Molecular Mechanism of Disease Course HSS 2305 Lecture Notes PDF

Course HSS 2305 A Molecular Mechanism of Disease Lecture-22 Friday, Dec 1, 2023 Drug Design, Therapeutics and Epigenetics Ajoy Basak, Ph. D. Adjunct and Part-time Professor, Pathology and Laboratory Medicine, Faculty of Medicine, U Ot...

Course HSS 2305 A Molecular Mechanism of Disease Lecture-22 Friday, Dec 1, 2023 Drug Design, Therapeutics and Epigenetics Ajoy Basak, Ph. D. Adjunct and Part-time Professor, Pathology and Laboratory Medicine, Faculty of Medicine, U Ottawa, Roger Guindon Building 451 Smyth Road Ottawa, ON K1H 8M5 Tel 613-878-7043 (Cell) E-mail: [email protected] Alternate: [email protected] Affiliate Investigator, Chronic Disease Program, Ottawa Hospital Research Institute Web: https://med.uottawa.ca/pathology/people/basak-ajoy 1 2 ❖ Drug Design & Therapeutics ❖ Strategies ❖ Various Steps and Stages ❖ Challenges ❖ Various Issues ❖ Approval ❖ Summary ❖ Epigenetics/Epigenome ❖ Definition ❖ Causes ❖ Role in diseases 3 Drug Development Process/Stages ❖ Disease ❖ Understand The Mechanism ❖ Identify Target ❖ Design Molecules For Intervention (In vitro study) ❖ Select The Most Potent Molecule ❖ Design Strategy For Cellular Delivery For Intracellular Target ❖ Demonstrate Efficacy in Cell Culture (Ex vivo work) ❖ Testing In Animal (In vivo Study) ❖ Toxicity Test ❖ Clinical (Human) Trial (4 Phases) ❖ Mode of Administration: Oral, Intravenous Injection, Inhalation or External application ❖ Safety Study / Side Effects (Short and Long Term) ❖ FDA (Food and Drug Administration) Approval  Clinical Trials For Drug Development PHASE 1: ~ 20-100 Subjects (Healthy volunteers or People with Disease Study Length: Several months Purpose: Safety and dose Percent of Drugs going to the next phase: ~70% PHASE 2: ~ Several hundred subjects with the disease Study Length: Several months to 2 years Purpose: Efficacy and Side Effects Percent Drugs moving to the next phase: ~33% PHASE 3: ~300-3,000 volunteers with the disease/condition Study Length: 1-4 years Purpose: Efficacy and Monitoring Adverse Reactions Percentage of Drugs that Move to the Next Phase 25-30% PHASE 4: Several thousand volunteers with Disease/Condition Purpose: Safety and Efficacy From Drug Invention To Market: ~ 12 Years (Average Time) 5  THE RULE of FIVE (Lipinski’s or Pfizer’s): FORMULATION in DRUG DISCOVERY ❖ In 1997 Christopher Lipinski from Pfizer found a simple rule which he called the "Rule of 5" that determines the drug like property of a compound. ❖ It is so called because the parameter cut-off values all contained 5's. ❖ This was a major breakthrough ❖ Describes molecular properties best suited for a drug's pharmacokinetics in the human body that includes parameters like Absorption, Distribution, Metabolism, and Excretion ("ADME"). ❖ Cannot predict if a compound is pharmacologically active. ❖ The Rule states that an orally active drug compound never violates more than one of the following 4 rules: Poor absorption or permeation of a compound occurs when there are: 1) More than 5 H-bond donors 2) The molecular weight is over 500 3) The Log P is over 5 (Lipophillicity Value) 4) The sum of Amine & Hydroxyl groups (N+O) (hydrogen bond acceptors) is over 10. P = Octanol : Water Partition Coefficient  Small Molecule Chemical Entities as Drugs : Sources From January, 1981-October, 2008 (Cragg et al. Chemical Reviews, 2009, Vol. 109, No. 7) S (37%) N: An unmodified Natural product ND: A modified Natural product S: A synthetic compound with no natural product conception S/NM: A synthetic compound showing competitive inhibition of the natural product material (NM) substrate S*: A synthetic compound with a natural product pharmacophore S*/NM: A synthetic compound with a natural product pharmacophore showing competitive inhibition of the natural product substrate  Drug discovery can build on several sources Chemistry and Biology are complementary and co-dependent areas of science, similar to the Chinese concepts of Yin and Yang - one is not present or sufficient without the other. The discovery of new bioactive compounds depends on valid biological assays, while chemistry can make the discovery of new biological targets possible. Medicinal chemistry combines techniques from chemistry and from biology to facilitate new drug discovery. Note: HM=Herbal Medicine  (Anticancer) (Conraceptive pills, Anticancer also) (Antibiotic)  Value of natural products Compounds from natural sources play some significant roles in modern medicine: 1. They provide a number of extremely useful drugs that are difficult, if not impossible, to produce commercially by synthetic means. 