An introduction into phosphodiesterases and their

potential role as drug targets for neglected diseases

Chapter 1

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1.1 Human African Trypanosomiasis

Human African Trypanomiasis (HAT), also known as African sleeping sickness, is a deadly

infectious disease caused by the kinetoplastid Trypanosoma brucei (Figure 1A). The spread

of HAT is restricted to sub-Saharan Africa by the prevalence of the disease vector, several

species of tsetse fly (genus Glossina, Figure 1B).1, 2 As shown in Figure 1C, the tsetse flies

are found across 38 central African countries, where they are able to cause localized

epidemics following infection by feeding on the blood of infected humans, livestock or wild

animals.3 Following eradication efforts and given that not all tsetse flies become infected or

are able to become infected, 24 countries have reported recent cases of HAT, leaving an

estimated 70 million people at risk of infection.2, 4, 5 While the number of annually reported

cases has remained under 10,000 since 2009, the disease has had three major epidemics in

the last century.1, 6, 7 Furthermore, efforts to control the spread of the disease are hampered

by the remoteness of outbreaks and conflicts in several endemic regions, leading to

incomplete reporting of new HAT cases and an increased risk of another major epidemic.8

Figure 1: A) A stylized representation of Trypanosoma brucei infected blood based on

scanning and transmission electron micrographs.9 B) A tsetse fly drawing blood with its

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proboscis inserted in human skin.10 C) A map showing the spread of the distribution of the

tsetse fly across Africa overlaid with the cases of HAT reported over the period 2000-

2009.11 The images in A, B and C are reproduced without adaptation from the source

referenced.

Two different forms of HAT exist and these result from infection with either T.b. gambiense

or T.b. rhodesiense. The two strains have different epidemiologies, with T.b. gambiense

endemic to central and western Africa accounting for 97% of infections and T.b.

rhodesiense endemic to the eastern Africa.5 Disease progression and therapy are also

affected by the strain. Infection by T.b. gambiense progresses from the haemolymphatic

phase (stage 1) to the meningoencephalitis phase (stage 2) after 2-4 years. In a few cases

patients have cleared the parasites during stage 1 without treatment, however once a patient

passes to stage 2 the disease is invariably fatal without treatment.12 Infection by T.b.

rhodesiense is more aggressive with progression from stage 1 to stage 2 usually occurring

within 8 weeks and no cases of parasitological clearance without treatment have been

reported.13

The treatment options for HAT have improved in recent years. However significant issues

remain.14 For HAT caused by T.b. gambiense the first-line treatment for stage 1 is

pentamidine and the second-line treatment is suramin, for stage 2 the first-line treatment is

NECT (nifurtimox and eflornithine) and the second-line treatment is melarsoprol (Figure

2). For HAT caused by T.b. rhodesiense the first-line treatment for stage 1 is suramin and

the second line treatment is pentamadine, for stage 2 the first-line treatment is melarsoprol

and there is no second-line treatment.15 Each of these treatments are regarded as essential

medicines by the WHO and they are now available free of charge to endemic countries. The

introduction of eflornithine and later NECT to replace melarsoprol as the treatment for stage

2 HAT caused by T.b. gambiense, has improved treatment outcomes significantly, with

deaths following treatment dropping from about 6% to 1%.16, 17 The situation for stage 2

HAT caused by T.b. rhodesiense has not seen such an improvement as eflornithine and

NECT are not effective against this parasite strain. Given the similarities between the two

subspecies, this is an indication that resistance to drug treatments may be able to arise in

HAT caused by T.b. gambiense. Resistance to treatments is an ongoing threat for parasitic

 

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diseases and in the case of HAT, eradication of the disease is unlikely given the reservoir

of trypanosomes found in livestock and wild life.18 This necessitates the discovery of novel

drugs to prepare for the eventuality of resistance to current treatments. Given the areas to

which HAT is endemic, a further complication in treating HAT is that each of these

treatments requires either intravenous or intramuscular administration. Therefore, a key

feature sought in novel treatments is that they be orally available.

