advanced medicinal chemistry unit V topics

1. BRADYKININ RECEPTOR ANTAGONISTS AND
THROMBIN INHIBITORS
ADVANCED MEDICINAL CHEMISTRY-I
Presented by
T.Bhanusri
21031S0215
M.PHARM 1ST YEAR 1ST SEM
Pharmaceutical chemistry
IST(Centre of pharmaceutical
science),JNTUH
Under the guidance of
DR.S.Shoba rani
M.PHARM(Ph.D)
Associate professor
HOD of CPS, JNTUH
Former Additional controller of
examination, JNTUH

2. BRADYKININ RECEPTOR ANTAGONISTS

3. Bradykinin (in Greek brady- = slow; -kinin =
to move) is a peptide that promotes
inflammation.
Bradykinin is a 9-amino acid peptide chain.
Bradykinin is a linear nonapeptide messenger
belonging to the kinin group of proteins, with
amino acid sequence RPPGFSPFR.
The amino acid sequence of bradykinin is :
Arg-pro-pro-Gly-phe-ser-pro-phe-Arg.
The empirical formula is C50H73N15O11.
Introduction
Structure of
bradykinin

4. A class of drugs called angiotension
converting enzyme inhibitors (ACE
inhibitors) increase bradykinin levels
by inhibiting its degradation , thereby
increasing its blood pressure lowering
effect.
Enzymatically produced from kallidin
in the blood, it is a powerful
vasodilator that causes smooth muscle
contraction, and may mediate
inflammation .
It has a role as a human blood serum
metabolite and a vasodilator agent.
It is a tautomer of a bradykinin(2+).
Structure of
bradykinin

5. history
1920’s and
1930’s
Characterized a hypotensive substance in urine and
found a similar material in saliva,plasma and a variety
of tissues.
Established that kallikreins generate a
pharmacologically active substance from an inactive
precursor present in plasma
1948
Named the active substance kallidin and showed it to
be a polypeptide cleaved from a plasma globulin that
they termed as kallinogen
Frey
,kraut,
werle
Werle,
gotze,
keppler
Werle,
berek
-
Rocha e
silva and
associate
d
Reported that trpsin and certain snake venoms acted on plasma
globulin to produce a substance that lowered bp and caused
developing contraction of the gut.
1937

6. -
1960
1960
-
Elliot and
coworkers The nonpeptide bradykinin was isolated
Synthesized the nonpeptide bradykinin.
because of this slow response, they named
the substance bradykinin.
Boissonnas
and
associated

7. prekallikrein Tissue kallikrein
Plasma kallikrein Tissue kallikrein
HMWK LMWK
KININS
bradykinin
kallidin
Inactive
peptidases
Des-Arg-kallidin
Des-Arg-BK
B1
R
B2R
Inactive
peptides
Plasma(vasculature) Tissue (glandular /exocrine)
XIIa Autolysis/proteinase
kininases
Kininase I
aminopeptidases
kininases
Cellular responses
Inducible
receptor
Constitutive
receptor
Kinin-kallikrein system
kininase-I

9. Kinin receptors:
Existence of two types bradykinin receptor: B1 and B2.
Both are GPCR and mediate similar effects.
B1
Normally expresses at very low levels but
are strongly induced in inflamed ar
damaged tissues by cytokines such as IL-1
Respond to des-Arg9-bradykinin and des-
Arg9-kallidin but not to bradykinin itself.
Likely that B1 receptors play asignificant
role in inflammation and hyperalgesia
Constitutively expressed in most normal
tissues.
Selectively binds bradykinin and kallidin
and mediates the majority of their effects.
The B2 receptors activates PLA2 and
PLC via interaction with distinct G
proteins.
B2

10. Investigations
Ligand-
based
Receptor-
based
Solution
conformation of
bradykinin
Conformationally
constrained
bradykinin
antagonist peptides
Elucidation of an
agonist binding
site on the B2
receptor
Elucidation of an
antagonist site on
the B2 receptor

11. Solution conformation of bradykinin
2-D NMR with empirical energy calculation:
 on basis of spectral data , biological results from conformationally restricted analogs, as well as the
relationship between ordering in bradykinin and the dielectric environment of the solvent.
 In aqueous solution bradykinin is conformationally random, the biologically active form of the peptide is
likely ordered and stabilized within the lipid bilayer of the cell membrane prior to binding with its
receptor. Hence the nature of receptor might be hydrophobic.
 The appropriate solvent environment should be able to stimulate, at least in terms of hydrophobicity and
dielectric constant, the nature of a cell membrane, and a 90:10 d8-dioxane-H2O mixture was selected for
NMR experiments.
 It was anticipated that under these nonsolvating conditions the conformational diversity of bradykinin
might be severely restricted.
 The ultimate analysis of the two-dimensional NMR data collected at 500 MHz supported a single major
conformational species. There were five HN-CαH connectivities, one for each amide. This was confirmed
in the 13C NMR spectrum where only nine carbonyl resonances, one for each amino acid, were present.
3-D strctures: Not known the receptors and sructure of bradykinin

12. NOESY:
 Based on multiple observed long range amide-to-amide nucear overhauser effects
(NOEs), that it was indeed a single major conformational species.
 Although bradykinin contains three proline residues, the absence of any strong CαHi
-CαHj +, cross peaks in the nuclear overhauser enhancement spectroscopy (NOESY)
spectrum was taken as proof that all peptide bonds were trans.
 In total, 35 interproton distances were extracted from the NOESY spectrum and,
whenever possible, stereospecific assignments for pro-R and pro-S hydrogens were
made explicitly
Temperature dependant:
 A temperature-dependent study of the chemical shifts of the amide protons resulted in
a near-linear dependence suggesting no major conformational changes were
coinciding with the temperature change and thereby allowing a comparison of slopes
(∆δ/∆t).
 The lowest values obtained for these slopes corresponded to Phe8 and Arg9
suggesting solvent sequestering for these amides.

