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Final Exam Review Part II

• Thermodynamics and Kinetics• Catalysis• Drug Design/Drug Binding

Case Study: HIV Protease

Thermodynamics and Kinetics

A + B C + D

• Thermodynamics: How favorable?ΔG = ΔH – TΔS

ΔG = ΔGº +RT ln (([C][D])/([A][B]))• Kinetics: How fast?

rate = kforward[A][B]Keq = kforward/kreverse

k = e -RT/ΔG‡

kforward

kreverse

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Thermodynamics and Kinetics

• Thermodynamics: ΔG and ΔGº

Thermodynamics and Kinetics

• Kinetics: ΔG‡

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Thermodynamics and Kinetics

• Reaction Energy Diagrams

Thermodynamics and Kinetics

• Transition State:fleeting, nearlyimpossible toobserve directly,local energymaximum

• Intermediate: easierto isolate, localenergy minimum

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Thermodynamics and Kinetics

• Making life happen: thermodynamics

• What about kinetics?

Catalysis

• Lowering ΔG‡

– Proximity and orientation effects

– Nucleophilicity and electrophilicity

– Acid and base catalysis

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Catalysis

Catalysis

• Proximity effects:bringing reactantscloser togetherfacilitates reaction

• Orientation effects:forcing reactants intothe correct positionfacilitates reaction

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Catalysis

• Promixity/orientation effects in HIV Protease

Catalysis

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Catalysis

• Nucleophilicity:

Catalysis

• Electrophilicity:

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Catalysis

• How can a basecatalyze a reaction?

• Nucleophilicity is akinetic parameter

• A better nucleophilereacts faster

• Better nucleophileshave higher pKa ofconjugate acid

Catalysis

• How can an acidcatalyze a reaction?

• Electrophilicity is akinetic parameter

• A better electrophilereacts faster

• Better electrophilesare electron-poor

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Catalysis

• Acid/base catalysis in HIV Protease

Practice Question 1

i. Both kinetics and thermodynamics of the reaction.ii. Only the kinetics of the reaction.iii. Only the thermodynamics of the reaction.iv. Neither the kinetics nor the thermodynamics of the reaction.

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Practice Question 1

i. Both kinetics and thermodynamics of the reaction.ii. Only the kinetics of the reaction.iii. Only the thermodynamics of the reaction.iv. Neither the kinetics nor the thermodynamics of the reaction.

Practice Question 1

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Practice Question 1

Side chain A acts as a proton donor and thus this is an example of acidcatalysis. More specifically, it protonates the leaving group to avoid anunfavorable accumulation of negative charge on the boldfaced oxygenatom.

Practice Question 1

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Practice Question 1

Sidechain B draws a proton away from water, thus converting water into abetter nucleophile. This is an example of base catalysis.

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Drug Design

• Thermodynamics of binding– Enthalpy– Entropy

• Strategies in drug design– Hydrophobic pockets– H-bond complementarity– Transition state mimic– Rigidity– Water displacement

Drug Design

• Enthalpy changes during binding

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Drug Design

• Entropy changes during binding

Drug Design

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Drug Design

• Case study: HIV Protease Inhibitors

Drug Design

• Filling hydrophobic pockets: saquinavir

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Drug Design

• Filling hydrophobic pockets: ritonavir

Drug Design

• H-bond complementarity: saquinavir

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Drug Design

• Transition state analogs: saquinavir andritonavir

Drug Design

• Rigidity: Dupont-Merck inhibitor

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Drug Design

• Water displacement: Dupont-Merckinhibitor

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This group may dislodge the water molecule upon binding, thus increasingentropy of the system, and may also be able to reform a hydrogen bond tothe carbonyl oxygen in the water-binding pocket, making the enthalpy ofprotein-methotrexate interactions more negative.

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It would fit into the space and be able to form two hydrogen bonds (enthalpically favorable)–one between its carbonyl oxygen and the amine hydrogen of methotrexate, and otherbetween its amine hydrogen and the nitrogen of methotrexate in the six-memberedring.

HIV Protease:

Function & Structure

Chemical reactions and energies driving them

How enzymes accelerate chemical reactions

Molecular basis of substrate specificity

HIV Protease Inhibitors

Drug Development

Entropy/enthalpy in small molecule-protein

interactions

Saquinavir & ritonavir

Disease & Evolution

Natural Selection and Evolving host-pathogen

interactions in AIDS

Steady States and Equilibria

Evolution of Central Dogma

Deducing evolution from sequence information

Cancer

Germline and soma

Cell proliferation and Cancer

Epidemiology of Cancer

The Cell Division Cycle

Cell Cycle engine

Cell Cycle checkpoints

Mitosis & Cytoskeleton

Mitosis

Microtubules - chromosome capture & exploration

with selection

Cell Signaling and regulation of Gene Expression

Regulation of gene expression

Growth factors and cell signalling

Cancer: Kinases & Gleevec

CML

Protein Kinases

Kinases important in CML

Src family & regulation of Src family kinases

Abl regulation & Bcr-Abl misregulation

Gleevec Structure, selectivity and mechanism of

action

Le Chatelier’s Principle

Gleevec Resistance

Topics since Exam 2:

Note: the terms deleterious, neutral and beneficialare all relative (to the pathogen or host)

Beneficial to pathogen = Deleterious to host

In HIV, if the mutation is in a gene encoding viralprotein, it can be deleterious, neutral and beneficial(less frequent) to the virus.