Figure 2: The chemical structures of compounds that form part of the WHO recommended

essential medicines for the treatment of HAT, suramin, pentamidine, merlasoprol, and the

two active components of NECT treatment, nifurtimox and eflornithine. Two compounds

currently in clinical trials for the treatment of HAT, SCYX-7158 and fexinidazole.

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There are several promising drug candidates undergoing clinical trials for the treatment of

HAT. A promising compound is SCYX-7158, a benzoxaborole currently in phase 1 clinical

trials and showing the potency and pharmacokinetics consistent with a single dose oral

treatment for stage 2 HAT (Figure 2).19 This compound was discovered and developed using

phenotypic screening and the mode of action remains unknown. To find out if the mode of

action might be related to phosphodiesterase (PDE) inhibition, SCYX-7158 was

synthesized in our lab and found to inhibit human PDE4B with an IC50 of 80 µM and

TbrPDEB1 with an IC50 of >100 µM, ruling out PDE inhibition as a mode of action

(unpublished). Another compound in development for the treatment of HAT is fexinidazole

which is currently undergoing phase II/III clinical trials.20 This too would be an orally

available drug effective against both strains of trypanosomes causing HAT. While such

advances are needed, even with a new drug reaching the market the need for novel

treatments would remain, since resistance can be expected to arise. A further benefit to the

discovery of multiple novel drugs is that the process of resistance can be slowed

significantly by the use of multiple drugs with orthogonal modes of action. For these

reasons, developing new drugs with novel targets for the treatment of HAT remains a high

priority for institutes tackling neglected diseases.21 The drug target that was explored in the

work presented here is phosphodiesterases, specifically TbrPDEB1.

1.2 Phosphodiesterases

Phosphodiesterases (PDEs) play a key role in signaling cascades that involve the second

messenger molecules cyclic adenosine monophosphate (cAMP) and cyclic guanosine

monophosphate (cGMP). Through the hydrolysis of cAMP and cGMP to AMP and GMP

respectively, PDEs regulate the concentrations of the cyclic nucleotides and thereby signal

transmission. Cyclic nucleotide signaling in parasites shows potential as a drug target due

to the important role PDEs play in the life cycles of many parasites.22-24 The Trypanosoma

brucei cyclic nucleotide signaling pathway begins with the activation of membrane bound

adenylyl cyclases (ACs).25 The ACs are bound to the cellular surface with specific

distribution densities determined by localization (for example in flagellar ACs) and life

cycle stage.26 The ACs are activated by an extracellular receptor domain that passes a signal

 

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via a transmembrane domain to an intracellular catalytic domain.24, 27 The activated catalytic

domain converts ATP to cAMP passing the signal into cell and amplifying it though the

creation of many second messenger molecules. The ACs act as sensors, but also play a role

in inhibiting the innate immune response of the host.28 Downstream effectors of cAMP

signaling are still poorly understood, however cAMP dependent protein kinase As (PKAs)

have been identified.29 The concentration of cAMP determines signaling to downstream

effectors and signaling is regulated by the activity of PDEs that hydrolyze the cAMP into

AMP, reducing the cAMP concentration. The cyclic nucleotide signaling pathway has been

found to play important roles in motility and cytokinesis of Trypanosoma brucei.30, 31.

There are 5 trypanosomal PDE genes, A, B1, B2, C and D and the same classification of

genes is seen in leishmanial parasites. Plasmodial parasites also encode 5 PDE genes,

however they are classed, αA, αB, β, γ and δ. Not all of the parasite PDEs are drug targets.

In Trypanosoma brucei the inhibition of cytokinesis is observed when the activity of both

TbrPDEB1 and TbrPDEB2 has been inhibited through either RNAi or pharmacological

means.32-34 Together these form a target for the control of parasite proliferation, however

due to their structural similarity and the correlation in inhibitor activity between them, drug

discovery efforts have focused on TbrPDEB1.32, 33, 35, 36 Since TbrPDEA in not essential and

TbrPDEC is inactive, they are not considered to be drug targets, whereas it is not yet known

whether TbrPDED is essential.24, 37

In humans the signaling cascade usually begins when an extracellular stimulus is detected

by a transmembrane receptor that upon activation stimulates the activity of adenylyl cyclase

(AC) or guanylyl cyclase (GC). While active, AC continuously catalyzes the conversion of

ATP into cAMP and GC continuously catalyzes the conversion of GTP into cGMP. This

results in high concentrations of cAMP or cGMP and a strong amplification of the signal.