13. CHARMm – version 21:
 Utilizing custom routines written using the program CHARMm, version 21 [21], the
interproton distances were incorporated into the potential-energy expression in the form
of an additional potential-energy term.
Computational stratergy:
 Given the high Chou and Fasman probability of β-turns in the sequences Pro2-Pro3-Gly4-Phe5
and Ser6- Pro7-Phe8-Arg9 (3.79 × 10-4 and 1.99 × 10-4, respectively), the computational
strategy employed was to begin from two initial structures: (a) an extended β strand, and (b) a
structure containing these two predicted β turns.
NOE restrains:
 Analysis of the two average structures obtainedfrom the two unique starting points demonstrated
convergence to a similar conformational species.
 In each , the sum of the NOE restraints energy was less thab 4.7 kcal/mol and the RMS deviation
from the target distances was below Å.
 Similar results were obtained for each simulation when they were repeated without the
electrostatic term being included in the total potential-energy function.
 This important data lends credence to the hypothesis that the final structures are derived from
the NOE restraints and not by poorly represented electrostatic interactions.

14. A similar C-terminal turn structure was observed in an
analogous NMR study of a first-generation kinin antagonist,
NPC 567 (DArg0-Arg1-Pro2-Hyp3-Gly4-Phe5-Ser6-DPhe7-
Phe8-Arg9), although the type of turn was not the same.
 Our initial speculation was that this slight structural
difference might partially account for the functional
differences of bradykinin and NPC 567.
 These solution conformations, one of an agonist and the
other of an antagonist, were subsequently used to focus the
design and synthesis of conformationally constrained peptide
analogues of NPC 567.

15. Conformationally constrained bradykinin antagonist peptides
 The ligand based approach of conformationally constrained peptides has been widely
used.
 The process involves the incorporation of conformational constrains into the known
peptides, either agonist or antagonist , which enforce a predictable geometry.
 A series of peptides containing these types of constraints can be useful for
extrapolating the steric and electronic environment of a given binding site.
 The structural information can be derived regardless of whether or not the constrained
peptide binds to the target receptor.
 Since peptides can be prepared rapidly , it is typical to establish a structure activity
relationship using them and then at some time later transpose that information onto a
nonpeptide lead molecule in an attempt to improve its potency.
 As part of an expansion upon the hypothesis that a C- terminal β turn was a structural
prequisit to high affinity antagonist binding, novel series of constrained decapeptides
was prepared.
 These peptides are of the sequence:

16. DArg-Arg-pro-Hyp-Gly-Phe-Ser-DHype-Y-Arg
Either
tetrahydroquinoline-3-
carboxylic acid(Tic) or
octahydroindole-2-
carboxylic acid (Oic)
An organic ether of
D-4-hydroxyproline
in either the cis or
trans geometric form

17. Lowest 5 kcal mol-1 of the calculated overall potential
energy surface for a model peptide of Ser-DHype(trans
propyl)-Oic-Arg. The contour interval is 0.5 Kcalmol-1
and the highest (outermost) and lowest contour energy
values are labeled. Superimposed on the contour plots are
values for ψi+1 and ψi+2 from each of the thirty structures
generated from the NMR data corresponding to the
tetrapeptide Ser-DHype(trans propyl)-Oic-Arg
 The C-terminal portion of a
representative member of
this class of peptides was
shown—first by empirical
calculation , then by NMR
at 600 MHz—to adopt a β
turn nearly unambiguously
(figure ).
 Moreover, it was shown by
calculation that the turn
was adopted regardless of
the nature of the ether
group (alkyl, aryl, etc.) or
its geometry (cis or trans).

18.  Hence, a diverse series of these peptides was initially
used as a tool to probe the steric and electrostatic
topology of an antagonist binding site on the
bradykinin B2 receptor in the guinea pig ileum.
 The cis ethers, in all cases, bound to the receptor
with significantly lower affinity than did the trans.
 A more complete listing of the peptides used in the
study is shown in Table 1.
 These results support the hypothesis that the domain
of the receptor that binds these antagonist ligands is
partly made up of a hydrophobic cavity about one
side of the C-terminal.
 However, adjacent to the other side of the turn, there
appears to be same type of steric interference ( or
lack of a pocket) that might otherwise accommodate
the ethers of the cis configuration

19. Receptor binding curves for the binding of NPC
17410 and NPC 17643 to B2 receptors from the
guinea pig ileum and cloned rat and human B2
receptors. Legends are noted on the figure.
 More recently, bradykinin B2 receptors
have been cloned from both rat and
human sources.
 In receptor-binding experiments using
these new receptors, selected members
of the DHype-containing decapeptides
were used to probe these receptors, a
representative sample of the data is
shown in Figure.
 NPC 17643( a trans propyl ether at
position 7) and NPC 17410( a cis
propyl ether of D-4-hydroxyproline at
position 7) were used.

