The Protein

Presented By
Dr. Shazzad Hosain
Asst. Prof. EECS, NSU
 Proteins
Proteins do the nitty-gritty jobs of every living cell.

• Proteins are made of

long strings of
individual building
blocks known as
amino acids.
Connection between DNA and Protein

Exon
Intron
 DNA Transcription and Translation
mRN
A

tRN
A

mRNA

mRNA is read in triplets, called codon

Four different nucleotides, thus 43 = 64 possible codon
However, there are only 20 different amino acids
Thus genetic code is degenerate i.e. multiple codon produce same protein
The Genetic Code of RNA

The codon AUG for Methionine acts as start codon

Protein Functions
Proteins
• Make up about 15% of the cell
• Have many functions in the cell
– Enzymes
– Structural
– Transport
– Motor
– Storage
– Signaling
– Receptors
– Gene regulation
– Special functions
Folded proteins are placed into two
general categories

Fibrous proteins globular proteins

Fibrous proteins have polypeptide chains
organized in long fibers or sheets

Water insoluble
Very tough physically, may
be stretchy
 Functions of fibrous proteins

Structural proteins function

in support
Insects and spiders use silk
fibers to make cocoons and
webs
Collagen and elastin are
used in animal tendons and
ligaments
Keratin is the protein in
hairs, horns and feathers
Functions of fibrous proteins
Contractile proteins
function in movement

Actin and myosin

contract to create the
cleavage furrow and
to move muscles

Contractile proteins
move cilia and flagella
Globular proteins have their chains folded into
compact, rounded shapes

Easily water soluble

 Functions of globular proteins
Storage proteins function in the
storage of amino acids
Ovalbumin is the protein in egg
whites

Casein is the protein in milk,

source of amino acids for baby
mammals
Functions of globular proteins
Transport proteins function in the movement of other
substances

Hemoglobin, the iron containing protein in blood,

transport oxygen from lungs to other parts of the body
(C3032H4816O872N780S9Fe4)

Membrane transport proteins such as channels for

potassium and water
Functions of globular proteins
Hormone proteins function as cellular messenger
molecules that help maintain homeostasis

Insulin: sends message “allow sugar into cells” (when blood

glucose levels are high, cells will transport glucose into the
cells for use or storage)

Glucagon: sends message “we need more sugar in the

blood” (when blood glucose is too low, cells will release
glucose)
Functions of globular proteins
Receptor proteins allow cells to respond to
chemical stimuli

Growth factor receptors initiate the signal

transduction pathway when a growth hormone
attaches
Functions of globular proteins
Protective proteins function as protection
against disease
Antibodies combat bacteria and viruses
Functions of globular proteins
Enzymes speed up chemical reactions
Amylase and other digestive enzymes hydrolyze
polymers in food

Catalase converts hydrogen peroxide H2O2 into

water and oxygen gas during cellular respiration
Protein Structures
Peptide Bonds join amino acids

It’s a condensation reaction

(meaning that H20 (or some other small
molecule) is released when the
bond is formed).

Two amino acids form a

DI-PEPTIDE

POLYPEPTIDES
are formed from more
than two amino acids
bonded together
Non-polar R groups make the amino acid hydrophobic
Polar R groups make the amino acid hydrophilic
Ionic R groups make the amino acid hydrophilic
Polar vs Nonpolar Amino Acids

Hydrophilic Hydrophobic
There are 20 commonly occurring amino
acids that are found in proteins
 alanine - ala - A
• leucine - leu - L
 arginine - arg - R ***
• lysine - lys - K
• methionine - met - M
 asparagine - asn - N • phenylalanine - phe -
 aspartic acid - asp - D F
 cysteine - cys - C • proline - pro - P
 glutamine - gln - Q • serine - ser - S
 glutamic acid - glu - E • threonine - thr - T
 glycine - gly - G • tryptophan - trp - W
 histidine - his - H *** • tyrosine - tyr - Y
 isoleucine - ile - I • valine - val - V

“Essential Amino Acids”

are those that must be ingested in the diet
(our body can’t make them)
*** essential in certain cases
Proteins have four levels
of organization
Primary structure
is the
amino acid
sequence
The amino acid
sequence is
coded for by
DNA and is
unique for
each kind of
protein
The amino acid
sequence
determines how
the polypeptide
will fold into its
3D shape
Even a slight change in the amino acid sequence
can cause the protein to malfunction