A virus with a beneficial mutation has a selectiveadvantage over another virus with a deleteriousmutation. A virus with neutral mutation does as wellas a normal (wt) virus.

Mutations can soon lead to drug-resistant strains

Disease & Evolution

Q: Consider the following situation of bacteria and antibiotic resistance. If a patientis taking an antibiotic to clear an infection and they do not properly take theirmedication, a single bacterial cell can develop a mutation that confers resistance tothis antibiotic.(a) Is this mutation beneficial to the bacterium?Yes

(b) To the patient?No

(c) Can this single bacterium change the outcome of the infection in the patient?Explain.Yes. If the patient continues taking the antibiotic, it would select for theresistant strain of bacteria. Eventually this strain will takeover the bacterialpopulation and the antibiotic will be ineffective against it.

Mutants already exist early in the infection cycle,but at levels of under 3% at the 25th cycle, areundetectable. But beyond this cycle, they takeover.

as the wt

The mutant has reduced sensitivity

to protease inhibitor and reduced catalytic activity.

Q: What happens to the HIV population if protease inhibitorcontinues to be administered?

Selects for the resistant strain as it has a selectiveadvantage over the wt in the presence of proteaseinhibitor.

Q: What happens to the HIV population if protease inhibitoradministration is stopped?

Selects for the wt strain as it has a selective advantageover the resistant in absence of protease inhibitor.

RT makes a DNA strand complementary to theviral RNA sequence. RT is sloppier than otherDNA polymerases.

In normal cells, constitutional isomers, such asG isomer (occurring only 0.1 - 1% of the time),is incorporated into the newly polymerized DNAsequence. This abnormal G base pairs with Tinstead of C. Following replication, DNA strandswill receive either the G (correct) or T(incorrect).

Cells possess 2 defenses against wrong baseincorporation:

1. Proof-reading by DNA Polymerase - checks afterinserting each new base. If it is incorrect, the baseis removed and puts in the correct one.

2. Mismatch repair - incorrect (e.g., G:T) pairingcreates a bulge in DNA helix. The incorrect base isremoved and a correct one put in.

Result:

In HIV, 1 mutation in every 30,000 bases.

In cellular DNA (w/proofreading & mismatch repair),1 mutation in every billion to 1 in 10 billion bases.

HIV’s high rate of mutation makes AIDS such adifficult disease to cure

Probability of HIV strain getting both mutations =

Probability mutation 1 X Probability mutation 2

Combination therapy requires two simultaneousmutations (to counter the therapies) to produceresistance, a very rare event. What is the probability ofthrowing 6 on two throws?

Multiply the number of new cells infected per day by theprobability of both mutations occurring at the same time (9X10-10)to obtain number of newly infected cells

Differences in patients’ response include ability to stick tomedication schedule, toxicity of drugs and ability to metabolizedrugs (varies between individuals due to differences in theirgenomes) etc.

HIV can’t be completely eliminated by combination therapy:

1. The virus already present in host cells cannot bedestroyed

2. Combination therapy only slows new infections (resistantvirus ultimately arise and take over)

In equilibria: No net chemical change, as forward =backward reaction, and no energy is consumed orreleased (∆G=0)

In steady state:

1. Rates of opposing processes equal each other (likein equilibrium)

2. Backward and forward reactions take differentpath (not in equilibrium)

3. Energy input is necessary to keep reactions fromapproaching equilibrium

Initially in primary infection, there are more uninfectedT-cells than HIV (rate of infection >> T-cell birth)

In acute phase, the number of uninfected T-cells falls asmore are infected then uninfected. This leads todecline in rate of infection as uninfected T-cells areharder to find. Eventually a steady state is reached inwhich rate of T-cell production = rate of infection

This is steady state, NOT equilibrium

(the patient will eventually succumb to infectiousdiseases as T-cell numbers are too low)

HIV therapy tries to raise the number of uninfectedT-cells so patients can fight off infectious diseases.By targeting RT and HIV protease, it lowers infectionrate, thereby raising the number of uninfected T-cells. However, this triggers increased T-cell infection(but slower than T-cell are born), establishing a newsteady state. If the patient stops therapy, he/she willreturn to the previous steady state (higher T-cellinfection rate) and succumb to infectious diseases

In equilibrium, the reaction proceeds until the rates offorward and backward reactions are equal. When thishappens, ∆G=0

In steady states, both forward and backward reactionsare coupled to ATP hydrolysis, and free energy isstrongly negative (∆G<0) (i.e., far from equilibrium).Rates of forward and backward reactions are dependenton kinetics and not thermodynamics. Continual inut ofenergy is required.