20. NPC17643
NPC17410
Behave similarly in binding assays directed toward the bradykinin B2
receptors in guinea pig,rat and human.
Bound with similar affinity to both the guinea pig and rat bradykinin B2
receptors, but had an appreciably higher affinity for the human B2
receptor
 The des-Arg9 forms of these peptides have also been shown to have high affinity for
the recently cloned human B1 receptor.
 An extension of the work described herein would be to use a more complete series of
des-Arg9 DHype-containing nonapeptides to probe the binding site of this new receptor
where other interesting pharmacological differences are likely to exist since the B1
receptor is only 33% homologous to the human B2.

21. DHype
1st 2nd 3rd
Incorporated a novel β-turn
mimetic that was
alternatively functionalized
and used to probe the
unknown topology of the
guinea pig, rat, and human
bradykinin B2 receptors.
subsequent molecular
biological and
computational procedures
in the elucidation of an
antagonist binding site
certain members of
this series of
decapeptides contain
alkyl ethers of D-4-
hydroxyproline at
position seven.
Experimentalexperimen
tal evidence that high-
affinity decapeptide
bradykinin receptor
antagonists adopt a C-
terminal β turn in the
receptor-bound
conformation

22.  Finally, several members of the series (i.e., NPC 17731, NPC 17761, NPC 17974) are among the
most potent antagonists for this receptor yet reported.
 Hence, there may be applications for these compounds as human therapeutics.
 Several “second generation” decapeptide antagonists have been reported, but the prototype from
the class, which was first to be reported, is HOE 140 (DArg0-Arg1-Pro2-Hyp3-Gly4-Thi5-Ser6-
DTic7-Oic8- Arg9).
 This decapeptide has also been shown to preferentially adopt a C-terminal β turn.
 There are two factors that must be considered when applying structure-activity-relationship (SAR)
information from a series of peptides toward the design of nonpeptide mimetics and putative
library scaffolds.
 One is in regard to the backbone conformation that primarily serves as a structural scaffold upon
which the various functionalities (side chains) are attached.
 The other factor is the side chains themselves whose spatial positions are primarily dictated by the
backbone structure.
 With the exception of the Cα-methyl-Phe5-containing peptide (NPC 18540), each conformational
constraint caused a significant, at least 1000-fold, loss in binding affinity with respect to the
unconstrained parent peptide, NPC 18545.

23.  A systematic study of the relative importances of amides and side chains in a prototypical
second generation antagonist, NPC 18545 (DArg0-Arg1-Pro2-Hyp3-Gly4-Phe5-Ser6-DTic7-
Oic8-Arg9) .
 The elimination of all chirality and sidechain moieties in the segment Arg1-Pro2-Hyp3-Gly4-
Phe5 via replacement by Gly1-Gly2-Gly3- Gly4-Gly5 (NPC 18152), led to a peptide that no
longer binds the receptor.
 NPC 18149 (DArg0-Arg1-Gly2-Gly3-Gly4-Phe5-Ser6-DTic7-Oic8-Arg9; Ki = 13.7 nM;
Guinea pig ileum) was taken as the lead peptide, the relative contributions to binding affinity
from each amide bond in the segment Arg1-Gly2-Gly3-Gly4-Phe5 were examined.
 The conclusions drawn from the data are that in terms of structural or electrostatic interactions
with this antagonist site on the receptor, the amide bond linking residues two and three may not
be as critical as those linking residues three to four and four to five.
 Each of these investigations was aimed toward an understanding of either the backbone
conformation of this prototypical decapeptide or the relative importance of the functional
groups in the side chains that make significant contributions to receptor affinity.
 From the former, nonpeptide frameworks and scaffolds can be imagined. From the latter,
insights into which functionality is required for high-affinity binding is derived. The remaining
challenge is to reassemble these fragments onto synthetically feasible nonpeptide frameworks as
potential new lead compounds.

24. Elucidation of an agonist binding site on the B2 receptor
 Receptor binding site on the basis of receptor binding data from conformationally
constrained ligands.
 G-Protein-coupled receptors do not lend themselves to analysis by either NMR or x-ray
crystallography due to their structural dependence on an intact cell membrane.
 In our laboratories we pursued this valuable structural information by utilizing a
combination of structural homology modeling, molecular dynamics, systematic
conformational searching methods, and mutagenesis experiments.
 The combination of these techniques led to a proposed model of bradykinin bound to the
agonist site on its receptor.
 A hydrophobicity (Kyte-Doolittle) calculation [42] on the amino acid sequence of the rat
bradykinin receptor yielded seven segments, each of which were 21 to 25 contiguous
residues with predominantly hydrophobic side chains.
 These were presumed to be the seven transmembrane portions of the receptor.
 Cartesian coordinates of the backbone atoms within each of these seven segments were built
by structural homology from the cryomicroscopic structure of the analogous segments of
bacteriohodopsin.