For example,
mis-formed hemoglobin causes sickle cell disease
Proteins have four levels
of organization
Secondary structure results from hydrogen bonding between
the oxygen of one amino acid and the hydrogen of another
Red = oxygen
Black = Carbon
Blue = Nitrogen
Green = R
The alpha helix is a coiled secondary structure
due to a hydrogen bond every fourth amino acid
The beta pleated sheet is formed by hydrogen
bonds between parallel parts of the protein
A single polypeptide may have portions with
both types of secondary structure
Proteins have four levels
of organization
Tertiary structure depends on the interactions
among the R group side chains
Types of interactions
Hydrophobic interactions: amino acids with
nonpolar side chains cluster in the core of the
protein, out of contact with water

= charged

= hydrophobic
Types of interactions
Hydrogen bonds between polar side chains
Types of interactions
Ionic bonds between positively and negatively
charged side chains
Types of interactions
Disulfide bridge (strong covalent bonds) between
sulfur atoms in the amino acid cysteine

Link to video
Keratin is a family of fibrous structural proteins
Proteins have four levels
of organization
Quaternary structure results from
interactions among separate polypeptide
chains into a larger functional cluster
For example, hemoglobin is composed of
4 polypeptide chains
Proteins have four levels
of organization
The folding of proteins is aided by other
proteins, called chaperones

Act as temporary braces as proteins fold into their

final conformation

Research into chaperones is a

area of research in biology
Denaturation results in disruption of the secondary,
tertiary, or quaternary structure of the protein
Denaturation may be due to changes in
pH, temperature or various chemicals
Protein function is lost during denaturation,
which is often irreversible
Protein Folding
 What is Protein Folding ?
 Protein folding is the process by which a protein
assumes its functional shape or conformation.

Random Coil Native conformation

 Why is the “Protein Folding” so important
 Most of the proteins should fold in order to function
 Misfolding cause some diseases.
 Cystic Fibrosis, affects lungs and digestive system
and cause early death
 Alzheimers’s and Parkinson's disease
 It may help us to understand the structure of proteins
which has not been known
 LEVINTHAL PARADOX

Let have Protein composed of 100 amino acids.

Assume that each amino acid has only 3 possible
conformations.
Total number of conformations = 3100 ~= 5x1047 .
If 100 psec (100x10-12 sec) were required to convert from
a conformation to another one, a random search of all
conformations would require
5x1047 x 10-10 sec = 1.6 x 1030 years.
However, folding of proteins takes place in msec to sec
order.
 Forces that stabilize protein structure
Interactions between atoms within the protein chain
Interactions between the protein and the solvent

Electrostatic Interactions
Interaction of charged side chain with the
opposite charged side chain.
Hydrogen Bonds & van der Waals forces
Hydrophobic interactions
The kinetic Theory of Protein Folding
 Folding proceeds through a definite series of steps or a
Pathway.
 A protein does not try out all possible rotations of
conformational angles, but only enough to find the
pathway.
Energy Landscape
Protein folding models
The Framework Model
Hydrophobic collapse
Nucleation Model
 The Framework Model
Local interactions are main determinants of protein
structures

native state
unfolded state Transition state
 Hydrophobic Collapse
Hydrophobic core forms first.

native state
collapse
unfolded state
 Hydrophobic Collapse
Formation of hydrophobic globule may hinder the
reorganization of both side chains and whole protein
 Nucleation Model
Unites hydrophobic collapse and frame work model

formation of
a nucleus
unfolded state native state
 Nucleation Model
Substantial expulsion of water from the burial of non
polar surfaces
Good correlation between decrease in hydrodynamic
volume and increase in secondary structure
Disease caused by Protein Mis-folding
Diseases caused by the defect in protein folding:

Cystic fibrosis: Defect in the folding of cystic fibrosis Tran membrane

conductance regulator protein.

Diseases caused by misfolding of Prion proteins:

Kuru Disease
Creutzfedlt-Jakob Disease
Scrapie Disease in sheep
Mad cow disease

Misfolded prion protein act as infectious agents.

They act as chaperons which can multiply by binding to normal PrP

and folding it to dangerous form similar to itself.