2. Natural sources also supply basic compounds that may be modified slightly to render them more effective or less toxic. 3. Some natural products contain compounds that demonstrate little or no activity themselves but which can be modified by chemical or biological methods to produce potent drugs not easily obtained by other methods (Leaves of Taxus Baccata (Yew plant)) or Baccatin III → Taxol One Story of Natural Drug: TAXOL  Taxol Development 1989 Bristol-Myers Squibb (BMS) 1991: Activity observed against metastatic breast cancer 1992: NDA approved by FDA for treatment of refractory ovarian cancer 1994: FDA approval for treatment of refractory breast cancer Now used for Lung and other Cancers Produced by Cell culture methods Major Challenges in Drug Development Process  ❖ Cell Permeability (How to Make a Bioactive Drug Compound Cross Cell Membrane in order to target intracellular molecule ??) ❖ Bio-availability and Stability (How to Make the Drug stable and survive for a long time before degradation in harsh physiological condition ??) 18th APS, July 19-23, Boston, USA 15  List of Common Cell Permeable or Penetrating Peptides (CPP) (They Cross Cell membrane Without The Help of Receptor) Peptides Characteristic Features - HIV Tat-Domain Peptides Highly Basic Peptides (Contain Arg, Lys, His) - Nuclear Localization Signal (NLS) Peptides Highly Basic Peptides - RNA-binding Peptides Basic Peptides - DNA-binding Peptides Basic Peptides - Poly-Arginines 6-8 Arginines (All Dextro-Peptides: Even Better) 23 R12 - Trptophan (W) -rich + Neutral + Basic Domain KETWWETWWTEW-SQP-KKKRKV (Designed Peptide, Most Potent so Far) (PEP-1) (Trp-rich) (Neutral) (Basic) - Pro-and Arg rich Peptides 26) RRIRPRPPRLPRPRPRPLPFPRPG - Poly Lys - dendrimeric Poly Lysm-(X)n m=4-10, n=2-4, X=di, tri or tetra peptides - Pro-rich + a nearby Cys IE (+ Palmitoylation or Other Fatty Acid Chains) Potential Mechanism Of Cell Entry of Cell Penetrating Peptides (CPP) The peptide recruits negatively charged phospholipid (filled circles) and induces the formation of an inverted micelle, the Hydrophilic Cavity of that accommodates the peptide and its cargo that can be released into the cytoplasmic compartment Novel Strategy For Design of Cell Permeable Drugs Using “CPP” Example: ❖ Furin Inhibitors Exhibit Anticancer & Anti-viral Activity in Cell/Animal Models ❖ One Can Attach “CPP” to Furin Inhibitors to Make them Cell Permeable ❖ Using Fluorescent and CPP Labeled Peptide One can monitor cell entry Linker Bioactive Molecule FL Cell Permeable Peptide (Drug) List of Various Fluorescent Molecules (FL) and their Excitation (lex) and Emission wavelengths (lem)  List of cell permeable fluorescent PCSK1- Inhibitory Peptides Attached “CPP” Fluorescent Cell Permeable Peptide Sequence 1 R8 - RRRRRRRR-Ln- VLGALLRVKR 2. Control - Ln- VLGALLRVKR 3. Tat48-64 - GRKKRRQRRRPPQ-Ln- VLGALLRVKR 4. Pep-1 KETWWETWWTEW-SQP-KKKRKV-Ln- VLGALLRVKR = Fluorescein Group, Ln = A linker chain (e-Amino hexanoic acid); R = Dextro-Arginine VLGALLRVKR = (SAAS10): This Peptide sequence was derived from “SAAS” protein and it has been shown to be an inhibitor of PCSK1 enzyme  Cellular uptake of various fluorescent labeled PCSK1 inhibitors in Human Pituitary AtT20 cells Fl-dR8-Ln-SAAS10 Fl-Tat48-64-Ln-SAAS10 Fl-Ln-SAAS10 (Control) Fl: Fluorescent group; dR8: 8 Dextro Arginine containing peptide (A cell penetrating peptide “CPP”), Tat48-64: Peptide from HIV Tat region (Another “CPP”); SAAS10: A 10 Amino Acid Peptide from SAAS protein Which Inhibits PCSK1 activity Note: Cell Nuclei are stained with fluorescence (Left Two Panels) when a “CPP” is attached, Compare with Control (Right), No Nuclei staining with Fluorescence  Effect on POMC processing in AtT20 cells by Cell Penetrating Labeled SAAS peptide. Efficient blocking of POMC processing by PCSK1 as confirmed by Western blot Analysis 0.5 (0.51mM) Pep1-SAAS Control (0.5mM) Cleavage of POMC protein by Pep1-SAAS SASS b-LPH/POMC ratio (1mM) Pep1-SAAS SASS 0.4 Pep1-SAAS PCSK1 leading to the formation (1mM) (10mM) (20mM) 0.3 of ACTH & beta LPH 0.2 0.1 AQRR AE EFKR EL 0 1 2 3 4 5 6 7 POMC kD 50 PCSK1 36 POMC ACTH b-LPH 22 A non-specific band 16 b-LPH Cell permeability of PCSK4 inhibitor in human trophoblast cell line (HTR8/SVneo) at 30 mM concentration following incubation Note: Fluorescent stained nuclei (Left) with “CPP” (R8) derivatives FLuorescyl-Ln-PCSK475-84 FLuorescyl-R8-Ln-PCSK475-84 (control) PCSK475-84 Peptide is an inhibitor of PCSK4 enzyme Cell Impermeable Bioactive Compounds (Drugs) Applications: ❖ Needed when the drug is targeting a molecule on Cell surface or in the Extracellular Space ❖ Will not affect Intracellular Target ❖ Selective Target ❖ Cell Impermeable Furin Inhibitors may find Therapeutic Applications for Intervention of Viral Infections  Fig 1A Schematic presentation of HA proteins of H5N1, H1N1 (less and more virulent types) Cell Impermeable Furin-Inhibitors Can be Deveoped by Attaching Cholyl, 6- Benzyle Guanine, Amino undecanoyl or even Acetyl group Cholyl H 6-O Benzyl guanine (Bg) CH3-CO- NH2-H Acetyl group Amino undecanoyl Remacle et al The Int J Biochem & Cell Biol, 42:987-995, 2010 EPIGENETICS 25 Epigenetics – Ch 11 and Ch12 Learning objectives 26 Debate between Nature and Nurture 27 28 What is Epigenetics ?? 29  What is Epigenetics ?? Causes for Epigenetic Changes 30 What is Epigenetics ?? 31 What is Epigenetics ?? 32 What is Epigenetics ?? 33  What is Epigenetics ?? Morphogenesis is the biological process that causes a cell, tissue or organism 34 to develop its shape Epigenetics : What is the Proof ?? 35 Epigenetics : What is the Proof ?? 36 Epigenetics : What is the Proof ?? 37 Mechanisms of Epigenetics 38 Mechanisms of Epigenetics : DNA methylation 39 Mechanisms of Epigenetics : DNA methylation 40 Mechanisms of Epigenetics : DNA methylation 41 Mechanisms of Epigenetics : DNA methylation 42  DNA methylation It is a crucial part of normal organismal development and cellular differentiation in higher organisms. It involves the introduction of a methyl group to position 5 of CYTOSINE (C) pyrimidine ring or the number 6 nitrogen of the ADENINE (A) purine ring. C and A are 2 of the 4 bases of DNA that can undergo methylation. This modification can be inherited through cell division. DNA methylation stably changes the gene expression pattern in cells such that cells can "remember where they were" or decrease gene expression. 43  DNA modification DNA methylation Occurs mostly at Cytosine 5-position and is a common event. Between 60% -90% of all CpGs (-Cytosine-phosphate-Guanine- site) are methylated in mammals. Methylated-C can spontaneously deaminate to form Thymine over evolutionary time. So CpG may transform into TpG sequence Deamination (over generations) Thymine 44 DNA is complexed with histones to form chromatin. Histones are proteins that the DNA coils around to become more condensed. The chromatin then becomes coiled upon itself, which ultimately forms chromosomes. Illustration of a DNA molecule that is methylated at the two center cytosines 45 (a) DNA Methyl Transferases (DNMT) (For methylation) DNA methylation is carried out by two general classes of enzymatic activities (i) Maintenance methylation (ii) de novo methylation Maintenance methylation activity is necessary to preserve DNA methylation after every cellular DNA replication cycle and the enzyme responsible is DNMT1. It is thought that DNMT3a and DNMT3b are the de novo methyltransferases that set up DNA methylation patterns early in development. (b) DNA Demethylases (removes methyl group) 46  Adenine Methylation N6-Methyladenine (m6A) has been found in DNAs of various eukaryotes (Algae, Fungi, Protozoa, and Higher Plants). Like bacterial DNA, DNAs of these organisms are subject to enzymatic modification (methylation) not only at Cytosine, but also at Adenine bases. There is indirect evidence that Adenine Methylation of the genome occurs in animals as well. The first higher-eukaryotic Adenine DNA N6- methyltransferase was isolated from vacuolar vesicles of wheat coleoptiles The enzyme helps to methylate the first adenine of the sequence TGATCA in single- and double-stranded DNAs 47 Mechanisms of Epigenetics : Histone modification 48 Mechanisms of Epigenetics : Histone