25.  Subsequently, side chains were added to these seven segments as appropriate for the rat
bradykinin receptor, and the resulting geometry was optimized via constrained energy
minimization to alleviate bad contacts.
 Extracellular and intracellular loops were extracted from the Protein Data Bank library,
following a geometric search based upon a vector defined by terminal alpha carbons in
adjacent helices.
 The model was subsequently subjected to a series of constrained and unconstrained energy
minimizations as well as molecular dynamics simulations. The resulting structure of the
receptor was used in a novel two-step docking procedure.
 bradykinin adopts a C-terminal β turn upon complexation with the receptor, the φ, ψ
backbone dihedral angles in the tetrapeptide corresponding to the C-terminus of
bradykinin (Ser-Pro-Phe-Agr) were constrained in a harmonic fashion (force constant = 15
Kcal Å-1 mol1) to values that define a type II' β-turn.
 This tetrapeptide probe was then systematically translated about the interior of a
theoretical box inscribing the rat receptor model.
 The translations were such that the tetrapeptide probe molecule was incrementally
repositioned within the receptor by following a 3 Å × 3 Å × 3 Å grid pattern.

26. Complete group of contour plots showing energy of
interaction between probe and receptor. Each contour plot
corresponds to a different horizontal slice as part of the
first stage in the conformational search. Darker gray
indicates most favorable interaction and the light shades
represent least favorable interactions
 At each new position, both the probe
and receptor were reset to their initial
conformations, then the geometry of
the complex was optimized using
200 steps of steepest descent
followed by 500 steps of Adopted-
Basis Newton-Raphson energy
minimization.
 Subsequently, the sum of the steric
and electrostatic contributions to the
overall potential energy (interaction
energy)—as measured only between
the tetrapeptide probe molecule and
the atoms of the receptor—were
calculated.

27. Several mutant receptors were made such that each contained either a point mutation or a small
cluster of point mutations, wherein native residues, having negatively charged side chains (Asp,
Glu), were replaced by alanine(s). Table 2 lists the initial cluster mutations (rat) that were
prepared as well as the follow-up single point mutations (rat).Therefore a receptor containing a
double mutation (Asp268,286 rarrow.gif Ala268,286) would be expected to show a much more
dramatic loss in affinity for bradykinin than would receptors containing the individual point
mutations. The appropriate double mutation experiment confirmed this by causing a 500-fold loss
in affinity for bradykinin, as predicted (Table 2)

28. Proposed model of bradykinin bound to the rat B2
receptor at the agonist binding site. Only the upper
portion of the receptor is shown as gray helical ribbons.
Bradykinin backbone and side chain atoms are shown as
thick white licorice. Positions of point mutations having
no significant adverse effects on bradykinin binding are
shown as light gray spheres. Positions of mutations
affecting bradykinin binding are shown as dark gray
spheres
 Figure shows a stereoview of
the selected ligand-receptor
complex chosen on the basis
of best agreement with the
results of these mutagenesis
studies.
 These residues are remotely
situated with respect to the
proposed site of bradykinin
binding and are colored light
gray in Figure 5.
 The mutagenized residues of
this double mutant B2
receptor are colored dark
gray in Figure 5.

29. Furthermore, in contrast to the functional activity of NPC 18325 at the human B2 receptor, the
compound is a functional antagonist as measured against bradykinin-induced contraction of the
isolated guinea pig ileum (pA2 = 5.5). These findings are in agreement with the concept that as a
ligand is made smaller (i.e., fewer contact points possible with the receptor), the subtle structural
differences in the binding sites on species variants of the same receptor become amplified. This
observation further supports a cautionary posture toward developing nonpeptide antagonists for use
in human diseases on the basis of results obtained in some animals including the guinea pig. Taking
this new molecule as a lead structure, together with the receptor model and structure-activity
relationship associated with related peptides including cyclic antagonists, the pursuit of several
related pseudopeptides was undertaken.

30. Elucidation of an antagonist site on the B2 receptor
 There have been a variety of single alanine point mutations experimentally
introduced into both rat and human bradykinin B2 receptors.
 Several of these have been shown to decrease the affinity of bradykinin to the
receptor and have been implicated structurally near the agonist binding site. In
contrast, at the time of this manuscript, there have been no mutations reported that
adversely affect the ability of any peptide antagonists to bind to the receptor.
 Furthermore, antibodies raised against the certain extracellular domains of the
kinin receptor compete with bradykinin for binding to the receptor but have no
inhibitory action on the binding of antagonist peptides.
 In addition, it has been shown that bradykinin can be covalently crosslinked to
the B2 receptor while antagonists cannot.
 These observations have fostered the belief that the agonist and antagonist
binding sites of the receptor are not the same.

31. specific groups of contiguous residues within
the receptor were identified as possible
contributors to an antagonist binding site. The
NPC 17410 binding to chimeras III, IV, and
VIII showed rat-like pharmacology (low NPC
17410 affinity). The NPC 17410 binding to
chimeras I, II, VI, and VII showed human-like
NPC 17410 pharmacology (high receptor
affinity). Binding to chimeras V and VIII,
however, was similar to rat-like NPC 17410
pharmacology, but the affinity of the compound
was slightly shifted back toward human-like
results.