The cyclic nucleotides then pass the signal on, usually to protein kinase A (PKA) for cAMP

or protein kinase G (PKG) for cGMP. In turn these protein kinases activate proteins further

down the signaling cascade. The specific roles of cAMP and cGMP are dependent on,

amongst others, the signaling cascade, the cell type and the location of the cyclic nucleotides

in the cell. The concentration of the cyclic nucleotides is reduced by PDEs and there are 21

genes that encode human PDEs. These are divided into 11 gene families that encode

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structurally related PDE subtypes. This diversity allows PDEs to regulate a diverse range

of signaling outcomes.38-40

Each PDE contains a catalytic domain, a lengthy N-terminus that may contain one or more

structured domains and an unstructured C-terminus (Figure 3).41 Multiple isoforms

(resulting from splice variants) of the PDE genes result in differences in the PDE sequence,

or truncation of the PDE sequence when compared to the canonical sequence. The

truncations typically occur at the carboxy-terminal unstructured region or at the amino-

terminal region where one or more structured domain may be absent. Differences in the

sequence of the catalytic domain are not seen and isoforms without a catalytic domain will

be inactive. However PDEs resulting from alternative spicing may show altered catalytic

activity due to the influence of the amino-terminal and carboxy-terminal regions on the

substrate access to the catalytic site. Sequence differences in, or truncation of, amino-

terminal domains may also affect the localization and activation mechanism of a PDE and

generating inhibitors selective for specific isoforms is of interest for drug discovery.42

The structured regions found in the N-termini of PDEs have diverse roles and the role of

the same domain family may vary between PDE families. The GAF domains have been

shown to modulate PDE activity, play a role in PDE dimerization and are able to bind the

cyclic nucleotides cAMP and cGMP.43 Differences in the GAF-A and GAF-B domains of

PDE2, PDE5, PDE6, PDE10, PDE11 and parasite PDEB families influence the specific

substrate binding and dimerization roles of the GAF domains in each case.44, 45 The UCR

domains regulate PDE4 activity and crystallography has shown direct contact between UCR

and inhibitors bound to the active site of PDE4D.46 The activation of PDE1 by Ca2+-

calmodulin through interaction with the CaM binding domain regulates PDE1 activity.47

The PDE amino-terminal domains influence PDE localization, oligomerization and activity,

alternative splicing of the N-terminus further diversifies the influence of PDEs on signaling

pathways.48

 

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Figure 3: The structures and domains of the 11 human PDE families are shown along with

the 3 parasite PDEs. The amino-terminal domains may regulate the activity of the

conserved catalytic domain, play a role in the localization of PDEs or the interaction with

protein partners. The amino-terminal domains include the CaM-binding domain (CaM),

GAF domains, transmembrane domain (TM domain), targeting domain (TD), upstream

conserved regions (UCRs), signal regulatory domain (REC), PAS domain, Pat7 nuclear

localization, FYVE-type domains and coiled coil regions. The names of PDE subtypes are

given in blue in cases where crystal structures of the PDE have been published and the

specific domains that have been crystalized are shown in blue.

The catalytic activity of PDEs in the hydrolysis of cAMP and cGMP is dependent on the

PDE family and to a lesser extent the PDE subtype. An overview of the selectivity of PDEs

for the hydrolysis of cAMP, cGMP or both cAMP and cGMP is provided in Figure 4. The

parasite PDEs of greatest interest as drug targets are shown along with the human PDEs. Of

the trypanosomal PDEs, TbrPDEA and both TbrPDEB1 and TbrPDEB2 hydrolyse cAMP,

while TbrPDEC is enzymatically inactive and the enzymatic activity of TbrPDED is

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unknown.24, 37 Although rates of catalysis may vary across PDE subtypes, the substrate

selectivity remains consistent within PDE families.

