Chapter 2 Drug Discovery,Design,and Development 12 CH diazepam 2.7 The initial difficulty arises in the discovery of the lead compound.Several approaches can be taken to identify a lead.The first requirement for all of the approaches is to have a means to assay compounds for a particular biological activity,so that researchers can tell when a compound is active.A bioassay (or screen)is a means of determining in a biological system,relative to a control compound,if a compound has the desired activity. and,if so.what the relative potency of the compound is.Note the distinction between the terms activiry and porency.Activiry is the particular biological or pharmacological effect (for example,antibacterial activity or anticonvulsant activity):potency is the strength of that effect. Some screens are in vitro tests,for example,the inhibition of an enzyme or antagonism of a receptor;others are in vivo tests,for example,the ability of the compound to prevent an induced seizure in a mouse.In general,the in vitro tests are quicker and less expen- sive.Currently,high-throughput screens(HTS)very rapid and sensitive in vitro screens initially developed about 1989-1991,that now can be carried out robotically in 1536-or 3456-well titer plates on small(submicrogram)amounts of compound(dissolved in submi- croliter volumes)are becoming universally used.With these ultra-high-throughput screening approaches,it is possible to screen 100,000 compounds in a day!As we will see below, combinatorial chemistry(see Section 2.2.E.5,p.34)can supply huge numbers of compounds in a short period of time,which,theoretically,should provide an increased number of hits, i.e.,compounds that elicit a predetermined level of activity in the bioassay and.therefore, provide more leads.According to Drews,2 the number of compounds assayed in a large pharmaceutical company in the early 1990s was about 200,000 a year:that number rose to 5-6 million during the mid-1990s,and by the end of the 1990s it was>50 million!How- ever,the increase in the assay rate did not result in a commensurate increase in research productivity,as measured by new compounds entering the market.Of course,it can take 12-15 years for a drug to reach the market,so productivity in the early part of the 21st century should provide a more accurate ruler for success of drug discovery changes made at the end of the 20th century.Currently.HTS appears to have resulted in an increase in the number of hits,but this may be because more lipophilic compounds,which may have more drug-like properties (see Section 2.2.F.2,p.53),can be tested by dissolving them in dimethylsulfoxide DMSO)rather than in water.Nonetheless,it is not yet clear if this increase in hit rate is translating into a much greater number of leads and development compounds.[13] An exciting approach for screening compounds that might interact with an enzyme in a metabolic pathway was demonstrated by Wong.Pompliano,and coworkers for the discov- ery of lead compounds that block bacterial cell wall biosynthesis(as potential antibacterial agents)Conditions were found to reconstitute all six enzymes in the cell wall biosynthetic
Section 2.1 Drug Discovery 13 pathway so that incubation with the substrate for the first enzyme leads to the formation of the product of the last enzyme in the pathway.Then by screening compounds and looking for the buildup of an intermediate,it is possible to identify not only compounds that block the pathway(and prevent the formation of the bacterial cell wall),but to determine which enzyme is blocked(the buildup of an intermediate means that the enzyme that acted on that intermediate was blocked). Compound screening also can be carried out by electrospray ionization mass spectro- metryl15](the technique for which John Fenn received the Nobel prize in 2002)and by NMR spectrometry.16 Tightly bound noncovalent complexes of compounds with a macromolecule (such as a receptor or enzyme)can be observed in the mass spectrum.The affinity of the ligand (a small molecule that binds to a receptor)can be measured by varying the collision energy and determining at what energy the complex dissociates.This method also can be used to screen mixtures(a library)of compounds,provided they have different molecular masses and/or charges,so the m/z for each complex with the biomolecule can be separated in the mass spectrometer.By varying the collision energy,it is possible to determine which test molecules bind to the biomolecule best.TheHNMR method exploits changes in either relaxation rates or diffusion rates of small molecules when they bind to a macromolecule. This method also can be used to screen mixtures of compounds to determine the ones that bind best. Once the screen is developed,a variety of approaches can be taken to obtain a lead.As we will see below,the typical lead compound for a receptor or enzyme is the natural ligand for the receptor or substrate for the enzyme.Another good source of lead compounds is marketed drugs.17 In this case the target will generally be wellestablished,and the lead structure will be known to bind well to the target and to have good absorption properties.The main stumbling block to the use of marketed drugs as leads may be patent issues for commercialization.If the target macromolecule is not known or if no new leads have come from a marketed drug, other