32. Mutagenesis experiments have been done on this
pair in the rat B2 receptor with interesting results.
Mutations in Thr263 only affect agonist binding,
not antagonist. Mutations in Gln260 affect
binding of bradykinin and first generation
antagonist peptides.
As depicted in the figure, it is possible that the
agonist and antagonist binding sites have
domains on opposite sides of the helix that makes
up TM 6, with Gln260 being situated partly in
both.
Schematic of the primary amino sequence of the human B2 receptor. Shown in black are
residues experimentally identified as contributing to an agonist binding site. The dark gray
residues are suspect positions for contributing to an antagonist site. The residues colored light
gray have been mutagenized only in the rat B2 receptor, but they are conserved in the human.
The Thr263 rarrow.gif Ala mutation interferes with agonist binding only, while Gln260
partially interferes with agonist and first generation antagonist binding.

33. Design and combinatorial synthesis of nonpeptidic antagonists
 a significant body of information was generated that provides insights into the key structural
features of bradykinin receptor binding sites and the residues that participate in ligand binding.
 In addition, from the ligand-based studies, knowledge about relevant structure-activity
relationships was acquired.
 Our modular synthetic strategy was based primarily upon the recognition that high-affinity
ligands appear to be comprised of three domains.
 These domains are (1) a positively charged N-terminal segment, (2) a midsection containing a
bend or twist with some hydrophobic substituent attached and, (3) a C-terminal segment of
appropriate hydrophobicity and structurally simulating a type II' β turn.
 Models of potent cyclic and linear peptide bradykinin receptor antagonists (described previously)
were used in a comparative fashion to select nonpeptide ring systems from a database of chemical
structures fine chemicals database.
 For each, some degree of chemical diversity was achieved by altering one of several parameters
including, o, m, or p substitution of an aromatic ring or nature of alkyl substituent(s) as well as
point(s) of synthetic attachment.

34. Composition of ten original
nonpeptidic libraries of the
sequence DArg-Arg-X-Y-Arg.
X and Y were selected from
the set of scaffolds shown in
Table 1. Also shown are the
subsequent breakdown
libraries from original library
number 1. Two-letter codes
used in the figure correspond
to the different nonpeptide
moieties described in Table 1.
Specifically, PH =
phenanthridinone, CB =
carboline, SP = spirocycle, SC
= Straight chain, CN =
cinnamic acid

35. Lead optimization
 We have previously reported that the C-terminal guanidinyl moiety of Arg [9] in
prototypical peptide bradykinin antagonists is likely to behave more as an aromatic
functional group rather than a hydrogenbond donor/acceptor.
 This speculation was based on proposed models of the agonist and antagonist binding
sites of this receptor that have been elucidated using molecular biological and
computational procedures.
 On this premise, the newly discovered lead compound, I, was altered such that the
Cterminal arginine was replaced by 3',5'-dimethylpyrimidylornithine in an attempt to
increase potency.
 This known mimetic of arginine contains an aromatic 3',5'-dimethylpyrimidyl ring in the
side chain rather than the guanidino group on naturally occurring arginine.
 The results of the receptor binding assay performed using this compound, IA, are shown
in Table 4 where it is clear that affinity to the human B2 receptor is improved with
respect to compound I.

36.  This data is supportive of the notion that the C-terminal residue(s) in this new series of
bradykinin antagonist compounds interact with a hydrophobic environment, perhaps
within the transmembrane domain of the receptor as previously suggested.
 The discovery of I and IA is significant in many regards.
 First, they are highly nonpeptidic lead compounds that could be further modified to
improve potency and/or reduce molecular weight.
 Such improvements might lead to novel therapeutic agents for the treatment of
inflammatory diseases.
 Thus far in the kinin antagonist literature there is significant evidence showing that,
for compounds containing a C-terminal arginine residue, removal of that arginine
generally yields compounds that are antagonists of the B1 subtype of the bradykinin
receptor.
 Following a similar strategy with compound I could lead to the discovery of a novel
series of nonpeptidic B1 receptor antagonists, although this remains to be
demonstrated

38. Thrombin inhibitors

39. I.ROLES OF THROMBIN IN HEMOSTASIS AND THE
THERAPEUTIC UTILITY OF THROMBIN INHIBITORS:
Thrombin is a serine protease.
It plays critical roles in both anticoagulation and blood cloat formation.
In penultimate step of the coagulation cascade;
Soluble fibrinogen
cleavage
Insoluble fibrinogen
thrombin

40. Thrombin activates Coagulation factor (XIII)
stabilize
Fibrin thrombus
Additional clot formation, thrombin participates in anticoagulation functions.
Most drug design efforts focus on thrombin inhibition as a means to prevent
the serious consequences of thrombus formation in myocardial infraction and
stoke.
Thrombin inhibitors clot formation in patients prone to deep vein
thrombosis or repeat hear attack.
Thrombin inhibitors may decreases the incidence of reocclusion , to release
active clot-bound thrombin
Prevents

41. II. STRUCTURE OF THROMBIN
Thrombin consists of two polypeptides, an
A chain of 36 residues and a 259-residue B
chain,linked by a disulfide bond.
The crystallographic structure of thrombin
reveals a globular protein organized about
two ß barrels with overall folding pattern
of the chymotrypsin serine protease
family.
Human thrombin large (red) and
small (green) subunits complex
with prolinamide derivative (PDB
code 1ppb

42. Figure 1 Stereoscopic view of the
crystallographic structure of thrombin
complexed with N-acetyl-(D-Phe)-Pro-
boroArg-OH. Helical regions are
represented in the standard way and
arrows indicate regions of β sheet. Solid
lines show the thrombin bound
conformation of N-acetyl-(D-Phe)-Pro-
boroArg-OH . Active-site residues, His57
and Ser195, are shown with a ball-and-
stick representation.