Figure 4: The substrate selectivity of PDEs, PDEs listed on the left selectively hydrolyze

cGMP to GMP, those in the middle display a dual selectivity and hydrolyze both cGMP to

GMP and cAMP to AMP and those on the right selectively hydrolyze cAMP to AMP.

The catalytic domains of phosphodiesterases contain a conserved substrate binding site

where the hydrolysis of cyclic nucleotides is catalyzed. The binding of the cyclic nucleotide

substrate is stabilized by the combination of a narrow hydrophobic region, dubbed the

hydrophobic clamp, and multiple hydrogen bonds to a conserved glutamine residue. The

phosphate group of the substrate is positioned in close proximity to the metal binding region

of the pocket. Two metal ions are bound to phosphodiesterases, one of which is a Zn2+ ion

and the second of which is most often Mg2+, but may be Mn2+ or another metal ion.49, 50 The

 

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metal ions coordinate the phosphate group of the substrate and an activated water molecule

attacks the phosphate breaking a phosphate ester bond. The binding and hydrolysis of

cAMP and AMP in PDE4 is shown in Figure 5, which reveals a conserved binding mode

for the substrate and product.51 The hydrolysis of cGMP to GMP by other PDEs follows a

similar mechanism.

Figure 5: A-B) The binding mode of cAMP (cyan, PDBcode: 2PW3) and AMP (magenta,

PDBcode: 1ROR) bound to PDE4 (gray, PDBcode: 1ROR) highlighting the key residues in

substrate stabilization and metal binding. The key residues, which interact with the

substrate, are a conserved glutamine residue (red), two hydrophobic residues which form

a clamp (yellow), a phenylalanine and an isoleucine (which may be a leucine or valine in

other PDEs), and a phenylalanine (which may be a tyrosine in other PDEs). The metal ions

zinc and magnesium are shown along with a key residue in metal binding, a conserved

aspartate (blue). C) The hydrolysis of cAMP to AMP with the bond broken indicated in red

and the added water in blue.

Through the analysis of PDE crystal structures published in the Protein Databank (PDB), a

set of 57 residues were identified as the PDE pocket residues. A novel nomenclature for

PDE pocket residues was devised, which combines the amino acid code, isoform residue

number, pocket region and pocket residue number (Figure 6A). The residues were divided

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into 10 pocket regions, Q, Q1, Q2, HC, HC1, HC2, S, MB, MB1 and MB2 (Figure 6B). The

Q region refers to the conserved glutamine, QQ.50, which is flanked by the Q1 and Q2

regions. The HC region refers to the hydrophobic clamp made up of a conserved

phenylalanine at FHC.52 and a hydrophobic residue at (I/L/V)HC.32 which may be isoleucine,

leucine, or valine. The HC region is flanked by the HC1, HC2 and S regions, S region refers

to a region exposed to bulk water that may contain multiple water filled subpockets. The

MB region refers to the metal binding region in which two metal ions are coordinated by

several conserved residues. The Zn2+ ion is coordinated to HMB.03, HMB.04, DMB.05, DMB.22 and

two water molecules and the second metal ion is coordinated with DMB.05 and 5 water

molecules.

An estimate of importance of the PDE pocket residues to ligand binding can be derived

from the percentage of ligands, which show interactions with the residues in crystal

structures. Using interaction fingerprints (IFPs) to identify interactions, these percentages

are shown as color coding in Figure 6C (a complete analysis of this data is provided in

Chapter 2). From this figure it is clear that of the 57 PDE pocket residues, 16 pocket

residues do not form interactions with ligands (17 including Q2.44) and a further 12 form

interactions with 3 ligands or less. A significant number of interactions are formed between

ligands and 28 residues, and of these 7 residues form interactions with over 75% of ligands

and can be described as key ligand binding residues, (I/L/V)MB1.17, (I/L/M)HC1.23,

(I/L/V)HC.32, (F/Y)S.35, (F/G/L/M/V)S.40, QQ.50 and FHC.52. The generic pharmacophore for

PDE inhibitors is determined by these interactions, chiefly the interactions with the

hydrophobic clamp residues, (I/V/L)HC.32 and FHC.52, and (F/Y)S.35 that together form a

hydrophobic cleft invariably occupied by aromatic or fully conjugated ring systems, and the

adjacent QQ.50 with which over 90% of PDE inhibitors form hydrogen bonds.