Mechanisms of the functions of normal prions and the dangerous ones

are still not clear.
Stained section from the cerebral cortex from a patient with Creutzfedlt-Jakob
disease indicating spongiform patahlogy
Non-local contacts = High contact order
contacts between residues in the primary sequence:

NEARBY FAR APART

A A B
B

A
B
B
A

ordering many more residues at once

= selecting from more conformational states
-> How is aggregation avoidance encoded?
How do high CO structures form co-translationally?
in vitro: in vivo:

ribosome

A B

• What conformations does A adopt

A before B appears?
• How much native structure can be
B
formed co-translationally?

ordering many more residues at once

= selecting from more conformational states
-> How is aggregation avoidance encoded?
 Drug Discovery

How are drugs discovered and developed?

Choose a disease
Choose a drug target
Identify a “bioassay”
bioassay = A test used to determine biological activity.
Find a “lead compound”
“lead compound” = structure that has some activity against
the chosen target, but not yet good enough to be the drug
itself.
If not known, determine the structure of the “lead
Synthesize analogs of the lead
Identify Structure-Activity-Relationships (SAR’s)
Synthesize analogs of the lead
Identify Structure-Activity-Relationships (SAR’s)
Identify the “pharmacophore”
pharmacophore = the structural features directly
responsible for activity
Optimize structure to improve interactions with target
Determine toxicity and efficacy in animal models.
Determine pharmacodynamics and pharmacokinetics of the
drug.
Pharmacodynamics explores what a drug does to the body,
whereas pharmacokinetics explores what the body does to the
drug.
Patent the drug
Continue to study drug metabolism
Continue to test for toxicity
Design a manufacturing process
Carry out clinical trials
Market the drug
Choosing a
Disease

Pharmaceutical companies are commercial enterprises

Pharmaceutical companies will, therefore, tend to
avoid products with a small market (i.e. a disease
which only affects a small subset of the population)
 Choosing a Disease

Pharmaceutical companies will

also avoid products that would
be consumed by individuals of
lower economic status (i.e. a
disease which only affects third
world countries)
 Choosing a Disease (cont.)
Most research is carried
out on diseases which
afflict “first world”
countries: (e.g. cancer,
cardiovascular diseases,
depression, diabetes, flu,
migraine, obesity).
 The Orphan Drug Act

The Orphan Drug Act of 1983 was passed to encourage

pharmaceutical companies to develop drugs to treat
diseases which affect fewer than 200,000 people in the US
Under this law, companies who develop such a drug are
entitled to market it without competition for seven years.
This is considered a significant benefit, since the standards
for patent protection are much more stringent.
 Identifying a Drug Target

Drug Target = specific macromolecule, or biological

system, which the drug will interact with
Sometimes this can happen through incidental
observation…
 Identifying a Drug Target (cont.)
Example: In addition to their being able to inhibit the uptake of
noradrenaline, the older tricyclic antidepressants were observed to
“incidentally” inhibit serotonin uptake. Thus, it was decided to prepare
molecules which could specifically inhibit serotonin uptake. It wasn’t clear
that this would work, but it eventually resulted in the production of
fluoxetine (Prozac).

HO

N NH2
F3C
HN

CH3 O
N N
H
H3C
serotonin prozac

Imipramine
(a classical tricyclic antidepressant)
 The mapping of the human genome should
help!
In the past, many medicines (and lead compounds) were
isolated from plant sources.
Since plants did not evolve with human beings in mind, the
fact that they posses chemicals which results in effects on
humans is incidental.
Having the genetic code for the production of an
enzyme or a receptor may enable us to over-
express that protein and determine its structure and
biological function. If it is deemed important to
the disease process, inhibitors (of enzymes), or
antagonists or agonists of the receptors can be
prepared through a process called rational drug
design.
 Simultaneously, Chemistry is Improving!
This is necessary, since,
ultimately, plants and natural
sources are not likely to provide
the cures to all diseases.
In a process called
“combinatorial chemistry” large
numbers of compounds can be
prepared at one time.
The efficiency of synthetic
chemical transformations is
improving.
 Selectivity is Important!

e.g. targeting a bacterial enzyme, which is not present

in mammals, or which has significant structural
differences from the corresponding enzyme in
mammals
The Standards are Being Raised

More is known about the biological chemistry of living

systems
For example: Targeting one subtype of receptor may enable
the pharmaceutical chemist to avoid potentially troublesome
side effects.
Problems can arise

Example: The chosen target, may over time, lose its

sensitivity to the drug
Example: The penicillin-binding-protein (PBP) known
to the the primary target of penicillin in the bacterial
species Staphylococcus aureus has evolved a mutant
form that no longer recognizes penicillin.
 Choosing the Bioassay
Definitions:
In vitro: In an artificial environment, as in a test tube or
culture media
In vivo: In the living body, referring to tests conductedin living
animals
Ex vivo: Usually refers to doing the test on a tissue taken from
a living organism.
Choosing the Bioassay (cont.)

