Drug Transporters: Pgp and BBB

Lecture 1: Transporters

• Drug efflux transporters, mainly Pgp, are located on the apical membrane of the BBB which prevents substrates from moving from the blood to the brain
• Pgp, an ATP-binding transport barrier, limits the brain's ability to absorb substrates
• It is possible to "tighten" the BBB Pgp transport barrier by increasing the amount of BBB Pgp, for example, by using rifampicin
• The barrier can be circumvented by using inhibitors (ABCB1) to block Pgp function
• Drug efflux transporters like Pgp and Bcrp are located on the apical membrane of small intestine enterocytes, which causes substrates to be pushed back into the lumen
• Pgp and Bcrp transport barriers limit the oral bioavailability of some substrates
• You can enhance the barrier by increasing the amount of Pgp, such as with rifampicin or St. John's wort
• The barrier can be overcome by blocking Pgp or Bcrp function with inhibitors
• Drug-drug interactions at transporters happen through the blocking and inducing of transport barrier proteins
• Transporter mutations can cause disease like the ABCA4 mutation that causes Stargardt (retinal disease) and multidrug resistance (MDR1)
• Efflux transporters can prevent chemotherapy drugs from penetrating tumor cells (ABCB1)
• A gene that has been rearranged, leading to promoter capture of MDR genes like MDR1 or ABCB1 will cause an increase
• Cytotoxicity serves as a proxy for transport, which SLC transporters impacting chemo drugs
• Ways to study transporters include; transwell transport, FRET probe, membrane vesicle, ATPase activity, and transportomics to identify endogenous substrates
• Assessing ligand binding to ABC transporters can be done with affinity probes and CETSA (cellular thermal shift assay), which stabilizes ligand-bound proteins and causes a Tm shift
• Detecting membrane proteins at the surface can be done through biotinylation

Lecture 2: Cell Therapies

• Transferring intact, live cells is cell therapy, which can be autologous (self) or allogenic (donor) with blood transfusions being the most common
• Types of cell therapies include; pluripotent stem cells (PSC), multipotent stem cells (MSC), and immune cell therapy
• PSC = ESC (IVF), iPSC (regenerative med), ntESC (nuclear transfer, regenerative med for immunodeficient), pES (parthenogenetic, source for cell therapies)
• MSC = HSC (hematopoietic, immune therapies), MSC (mesenchymal, tissue repair), NSC (neural, CNS), ESC (epithelial, cornea)
• Hematopoietic stem cell transplant (HSCT) involves transplanting hematopoietic stem cells to create bone marrow
• HLA matching is important for allogenic transplant to allow for graft vs. tumor and to restore the immune system
• Immune cell therapy with T cells has high targeting specificity, good biodistribution, multiple effector, self-amplification and is easy to manipulate
• Chimeric antigen receptor (CAR) consists of an antigen recognition domain, spacer, and endodomain
• An ideal CAR target would have efficient recognition + discrimination from itself, like IL13Ra2 in glioma, m47 scFv ectodomain
• CAR presence can be studied with flow cytometry, and functional studies can assess % lysis, cytokines, survival, and IVIS imaging (T cell persistence)
• To improve T cell persistence, IL15 and STAT5 activation are key
• Roadblocks to T cell therapy include:
• Antigen dilemma: to target multiple antigens look to CARTpool, or Bispecific CARTs
• Homing/penetration: improve delivery routes, overexpress chemokine receptors, use an inducible system expressed in specific tissue, and ECM degrading enzymes
• T cell fitness: use genetic manipulations, delete negative regulators, and overexpress cytokines
• Tumor microenvironment: resistance to inhibit cytokines + suppressive immune cells, adapt T cell metabolism, enhance T cell trafficking to tumor site, boost capacity to penetrate the tumor
• Safety factors include increased specificity, persistence, delivery routes, and chemo drugs to deplete T cells
• Toxicities include: OTOT (on-target off-tumor related toxicities), CRS (cytokine release syndrome), ICANS (immune effector cell associated neurotoxicity syndrome), and TIAN (tumor inflammation associated neurotoxicity), where CRS ICANS TIAN = immediate toxicity can be mitigated
• Long term toxicity is still in progress