In biology, many processes need to be maintained withina narrow range compatible with life. The steady state thatis created is often far from its equilibrium and in order tokeep it there, energy is needed. If a biological reaction isallowed to reach equilibrium, it often equals death.

Only this is inequilibrium

(HIV is an anomaly)

Self-splicing RNA do not require proteins to removeintrons. Hence the first life forms may not requireproteins for cellular functions. Also, the fact that theribosome (main function-to join amino acids) is mademainly of RNA, and not protein, supports this theory,that RNA was responsible for everything in theprimitive cell’s environment

Hence, RNA is believed to be the information carrierand catalyst. Self-splicing RNA & RNA active site inribosome are remnants of RNA-only world whichpreceeded DNA

Q: If RNA is so good for storing genetic information,why bother making DNA?

It is not as stable as DNA

Even though the life form based solely on RNA is self-sufficient (for metabolism, replication,energy harvesting etc), these are rather limited capabilities

Next, mRNA, rRNA and tRNA are used for protein synthesis. Now proteins can take over many ofthe catalytic functions of RNA molecules such as catalysts, regulators, & structural elements

Finally, DNA is preferred (over RNA) for storinggenetic information. DNA evolved as stable,specialized, information repositoryKey to evolution is the ability to reproducesuccessfully and leave lots of progeny

Q: Why is genetic variability is beneficial for aspecies?To better adapt to changes in the environment,surviving new diseases etc.

Q: (b) Why then, does a cell go to great lengths toassure the fidelity of DNA replication?

Too much variability can lead to deleteriousmutations, reduced fitness and death.

The Zuckerkandl & Pauling 1965 paperproposed that since DNA and proteinsevolved at certain fixed rates over time,sequence differences between species canbe used to create an evolutionary tree,demonstrating the existence of commonancestors

Why is it harder to find the cure forcancer than getting to the Moon?

Germ line: eggs, sperm, and the cells that give rise to themSoma: all other cells. Soma supports germ line because germ-line and

somatic cells are genetically identicalSomatic mutations: more innocuous, a small fraction can give rise to

cancer Q: Could a spontaneous mutation in a germ line cell be passed on to an organism’s progeny? Yes Q: Could a spontaneous mutation in a soma cell be passed on to an organism’s progeny? No

How multicellular organisms enforce strict rules governing cell growth, proliferation, and death

The human body is a cooperative of more than 100 cell types. Proliferation and death of each cell type must becarefully regulated: by matching supply and demand - small imbalances could lead to long term problems

Most cells in the human body are neither growing nor proliferating and if they are they obey strict rules. Cancercells ignore these rules to allow them to grow and proliferate out of control. These include:

1. Mutate so that it can grow & divide under conditions that normally it cannot.2. Ignore signals that tell the cell to commit suicide (apoptosis)3. Induce new blood vessels for tumor growth4. Acquire ability to metastasize (move around the body)5. Become genetically unstable so (increase rate at which it accumulates genetic changes)

Cancer is an evolutionary disease - it arises because a cell accumulates mutationsthat alter its behavior in a way that allow it to eventually proliferate uncontrollably

Best defense against cancer is to allow as few mutations to occur as possible (mostcancers involve >1 mutations in the genome)

Most deleterious human mutations are recessive to the wild type form of the gene

Number of mutations can be estimated from analyzing: (i) Dependence of cancer incidence on age

(ii) Tracking down and counting important mutations in cancer cells

Cancer has not been selected against because it occurs too late in human life

Q: Heavy smokers or industrial workers exposed for a limited time to a chemical carcinogen thatinduces mutations in DNA do not usually begin to develop cancers characteristic of their habit oroccupation until 10, 20, or more years after exposure. Suggest an explanation for this long delay.

Mutations are induced during exposure to the carcinogen, but the number of deleteriousmutations in any one cell is usually not enough to convert it into a cancer cell. Over time(years), the cells that have become predisposed to cancer through the induced mutationsaccumulate progressively more mutations. Eventually, some of them will turn into cancercells.

Environmental Factors Play a Role in Cancer as well

Different cancers have different mutations and destabilize genomes in different ways. HNPCC and APC bothcause colorectal cancer. Yet they are very different cancers:

HNPCC (mismatch repair defective) – increased frequency of point mutations

APC (a tumor suppressor gene – mutations in APC lead to problems with chromosomal segregation (cells withvariable chromosomal numbers) and tumors

This is question 2 from the extra Practice Problems Set 8. To download it and the answers, go to the coursewebsite, look under “Problems” and under “Problems for Practice”, click on “Practice Problem 8”

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