In vitro testing
Has advantages in terms of speed and requires relatively small amounts
of compound
Speed may be increased to the point where it is possible to analyze
several hundred compounds in a single day (high throughput screening)
Results may not translate to living animals
 Choosing the Bioassay (cont.)

In vivo tests
More expensive
May cause suffering to animals
Results may be clouded by interference with other
biological systems
Finding the Lead

Screening Natural Products

Plants, microbes, the marine world, and animals, all
provide a rich source of structurally complex natural
products.
It is necessary to have a quick assay for the desired
biological activity and to be able to separate the
bioactive compound from the other inactive substances
Lastly, a structural determination will need to be made
Finding the Lead (cont.)

Screening synthetic banks

Pharmaceutical companies have prepared thousands of
compounds
These are stored (in the freezer!), cataloged and
screened on new targets as these new targets are
identified
Finding the Lead (cont.)

Using Someone Else’s Lead

 Design structure which is similar to existing lead, but
different enough to avoid patent restrictions.
 Sometimes this can lead to dramatic improvements in
biological activity and pharmacokinetic profile. (e.g.
modern penicillins are much better drugs than original
discovery).
 Finding the Lead (cont.)
Enhance a side effect

O
H2N S NH2
O
sulphanilamide
(an antibacterial with the side effect of
lowering glucose levels in the blood and also
diuretic activity)

Cl N
O
O S O
O NH
NH S S
H2N O
NH O O
tolbutamide Chlorothiazide
(a compound which has been optimized to only (a compound which has been optimized to only display diuretic
lower blood glucose levels. Useful in the treatment activity.)
of Type II diabetes.)
 Use structural similarity to a natural ligand

NH2 N(CH3)2

H
HO N
H3C S
O O
N N
H H

5-Hydroxytryptamine (5-HT) Sumatriptan (Imitrex)

Serotonin (a natural neurotransmitter Used to treat migrain headaches
synthesized in certain neurons in the CNS) known to be a 5-HT1 agonist
 Computer-Assisted Drug Design
If one knows the precise molecular structure of the target
(enzyme or receptor), then one can use a computer to
design a perfectly-fitting ligand.
Drawbacks: Most commercially available programs do
not allow conformational movement in the target (as the
ligand is being designed and/or docked into the active
site). Thus, most programs are somewhat inaccurate
representations of reality.
 Serendipity: a chance occurrence
Must be accompanied by an experimentalist who
understands the “big picture” (and is not solely focused
on his/her immediate research goal), who has an open
mind toward unexpected results, and who has the ability
to use deductive logic in the explanation of such results.
Example: Penicillin discovery
Example: development of Viagra to treat erectile
dysfunction
 Finding a Lead (cont.)

Sildenafil (compound UK-92,480) was synthesized by a group of

pharmaceutical chemists working at Pfizer's Sandwich, Kent
research facility in England.
It was initially studied for use in hypertension (high blood
pressure) and angina pectoris (a form of ischaemic cardiovascular
disease).
Phase I clinical trials under the direction of Ian Osterloh
suggested that the drug had little effect on angina, but that it
could induce marked penile erections.
Pfizer therefore decided to market it for erectile dysfunction, rather than
for angina.
The drug was patented in 1996, approved for use in erectile dysfunction
by the Food and Drug Administration on March 27, 1998, becoming
the first pill approved to treat erectile dysfunction in the United States,
and offered for sale in the United States later that year.
It soon became a great success: annual sales of Viagra in the period 1999–
2001 exceeded $1 billion.
Finding a Lead (cont.)