43. Thrombin’s multionality and regulation of activity are
achieved by specialized subsites on the enzyme’s
surface(fig.2).
Fibrinogen cleavage, for example,involves interactions at the
primary specificity pocket, the extended fibrinogen
recognition exosite, and an additional specificity pocket.
 Subsite interactions differ for cleavage of other thrombin
substrates including the thrombin receptor and protein C.
Additional and overlapping subsites exist for thrombin
effector molecules including heparin, antithrombin III, and
heparin cofactor II .

44. Figure 2 Schematic representation of
Subsite Utilization in Thrombin
Complexes.
Fibrinogen interacts with three
thrombin subsites (here thrombin is
represented by a large oval and the
interconnected subsites by an irregular
three-armed shape). Physiological
effectors of thrombin and thrombin
inhibitors form distinct interactions at
these subsites. Additional subsites, such
as the heparin-binding site, exist on the
thrombin surface and are not indicated
here. The catalytic triad is represented
by three circles at the vertices of a
triangle.

45. III.THROMBIN INHIBITORS DIRECTED AT THE
FIBRINOPEPTIDE A BINDING POCKET
The majority of synthetic thrombin
inhibitors interact at the
fibinopeptide A binding
pocket,which include the catalytic
residues Ser 195 and
His57,hydrogen-binding
capabilities with in oxyanion
hole,peptide backbone functional
groups that hydrogen bond with the
peptide backbone of the substrate
and residues involved in amino acid
recognition.
fig 1
Schematic diagram of binding determinants
within the fibrinopeptide A binding pocket of
thrombin and their utilization by N-acetyl-(D-
Phe)-Pro-boroArg-OH

46. Many of the binding determinants are utilized by N-acetyl-(D-Phe)-Pro-
Arg-chloromethylketone (PPACK) and its boronic acid analog (DUP714).
The crystallographic structures of these molecules complexed with
thrombin have both served as starting points for structure-based drug
design and as reference structures for comparison of binding modes of
other inhibitors.
 The use of arginine boronate esters as transition-state mimetics results in
potent peptidyl thrombin inhibitors.
 These inhibitors, however, exhibit significant affinity for other serine
proteases that have in common a specificity for substrates with basic
residues at P1 (e.g. trypsin, Factor Xa, and plasmin).
 Earlier work demonstrated that neutral side chains of P1 boronate esters
impart greater selectivity for thrombin.

47. The boropeptide shown in Figure 2
was investigated as the prototype of
neutral side chain, tripeptide
thrombin inhibitors.
 It had a Ki against thrombin of
and shows selectivity relative to
other trypsin-like plasma proteases.
 Since these inhibitors have a
neutral residue at the P1 site,
Deadman and coworkers sought to
demonstrate the mode of binding to
thrombin in the absence of a salt
bridge with Asp189.
fig 2
Schematic representation of the active-
site orientation of a “neutral” P1 boronic
acid thrombin inhibitor.

48. Boron-11-NMR, a sensitive probe of the chemical environment
around boronate esters, can distinguish between trigonal and
tetrahedral forms of boron.
The 11B-NMR spectrum of this inhibitor complexed with
thrombin showed a single peak at -17 ppm that remained constant
for 7 hours.
The chemical shift suggests boron adopts a tetrahedral geometry
on binding to thrombin and is consistent with the orientation of the
inhibitor in the active site shown in Figure 2.
 While the 11B-NMR revealed an interaction within the catalytic
site, it could not distinguish between bonding with Ser195 or
His57

49. Kahn and coworkers recently
investigated the application of
synthesized peptidomimetics as novel
inhibitors of thrombin.
 Fibrinogen peptide A mimetic
(FPAM, Figure 3) incorporates a
bicyclic peptidomimetic within the
turn region of fibrinogen peptide A.
 The bicyclic peptidomimetic confers
conformational stability to the turn
region as suggested by x-ray crystal
structures of fibrinogen peptide
complexes as well as complexes of
BPTI with thrombin.
fig 3
Schematic representation of the principal
intermolecular interactions of a fibrinogen
peptide A mimetic within the active site of
thrombin

50. Fig 2
Complexed
with arginine
guanidium
Extensive
hydrophobic contacts
within the S2 apolar
binding site
Gly at P3 interact with thrombin via β-sheet-type
hydrogen bond with the carbonyl group gly
216.(Imp in the positioning of the bicyclic ring
corresponding to the β bend
Phenyl rings shows
hydrophobic contact with
11e174
This bicyclic ring,
although not
aromatic , forms
an edge to face
contact with
Trp215
Cyclic template having attachment sites
for three side chains that are
complementary to the S1,S2,S3 sites in
thrombin

