BPS2110 lecture 3-2014

Drug discovery and development

1. Natural product leads and characteristics

2. Case study of drug development: artemesisn

3. Anticancer drugs and biologics

Are natural products still viable sources of New Chemical Entities today?

• Despite growth of new products from combinatorial chemistry and “biologics” derived from molecular biology, 68% of NCEs in last 2 decades derived from or inspired by natural products.

• However, more drugs are now semisynthetic or synthetic mimics derived by medicinal chemistry

• Why? – Natural products evolved as natural defenses in plants, marine and microorganisms. Almost every biological activity has been invented in nature

– Natural products are small MW compounds that have drug like properties as defined in Lipinski’s rules. They can cross membranes and reach site of action where large MW biologics cannot reach

Characteristics of drug like molecules

( J. Nat. Prod. 2008, 71, 464–468)Lipinski’s rules:

To be drug-like, a candidate should• have less than five hydrogen bond donors (HBD),

• less than 10 hydrogen bond acceptors (HBA),

• a molecular weight of less than 500 Da,

• and a log partition coefficient (log P) of less than 5.( P= C octanol / Cwater )

Looking at 126 140 uniqueNatural compounds in The Dictionary of Natural Products 65% obeyLipinski’s rules

Drug development

WHO Drug development: antimalarial from a traditional medicine

Classic ethnobotanical discovery starting with traditionally used plant for treatment of fevers like china. Treatment for feavers. Started being used as malarial drug. It’s the only thing that works against malarial resistant compounds.

• bioassay• phytochemical isolation of active principle• animal trial• pharmacokinetics and toxicity studies• clinical trial ( phase I, II and III)• analoging and derivatives (QSAR)• recent worldwide distribution: effective against drug resistant cerebral malaria

Artemesia annuaSweet Annie

Sweet wormwood Annual wormwood

Qinghao

artemisinin

Improvement by medicinal chemistry

• Semisynthesis lead to optimized derivatives artemether or artesunate which has better pharmacokinetics (drug absorbtion), dihyroartemisinin is human metabolite : chemist have made arthemeter.

• .

Pharmacokinetics:

Triangles: artesunate conc, circles: dihydroartemsinin, sqaures: malaria paraisite Time (h) after drug administration

Conc(nmole/L)

Clinical trial in humans

Case: Anticancer drugs Current drugs• Podophyllotoxin • Vincristine• TaxolNewer approaches• Betulinic acid• Perveilline A• TIC10• Herceptin

May apple Podophyllum peltatum a native Canadian medicinal plant

• Used traditionally by Iroquois for treatment of stomach disorders & warts, but not for cancer

• Contains podophyllotoxin, a lignan with potent anticancer activity

• Phynolic compound. • Canadian plant produce ½% of drug.

• Very effect cancer agent. • Current commercial source is Indian Podophyllum emodi,

Anticancer activity of podophyllotoxin and etoposide

(Phytochemistry 54: 115-120.)• Podophyllotoxin has antineoplastic and antiviral activity

• derivative etoposide is a topisomerase leading to apoptosis (programmed cell death) of cancer cells

• used in combination therapy for testicular leukemia, stomach, ovarian, brain, breast, pancreatic and lung cancer

Cantheranthus roseus, Madagascar periwinkle

• Source of major 2 antileukemia drugs vincristine and vinblastine

• Identified through antidiabetic study in Canada at McMaster University which showed reduction in white blood cell counts

• Swoboda at Eli Lilly showed toxicity to leukemia cells and developed it as an antileukemia drug

Vinblastine is a complicated drug resulting from elaborate biosynthesis

2. Drug discovery strategies

• NCI screening • High throughput screening• Overcoming resistance• Finding less toxic treatments

National Cancer Institute (US)

• National Cancer Institute is one of the most successful drug program with a large biopospecting component

• They have produced 12 clinically tested drugs, several of which are major drugs

• (taxol, camptothecin etc with enormous value)

• Their success is due to:– Long term commitment (40years) due to public funding rather than short term venture capital

– Host country agreements– Public agency did not fall victim to “biopiracy”

National Cancer Institutelargest drug discovery program >108,000 extracts screened

4149 active extracts

2000 compounds isolated

95 special

11 clinical trialsCompounds: taxol, camptothecin, maytensine, homoharringtonin,bouvardin etc.