O
N
N
N

NH
O
O S
O
N

viagra
(Sildenafil)
 Structure-Activity-Relationships (SAR’s)

Once a lead has been discovered, it is important to

understand precisely which structural features are
responsible for its biological activity (i.e. to identify the
“pharmacophore”)
The pharmacophore is the precise section of the molecule that
is responsible for biological activity
 This may enable one to prepare a more active molecule
 This may allow the elimination of “excessive” functionality, thus
reducing the toxicity and cost of production of the active material
 This can be done through synthetic modifications
 Example: R-OH can be converted to R-OCH3 to see if O-H is
involved in an important interaction
 Example: R-NH2 can be converted to R-NH-COR’ to see if interaction
with positive charge on protonated amine is an important interaction
Link
Next step: Improve Pharmacokinetic
Properties
Improve pharmacokinetic properties.
pharmacokinetic = The study of absorption,
distribution, metabolism and excretion of a drug
(ADME).
Video
exercise=MedicationDistribution&title=Medicatio
n%20Absorption,%20Distribution,
%20Metabolism%20and%20Excretion
%20Animation&publication_ID=2450
 Metabolism of Drugs
The body regards drugs as foreign
substances, not produced naturally.
Sometimes such substances are
referred to as “xenobiotics”

•Body has “goal” of removing such xenobiotics from system by excretion in the
urine
•The kidney is set up to allow polar substances to escape in the urine, so the body
tries to chemically transform the drugs into more polar structures.
 Metabolism of Drugs (cont.)
Phase 1 Metabolism involves the conversion of
nonpolar bonds (eg C-H bonds) to more polar bonds
(eg C-OH bonds).
A key enzyme is the cytochrome P450 system, which
catalyzes this reaction:

RH + O2 + 2H+ + 2e– → ROH + H2O

Mechanism of Cytochrome P450
Phase I metabolism may either detoxify or
toxify.
Phase I reactions produce a more polar molecule that
is easier to eliminate.
Phase I reactions can sometimes result in a substance
more toxic than the originally ingested substance.
An example is the Phase I metabolism of acetonitrile
 Phase I HO Spontaneous O
H3C C N + H C N
H2C C N Fragmentation H H
Metabolism
Highly Toxic Metabolites
The Liver
Oral administration frequently brings the drugs (via
the portal system) to the liver
 Metabolism of Drugs (cont.)

Phase II metabolism links the drug to still more polar

molecules to render them even more easy to excrete

UDP Glucuronic Acid

Glucuronic Acid
HO O
O
P O
HO O
O P O O O
NH
glucuronosyltransferase O O
Drug
O O N enzyme HO R
HO HO O

OH HO OH
HO OH
OH
OH

More easily excreted than ROH itself

R OH

Drug
 Metabolism of Drugs (cont.)
Another Phase II reaction is sulfation (shown below)

NH2

N
O O N

O S O P O
O N
O-
N SO3-
R OH
O-
R O
Drug
Sulfated Drug
O OH (more easily excreted)
O P O-

O-

3'-Phosphoadenosine-5'-phosphosulfate
 Phase II Metabolism

Phase II reactions most commonly detoxify

Phase II reactions usually occur at polar sites, like
COOH, OH, etc.
 Manufacture of Drugs

 Pharmaceutical companies must make a profit to continue to exist

 Therefore, drugs must be sold at a profit
 One must have readily available, inexpensive starting materials
 One must have an efficient synthetic route to the compound
 As few steps as possible
 Inexpensive reagents
 The route must be suitable to the “scale up”
needed for the production of at least tens of
kilograms of final product
 This may limit the structural complexity
and/or ultimate size (i.e. mw) of the final
product
 In some cases, it may be useful to design
microbial processes which produce highly
functional, advanced intermediates. This
type of process usually is more efficient
than trying to prepare the same
intermediate using synthetic methodology.
 Toxicity
Toxicity standards are continually becoming tougher
Must use in vivo (i.e. animal) testing to screen for
toxicity
Each animal is slightly different, with different metabolic
systems, etc.
Thus a drug may be toxic to one species and not to another
 Example: Thalidomide
Thalidomide was developed by German pharmaceutical company
Grünenthal. It was sold from 1957 to 1961 in almost 50 countries
under at least 40 names. Thalidomide was chiefly sold and
prescribed during the late 1950s and early 1960s to pregnant women,
as an antiemetic to combat morning sickness and as an aid to help
them sleep. Before its release, inadequate tests were performed to
assess the drug's safety, with catastrophic results for the children of
women who had taken thalidomide during their pregnancies.