51. Obst et al.,departing from thee peptide
template, designed and synthesized novel
nonpeptide inhibitors of thrombin.
Using computational approaches (Insight
II/Discover/CVFF force field), possible
templates were modeled within the active
site of thrombin.
 These studies resulted in the synthesis of
thirteen analogs that shared a common
template.
The most active molecule (Ki = 90 nM,
8-fold selective versus trypsin) was
studied further by x-ray crystallography
(Figure 4).
fig 4
Schematic representation of the
principal intermolecular interactions of
a nonpeptide bicyclic inhibitor within
the active site of thrombin

52. +ve charged
benzamidine binds
into the S1 pocket of
thrombin forming a
bidentate hydrogen
bond with Asp189
The proximal
carbonyl of the
rigid template
acts as a
hydrogen-bond
receptor for the
amideNH of
Gly216
The
methylene
dioxybenzyl
group at P3
interact with
thrombin in
two ways
An edge to
face
interaction
was
observed
withTrp215
An oxygen of
methylenedioxy group
acts as an acceptor for a
hydrogen bond with the
OH hydrogen of
Tyr60A

53. Recent communications from Bristol-Myers Squibb [14,15]
describe peptidomimetic inhibitors (Fig5) that were designed
to bind thrombin with N- to C- polypeptide chain sense
opposite that of the substrate and form interactions similar to
those made by the 1st three residues of hirudin
(11e1,Thr2,Tyr3).
 In the x-ray crystal structure of BMS-183507 (Ki = 17.2
nM) with thrombin.

54. N-terminal
facing the
catalytic
site
Methyl ester is
exposed to
solvent
A bound water
molecule
hydrogen bonded
to the ser195
hydroxyl
Phe 1-O and the
phe3-NH from
hydrogen bonds
with Gly216
Phe1-NH hydrogen
bonds to the backbone
carbonyl of SER214
The retro –inhibitors
contain a 4-
guanidinobutanoyl
group that extends
into the S1
specificity site
Forming two hydrogen bonds
between the guanidine and
Asp189 in a similar manner to
PPACK.
BMS-183507 forms
only one ,The one
hydrogen bond being
directed to the
carbonyl oxygen of
Gly219

55.  cyclotheonamide A (CtA), a
macrocyclic marine natural
product derived from the
Japanese sponge,Theonella sp.,
inhibiys thrombin with an IC50
value of 100 nM and represents a
novel structural class of serine
protease inhibitors.
An x-ray crystal structure of CtA
complexed with thrombin was
used to determine the molecular
basis for this inhibition.(fig 6)
Figure 8 Schematic representation of the
principal intermolecular interactions of
cyclotheonamide A within the active site of
thrombin

56. The Arg-pro unit binds to the S1 and
S2 sites in a similar manner to the
Arg-pro of PPACK.
The Arg guandinium group forms a
bidentate hydrogen bond wth Asp
189 while the pro establishes a
βsheet interaction with the Ser214-
Gly216 backbone.
The α-ketoamide acts as a
transition-state mimetic forming
a tetrahedral hemiketal with the
hydroxyl of Ser195.

57. Starting with the known thrombin inhibitors Argatroban and
Nα(2-naphthyl-sulfonyl-glycyl)-DL-pamidinophenylalanyl-
piperidine (NAPAP), a group at Roche initiated a medicinal
chemistry program to develop thrombin inhibitors with reduced
toxicity and an improved hemodynamic profile.
 The discovery program proceeded in four iterative phases which
are shown in Table 1.
Initial screening of low molecular weight organic bases led to the
discovery of 1-amidinopiperidine (1–1) as a new surrogate for
the guanidine and amidine functionality in Argatroban and
NAPAP, respectively.
A distinct advantage of 1-amidinopiperidine is its intrinsic
selectivity for thrombin over trypsin.

59.  Application of three-fold iterative strategy of design involving synthesis, x-ray
crystallography, and molecular modeling, this group elaborated the 1-
amidinopiperidine from structures that inhibited in the micromolar range to
some inhibiting in the picomolar range.
 In doing so significant improvements in the selectivity of thrombin relative to
trypsin were also achieved.
In the case of the D-amino acid series (1–2), a “second inhibitor binding mode”
that differed from that of Argatroban was identified.
 In this new and unexpected binding mode, the S2 pocket is unoccupied and the
napthalenesulfonyl group fills the S3 site and overlaps the front of the S2 site.
 The benzyl group of the phenylalanine is oriented toward the protein surface
and is partially exposed to solvent.
 The Argatroban or “inhibitor binding” mode was favored by the more potent
L-amino acid series (1–3 and 1–4) where the piperidide (1–3) or Nbenzyl (1–4)
binds to the S2 site and the aryl groups are found in the S3 site.

60. IV.BIVALENT THROMBIN INHIBITORS DIRECTED AT THE
FIBRINOPEPTIDE A BINDING POCKET AND THE
FIBRINOGEN RECOGNITION SITE:
A strategy to prepare highly selective thrombin inhibitors involves linkage
of molecules capable of interacting at distinct subsites.
This approach should result in inhibitors more specific for thrombin: while
serine proteases possess common structural features related to catalysis and
some serine proteases—including the coagulation enzyme Factor Xa—also
exhibit primary substrate specificity for positively charged residues, only
thrombin possesses recognition subsites for fibrinogen and effector
molecules such as thrombomodulin.
Nature has used this strategy in the evolution of hirudin, the anticoagulant
protein produced by the medicinal leech.

