies for developing next-generation modulators?

Answer 20

Challenges with OPOG: It has high non-specific brain binding and low bioavailability.
Strategy: Development of drug-like molecules that target the same binding sites but with better pharmacokinetic profiles to ensure effectiveness and safety.

Question 21

Front: Summarize the key points about glycine neurotransmission and its modulation in neuropathic pain treatment.

Answer 21

Current treatments for neuropathic pain are often inadequate with significant side effects.
Glycine neurotransmission in the dorsal horn is disrupted in neuropathic pain.
Restoration of glycine neurotransmission is a potential therapeutic target.
Restoration can be achieved by inhibiting GlyT2 or stimulating glycine receptors.
Novel inhibitors and positive allosteric modulators of glycine receptors offer new avenues for analgesic development.

Question 22

Front: What are the limitations of current treatments for neuropathic pain?

Answer 22

Current treatments often have inadequate efficacy.
They can lead to considerable side effects, including drug dependency and tolerance.
There is a need for more targeted and effective therapies with fewer adverse effects.

Question 23

Front: How are neural pathways in the dorsal horn disrupted in neuropathic pain?

Answer 23

In neuropathic pain, glycine neurotransmission in the dorsal horn is impaired.
This disruption leads to decreased inhibitory signaling, resulting in increased pain transmission.
Loss of glycinergic inhibition contributes to chronic pain states.

Question 24

Front: What strategies can be employed to restore glycine neurotransmission?

Answer 24

Inhibiting GlyT2: Prevents the reuptake of glycine, increasing its availability at inhibitory synapses.
Stimulating Glycine Receptors: Enhances inhibitory neurotransmission, reducing pain signals.

Question 25

Front: What is the rationale for using glycine transport inhibitors and receptor stimulators in the treatment of neuropathic pain?

Answer 25

Glycine Transport Inhibitors: By inhibiting GlyT2, these drugs increase synaptic glycine levels, enhancing inhibitory signaling in the dorsal horn.
Glycine Receptor Stimulators: These drugs increase the efficacy of glycine at its receptors, further boosting inhibitory transmission and alleviating pain.