Random screening

NCI approach• Plants/ microorganisms/marine organisms are collected under agreement with host country

• For plants 1 kg is collected and dried• Sample is extracted in MeOH:dichloromethane (1:1), then partitioned with water to give a lpohilic and hydrophilic extract

• Prescreen with 3 cancer lines: lung CNS and breast

• Full screen with 60 cell lines for growth inhibition and toxicity.

NCI cell panel for anticancer screenings

• Taxol discovered by NCI

• Screening program headed by Monroe Wall discovered the anticancer drug taxol in Taxus brevifolia, the Pacific Yew

• Taxol prevents tubulin assembly • It has been found in clinical trials to be a very successful treatment for breast and ovarian cancer: widely used

Taxol is another complex biosynthetically derived NP

Analysis of taxol development shows 1) thousands of plants screened to discover one pharma drug

2) time line to clinical use: 30 years

Public funding through national cancer institute programsmade this drug possible. Venture capital cannot wait this long

Camptothecin, NCI anticancercompound from Camptotheca, of Chinese origin (also commonly used)

NN

OHO

CH 3O

O

CAM PTOTHECIN

Pervilleine A: Resistance modulator in cancer

J. Nat. Prod. 2011, 74, 1539–1555

• After several rounds of cancer therapy, tumors become drug resistant. (Mdr genotype)

• Potent selection on cells surviving due to increased expression of pgp, a natural toxin pump

• Requires 2 ATPs, no metabolism occurs

Some new approaches in cancer

Pervilleine A from Erythroxylon pervillei from Madagascar

• Inhibits pgp pump

• It is not a cytotoxic anticancer drug

• It restores sensitivity to vinblastine in MDR cancers

• Now slated for rapid development

Pisha et al., Nature medicine 1: 1046-10514. Antimelanoma drug in clinical development

Screening of plants for plants for antimelanoma activity led to discovery of activity in Ziziphus mauritania (fr Zimbabwe)

Whole plant

Hexane extract no activity

EtOAc extract active

Water extract inactive

10 fractions fraction #3

active

Related Compound inbirch

Columnchromatography

Betulinic acid has specific anticancer activity only with melanoma

Tumor developmentIn Mice injected withHuman melanoma

controlBA @ 50-500 mg/kg

controlDTIC

BA@ 250-500 mg/kg

Start 50mg/kg of BAControl group

5- 250 mg/kg

Development of DNA laddering (50 kb bands), a marker forApoptosis (programmed cell death) in melanoma cells

2 ug /mL of BA

High throughput screening of approach in “big pharma”

• Most major pharma companies use robotic screening of libraries of extracts and pure compounds (both natural and synthetic) in a battery of relevant assays.

• 100,000 -500,000 samples can be screened in a week using a data handling system and robotics

• Rate of screening is usually limited by cost of consumables and limited amount of compounds available

Typical high throughput assays

• Usually microplate in vitro assays with 384 wells

• Commercial libraries of thousands of natural and synthetic derivatives are available for screening new targets

• TIC10 is a compound discovered by this method

Trail is an endogenous anticancerprotein

• Trail is Tumour necrosis factor Related Apoptosis Inducing Ligand

• Kills tumours by upregualting TNFα• Natural immunity mechanism of cancer supression

• Lost during tumour progression• Upregulation of Trail could reduce tumours• Promising alternative to cytotoxic anticancer agents

• Inducing TRAIL with a small molecule could be easier than treatment with recombinant protein

TIC10 reported by NCI to induce Trail

TIC10

Caspase 3 (green) induced by TIC10Caspase is an enzyme involved in programmed

cell death

TIC 10 reduces xenograph tumours in mice

TIC10 is a library screening compound: Original proposed structure (linear) was made synthetically and was inactive, then

angular compound was made and found to be the correct active structure.

Herceptin, a monoclonal antibody for HER2 breast cancer

• HER receptors are proteins that are embedded in the cell membrane. In certain types of breast cancer HER receptors are overexpressed and cause cell proliferation

• A monoclonal antibody was made to the HER receptors by using the HER receptor as antigen

• The antibody, called herceptin is an effective non toxic anticancer agent, but must be used intravienously

• This is an example of personalized medicine made possible by human genome project: Herceptin is highly specific to one cancer while taxol inhibits many cancers

• Other cancer antibodies have been developed but are less effective than herceptin