Lecture 3: Radiopharmaceuticals

• X-ray and gamma rays have a high frequency and short wavelength, making them highly penetrating to tissue
• Molecular imaging modalities: optical imaging that uses electrons with low energy, x-ray CT, PET/SPECT, MRI, and ultrasound
• Imaging effect depends on what effect it has on the body and the type of tissue penetrance (absorption/loss of signal)
• Essential elements for nuclear/optical molecular imaging are: scanner/imaging (sensitivity, resolution), probe/tracer (penetration), and image reconstruction (quantitative)
• Fermi-Dirac statistics determines the probability of electrons occupying orbital energies
• PET imaging is positron emission tomography
• FDG contains unstable fluorine (18F) on glucose that decays by positron emission, creating a collision and photons which are then picked up by detectors
• Detectors have ring formation for detecting area in space of the positron emission
• Glucose transport is done via GLUT1
• PET reporters are a lock and key mechanism, labeling the molecule that activates the protein/enzyme/receptor
• The tracer principle states that a tracer must blend into the system under study and its behavior must be indistinguishable from the non-radioactive materials with some exceptions
• Radiopharmaceuticals participate in biological processes but do not alter the process
• Radioactive decay is the spontaneous release of energy or subatomic particles from the nucleus of an atom in an attempt to reach a more stable state
• A = atomic mass, Z = atomic number
• Alpha decay occurs in heavier nuclei and results in Helium emission
• Beta decay/electron capture accounts for the change in charge from neutron to proton, ejecting a neutrino and antineutrino
• Positron decay transforms a proton into a neutron, ejecting a positron
• Ideal physical characteristics of radioisotopes: inexpensive to produce using cyclotron or reactor, carrier-free, ideal half life, readily available with a stable daughter
• Molar activity is the amount of radioactivity per unit mass of a radionuclide or labeled compound
• Ideal biological characteristics of radiopharmaceuticals: high target:non-target ratio, ideal biological half-life, good metabolic properties, and synthetic accessibility
• Considers nonspecific binding, Blood-brain barrier permeability, excretion and clearance
• Isotope production is achieved via cyclotron (accelerated protons), linear accelerator, reactor and generator
• Iodine-131 – MIBG is used in detection and treatment of pheochromocytoma and neuroblastoma
• Carbon-11 does not change the chemical, has a 20 min half life, and 11C-choline is used in prostate cancer imaging
• Imaging can help with drug discovery/development, monitor treatment, and make more efficient decisions in the clinic
• The difference between drugs and radiotracers is that drugs modulate biological activity while tracers participate in biological activities
• Radiopharmaceuticals use radiation therapy, where the mechanism of action is direct damage to DNA, from beta and alpha emitters, where alpha is stronger with high energy and induces dsDNA breaks, along with low tissue penetration

Lecture 4: Drugging transcription

• Modes of genome binding are intercalation, minor groove docking, sequence selective binding, and shape selectivity, and can form hydrogen bonds where purines form more narrow minor groove
• Polyamide pairing rules engineer sequence specific genome targeting drugs
• Clustered sites of varying affinities better predict genomic occupancy in vivo
• Proximity determines cell-cell interactions, degradation, and gene expression. It can also induce proximity by targeted protein degradation, gene regulation, and innovative modalities
• Synthetic transcription factors: bind DNA, recruit chromatin modifying, transcription, elongation, or termination machinery
• Synthetic gene regulators, like in Fredricks ataxia with its repeating GAA sequence, cause Polll to not transcribe because of heterochromatin state; remedied by using JG1 to modify polymide on nucleosome to open the chromatin so transcription can occur
• Rewiring cellular processes involves bringing cells together, induce cell death, therapy, and changing cell fates

Lecture 5: Medicinal Chemistry

Lecture 6: Structure based drug discovery