modification 49 Mechanisms of Epigenetics : Histone modification 50 Mechanisms of Epigenetics : Long non-coding RNAS 51  Clinical consequences of epigenetic errors (DNA methylation and Histone acetylation) Following fertilization, the paternal genome undergoes rapid DNA demethylation and histone modifications. The maternal genome is demethylated gradually, and eventually a new wave of embryonic methylation is initiated that establishes the blueprint for the tissues of the developing embryo. As a result, each cell has its own epigenetic pattern that must be carefully maintained to regulate proper gene expression. Perturbations in patterns of DNA methylation and histone modifications can lead to congenital disorders and multisystem pediatric syndromes or predispose people to acquired disease states such as sporadic cancers and neurodegenerative disorders. 52  Epigenetic Therapies Epigenetic therapies are few, but several are being developed (in clinical trials) or approved for specific cancer types Nucleoside analogues such as azacitidine are incorporated into replicating DNA, inhibit methylation and reactivate previously silenced genes. Azacitidine has been effective in phase I clinical trials in treating myelodysplastic syndrome and leukemias characterized by gene hypermethylation. The antisense oligonucleotide MG98 that downregulates DNMT1 is showing promising results in phase I clinical trials Small molecules such as valproic acid that downregulate HDACs are being used to induce growth arrest and tumor cell death. Combination epigenetic therapies and chemotherapy may be more effective. 53 Since many tumor suppressor genes are silenced by DNA methylation during carcinogenesis, there have been attempts to re- express these genes by inhibiting the DNMTs. 5-Aza-2'- deoxycytidine (Decitabine) is a nucleoside analog that inhibits DNMTs leading to its degradation. However, for decitabine to be active, it must be incorporated into the genome of the cell, which can cause mutations in the daughter cells if the cell does not die. In addition, decitabine is toxic to the bone marrow, which limits the size of its therapeutic window. These pitfalls have led to the development of antisense RNA therapies that target the DNMTs by degrading their mRNAs and preventing their translation. However, it is currently unclear whether targeting DNMT1 alone is sufficient to reactivate tumor suppressor genes silenced by DNA methylation. 54 In Huntington's disease (HD), a fatal neurodegenerative disorder, transcriptional dysregulation is a key pathogenic feature. Histone modifications are altered in multiple cellular and animal models of HD suggesting a potential mechanism for the observed changes in transcriptional levels. In particular, previous work has suggested an important link between decreased histone acetylation, particularly acetylated histone H3 (H3-K9K14-ac), and down-regulated gene expression. 55  DNA methylation It is crucial for normal development & is linked to a no. of key processes. Its abnormality leads to (a) Genomic imprinting (b) X-chromosome inactivation (c) Suppression of repetitive elements (d) Carcinogenesis Methylation of CpG sites within the promoters of genes can lead to their silencing, a feature found in a number of human cancers (for example the silencing of tumor suppresor genes). In contrast, the hypomethylation of CpG sites has been associated with the over- expression of oncogenes within cancer cells 56  Histone acetylation Acetylation of the lysine residues at the N terminus of histone proteins removes positive charges, thereby reducing the affinity between histones and DNA. This makes RNA polymerase and transcription factors easier to access the promoter region. Therefore, in most cases, histone acetylation enhances transcription while histone deacetylation represses transcription. 57 Histone acetylation is catalyzed by histone acetyltransferases (HATs) and histone deacetylation is catalyzed by histone deacetylases (HDs or HDACs). Several different forms of HATs and HDs have been identified. Acetylation occurs on N-terminal tail Lys as well as nucleosome core surface. The source of the acetyl group in histone acetylation is Acetyl-Coenzyme A, and in histone deacetylation the acetyl group is transferred to Coenzyme A. 58 (H-2A/2B/3/4)