61. When this effective anticoagulant binds thrombin, the N-terminal
domain blocks the primary specificity pocket while the C-terminal
residues adopt an extended conformation and make multiple
interactions within the fibrinogen recognition exosite.
Guided by structural and biochemical information, small
molecules capable of simultaneous interactions with both the
primary specificity pocket and the fibrinogen recognition exosite
were designed and synthesized.
 These bivalent inhibitors are composed of three regions: a group
to block the primary specificity pocket, a sequence to bind the
fibrinogen recognition site, and a chemical linker.
The bivalent inhibitor approach was first executed with peptides.

63. In 1990, DiMaio et al. (3–3 [22]) used the peptide sequence from hirudin to link (d-Phe)-
Pro-Arg-Pro, known to bind at the primary specificity pocket [23], with hirudin C-
terminal residues, known to bind at the fibrinogen recognition site.
 Polyglycine linkers were also used to connect these sequences (Maraganore et al).
Among these hirudin analogs,the tetraglycine linker appeared optimal(3-8,ki=2.3nM).
Most of the peptide-based bivalent inhibitors were slowly cleaved by thrombin.
Incorporation of ketomethylene pseudo peptide bond resulted in a non cleavable bivalent
inhibitor that rretained high thrombin affinity.
Decreased proteolysis in bivalent inhibitors increasingly nonpeptide in character to be
observed.
Chemically simpler linkers were made using multiple methylene-containing glycine
variants.
This was confirmed in the crystal structure of hirutonin-6:thrombin complex (3–26 [26])
where continuous electron density was observed for the entire bivalent inhibitor including
the linker region. The extended nature of the fibrinogen recognition site complicates
attempts to reduce inhibitor molecular weight while maintaining affinity.

64.  Only seven residues are present in one of the smallest bivalent inhibitors.
Increasingly nonpeptide substituents have been incorporated into the primary
specificity pocket binding portion of the bivalent inhibitors.
Crystallographic analysis of its complex with thrombin showed the ketone carbonyl
becomes tetrahedrally coordinate by bonding to the side chain of thrombin's active
site residue, Ser195.
Substitutions of cyclohexylalanine for phenylalanine (3–4 compared to 3–5) and the
cyclohexylalanine-containing fibrinogen recognition peptide for the hirudin
sequence (3–17 compared to 3–18) also contribute to the increased affinity of this
bivalent inhibitor

65. Inactivated thrombin as an inhibitor of clot formation:
 A means to selectively inhibit thrombin’s role in coagulation
while preserving its anticoagulant functions involves site-directed
mutagenesis of thrombin itself.
 By introduction of a single mutation,altered thrombin’s relative
specificity for fibrinogen and protein C.
 The engineered thrombin’s increased activation of protein C over
fibrinogen cleavage offers the possibility of inhibiting clot
formation with a modified human protein , a molecule likely to
exhibit few side effects.

66. The role of
structural
information
discovery of thrombin
inhibitors has benefited
from available protein
structural information.
Models of the thrombin overall
structure and its active site
geometry, constructd from
available structures of related
serine proteases,aided in the
design of the mechanism-based
inhibitors such as PPACK and
its boroarginine analog
Unexpected, nonsubstrate
binding mode of early
thrombin inhibitors such as
NAPAP was revealed by x-
ray crystallographic analyses.
Structure-based design methods have
been critical in the optimization of
bivalent inhibitors and inhibitors directed
at the primary specificity
Structures of
inhibitor:thrombin
complexes are essential
for the optimization of
substituents forming
interactions within the
aryl-binding site of the
primary specificity
pocket.
In some cases, minor
alterations of the inhibitor can
result in dramatic changes in
the inhibitor’s overall
interactions with thrombin

67. Roles of
structural
information
Drug discovery efforts have also been
strongly influenced by results of
structural of thrombin complexed with
effectors and structural peptides
For example, recently
the structures of
thrombin complexed
with fibrinopeptide A
and human
prothrombin fragment
F1 have been
determined
In addition to their role
in design of high-
affinity inhibitors,
these structure provide
valuable insights for
design of drugs
specific for the various
subsites and
conformational states
of thrombin

68. VII. CONCLUSION:
Discovery of therapeutically effective thrombin inhibitors
involves issues such as affinity and selectivity,and formulation.
In addition to these relatively common concerns,the complex
invivo mechanisms designed to balanced its pro-and
anticoagulant activites present additional challenges in the
discovery of therapeutically effective thrombin inhibitors.

69. REFERENCES:
Structural based drug design by pandi veerapandion.
Wiliam o foye medicinal chemistry.
Berger’s medicinal chemistry and drug design 6th edition.
The enhanced permeability retention effect: a new paradigm for
drug targeting in infection Ernest A. Azzopardi1,2*, Elaine L.
Ferguson1 and David W. Thomas1 journal.

70. THANK YOU