Antiemetic = a medication that helps prevent and

control nausea and vomiting
 Birth defects
caused by use of thalidomide
 Example: Thalidomide

From 1956 to 1962, approximately 10,000 children were born with severe
malformities, including phocomelia, because their mothers had taken
thalidomide during pregnancy. In 1962, in reaction to the tragedy, the United
States Congress enacted laws requiring tests for safety during pregnancy before
a drug can receive approval for sale in the U.S.

N O

NH

O O

Thalidomide

Phocomelia presents at birth very short or absent long bones and

flipper-like appearance of hands and sometimes feet.
 Example: Thalidomide
Researchers, however, continued to work with the drug. Soon after its
banishment, an Israeli doctor discovered anti-inflammatory effects of
thalidomide and began to look for uses of the medication despite its
teratogenic effects.

He found that patients with erythema nodosum leprosum, a painful skin
condition associated with leprosy, experienced relief of their pain by
taking thalidomide.

Teratogenic = Causing malformations in a fetus

 Thalidomide
Further work conducted in 1991 by Dr. Gilla Kaplan at Rockefeller
University in New York City showed that thalidomide worked in leprosy
by inhibiting tumor necrosis factor alpha. Kaplan partnered with Celgene
Corporation to further develop the potential for thalidomide.
Subsequent research has shown that it is effective in multiple myeloma, and
it is now approved by the FDA for use in this malignancy. There are
studies underway to determine the drug's effects on arachnoiditis, Crohn's
disease, and several types of cancers.
 Clinical Trials
Phase I: Drug is tested on healthy volunteers to
determine toxicity relative to dose and to screen
for unexpected side effects
Clinical Trials
 Phase II: Drug is tested on small group of patients to see
if drug has any beneficial effect and to determine the dose
level needed for this effect.
 Clinical Trials
Phase III: Drug is tested on much larger group of
patients and compared with existing treatments and
with a placebo
Clinical Trials
 Phase IV: Drug is placed on the market and patients are
monitored for side effects
 Assigned Reading
 Haffner Marlene E; Whitley Janet; Moses Marie Two decades of orphan product
development. Nature reviews. Drug discovery (2002), 1(10), 821-5. Link
 Franks Michael E; Macpherson Gordon R; Figg William D Thalidomide. Lancet
(2004), 363(9423), 1802-11. Link
 Abou-Gharbia, Magid. Discovery of innovative small molecule therapeutics.
Journal of Medicinal Chemistry (2009), 52(1), 2-9. Link
 Paul, S. M. et al. How to improve R&D productivity: the pharmaceutical industry’s
grand challenge. Nature Reviews Drug Discovery (2010), 9: 203-214.
 Jorgensen, W. L. The many roles of computation in drug discovery. Science (2004)
303: 1813-1818.
 Butcher, E. C. et al. Systems biology in drug discovery. Nature biotechnology
(2004) 22(10): 1253-1259.
 Optional Additional Reading

 Bartlett J Blake; Dredge Keith; Dalgleish Angus G The evolution of

thalidomide and its IMiD derivatives as anticancer agents. Nature
reviews. Cancer (2004), 4(4), 314-22. Link
 Cragg, G. M.; Newman, D. J. Nature: a vital source of leads for
anticancer drug development. Phytochemistry Reviews (2009),
8(2), 313-331. Link
 Betz, U. A. K. et al. Genomics: success or failure to deliver drug
targets? Current Opinion in Chemical Biology (2005), 9: 387-391
 Sams-Dodd, F. Target-based drug discovery: is something wrong?
Drug Discovery Today (2005) 10: 139-147.
 Homework Questions
 What is an ‘orphan drug’. Why has the Orphan Drug Act been successful?
 Thalidomide is actually a mixture of two compounds. Draw their
structures and list the physiological effects of each.
 What does ADMET stand for?
 List several possible reasons for poor efficacy of drug candidates in in vivo
models.
 Explain how structure-based design was used to develop an inhibitor with
improved selectivity for TACE over MMP-1 and MMP-9.
 How can the pharmaceutical industry increase the probability of technical
success (p(TS))? What are the major causes of Phase II and III attrition?
 Reference
Mostly from Web