approaches can taken. B.1 Random Screening In the absence of known drugs and other compounds with desired activity,a random screen is a valuable approach.Random screening involves no intellectualization:all compounds are tested in the bioassay without regard to their structures.Prior to 1935(the discovery of sulfa drugs),this was essentially the only approach;today this method is still an important approach to discover drugs or leads,particularly because it is now possible to screen such huge numbers of compounds rapidly with HTSs.This is the lead discovery method of choice when nothing is known about the receptor target. The two major classes of materials screened are synthetic chemicals and natural products (microbial,plant,and marine).An example of a random screen of synthetic and natural compounds was the"war on cancer"declared by Congress and the National Cancer Institute in the early 1970s.Any new compound submitted was screened in a mouse tumor bioassay. Few new anticancer drugs resulted from that screen,but many known anticancer drugs also did not show activity in the screen used,so a new set of screens was devised that gave more consistent results.In the 1940s and 1950s,a random screen of soil samples by various pharmaceutical companies in search of new antibiotics was undertaken.However,in this case, not only were numerous leads uncovered,but two important antibiotics,streptomyein and the
14 Chapter 2 Drug Discovery,Design,and Development tetracyclines,were found.Screening of microbial broths.particular strains of Streptomyces. was a common random screen methodology prior to 1980. B.2 Nonrandom(or Targeted or Focused)Screening Nonrandom screening,also called targeted or focused screening,is a more narrow approach than is random screening.In this case,compounds having a vague resemblance to weakly active compounds uncovered in a random screen.or compounds containing different functional groups than leads,may be tested selectively.By the late 1970s,the National Cancer Institute's random screen was modified to a nonrandom screen because of budgetary and man- power restrictions.Also,the single tumor screen was changed to a variety of tumor screens because it was realized that cancer is not just a single disease. B.3 Drug Metabolism Studies During drug metabolism studies(see Chapter 7)metabolires(drug degradation products gen- erated in vivo)that are isolated are screened to determine if the activity observed is derived from the drug candidate or from a metabolite.For example,the anti-inflammatory drug sulin- dac (2.8.Clinoril)is not the active agent:the metabolic reduction product,2.9.is responsible for the activity COOH COOH CH sulindac 2.8 2.9 The nonsedating antihistamine terfenadine hydrochloride(2.10,Seldane)was found to cause an abnormal heart rhythm in some users who also were taking certain antifungal agents, which were found to block the enzyme that metabolizes terfenadine.This caused a buildup of terfenadine,which led to the abnormal heart rhythms.However,a metabolite of terfenadine, fexofenadine hydrochloride(2.11.Allegra),was also found to be a nonsedating antihistamine. but it can be metabolized even in the presence of antifungal agents.This,then,is a safer drug. Metabolites can be screened for other activities as well. CHy CH Ph HCI CHy HO OH terfenadine HCI 2.10
Section 2.1 Drug Discovery 15 CH: CO0H CH Ph HCI HO- OH fexofenadine HCI 2.11 B.4 Clinical Observations Sometimes a drug candidate during clinical trials will exhibit more than one pharmacological activity;that is,it may produce a side effect.This compound,then,can be used as a lead (or. with luck,as a drug)for the secondary activity.In 1947 an antihistamine,dimenhydrinate (2.12.Dramamine)was tested at the allergy clinic at Johns Hopkins University and was found also to be effective in relieving a patient who suffered from car sickness;a further study proved its effectiveness in the treatment of seasickness and airsickness.2 It then became the most widely used drug for the treatment of all forms of motion sickness dimenhydrinate 2.12 There are other popular examples of drugs derived from clinical observations.Bupro- pion hydrochloride(2.13).an antidepressant drug (Wellbutrin),was found to help patients stop smoking and is now the first drug marketed as a smoking cessation aid(Zyban).The impotence drug sildenafil citrate(2.14:Viagra)was designed for the treatment of angina and hypertension by blocking the enzyme phosphodiesterase-5.which hydrolyzes cyclic guano- sine monophosphate(cGMP).a vasodilator that allows increased blood flow 1211 In 1991 sildenafil went into phase I elinical trials for angina.In phase II clinical trials,it was not as effective against angina as Pfizer had hoped,so it went back to phase Iclinical trials to see how high of a dose could be tolerated.It was during that clinical trial that the volunteers reported increased erectile function.Given the weak activity against angina,it was an easy decision to try to determine its effectiveness as the first treatment for erectile dysfunction.Sildenafil works by the mechanism for which it was designed as an antianginal drug,except it inhibits the phosphodiesterase in the penis(phosphodiesterase-5)instead of the heart(Figure 2.1). Sexual stimulation causes release of nitric oxide in the penis. HN HCI bupropion HCI sildenafil citrate 2.13 2.14