Another way to assess the role of particular residues is to perform mutagenesis studies. As

an enzyme there are multiple outcomes that can be tested, the catalytic activity of the

mutated PDE, inhibitor binding to the mutated PDE, or the inhibitory activity of PDE

inhibitors against mutated PDEs. An overview of the mutagenesis studies and outcomes for

PDEs is provided in Figure 6D. From this overview it is clear that the identities of key

residues involved in ligand binding are important to PDE function and inhibition, as might

 

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be expected. There is a correlation for these residues with the residues found to form most

interactions with ligands (Figure 6C), however in the metal binding region mutating

residues has a significant impact on PDE activity, while these residues do not play a

significant role in ligand binding.

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Figure 6: A) A nomenclature is presented that combines the standard amino acid reference

containing the single letter amino acid code (red) and isoform specific residue number

(purple) with the PDE pocket residue region name (blue) and the PDE pocket residue

 

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number (green). When referencing PDE pocket residues of a subtype the isoform number

may be omitted (YHC1.01) and when referencing PDE pocket residues across the families the

amino acid code and isoform number may be omitted (HC1.01). B) The 57 PDE binding

site residues, defined, colored, and labeled according to the novel PDE pocket regions and

nomenclature presented in Chapter 2. The names of the residues are made up of the amino

acid code, regions of the PDE pocket (Q, Q1, Q2, HC, HC1, HC2, MB, MB1, MB2 and S)

and the relative position of the pocket residue in the PDE sequence (the isoform residue

numbers are not shown). C) The pocket residues are colored according to percentage of

PDE crystal structures in which the residues are found to form interactions with ligands,

using IFP analysis to determine interactions (Chapter 2). D) An overview of the mutational

studies performed on PDEs. The size of the spheres denotes the number of mutational

studies performed on a particular residue. The color denotes the average change in the

reported parameter of PDE function or inhibition.

1.3 Drugs targeting PDEs

PDE activity plays an important role in a wide variety of signaling pathways, making PDEs

attractive therapeutic targets. The value of modulating PDE activity is evident when

considering the 23 PDE inhibitors that are approved for use as drugs (Figure 7). This is an

impressive number of drugs for a single proteins class, although it is noted that some of

these compounds bind not only PDEs, but also other targets (consider for example the

multipharmacological profile of felodipine, levosimendan, ibudilast, dipyridamole, and the

xanthines). Several drugs which act through PDE inhibition were not known to be PDE

inhibitors during their development (amrinone,52 anagrelide,53 cilostazol,54 papaverine

[1848, Georg Merck],55 and enoximone56). Drotaverine is a derivative of papaverine

developed as No-Spa in Hungary in 1961 by Chinoin. However the rational of its design is

not reported.

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Figure 7: The drug molecules approved for therapeutic use which are known to inhibit PDE

activity.

The first drug developed with PDE inhibition known to be a probable mode of action was

the PDE3 inhibitor milrinone, a derivative of amrinone (Figure 8A). The identification of

milrinone as a clinical candidate followed phenotypic screening using inotropic activity to

identify hits.57 The discovery of roflumilast followed from the earlier discovery of the

selective PDE4 inhibitor rolipram (Figure 8B).58 An extensive structure-activity

relationship study around rolipram was published prior to the discovery of roflumilast, that

included piclamilast a PDE4 inhibitor with a 1500 fold improvement in potency over

rolipram.59 Roflumilast became the first drug designed as a PDE4 inhibitor to reach the

market in 2011, following the failures of rolipram, cilomilast and piclamilast in clinical

trials due to lack of efficacy or failure to show sufficient safety.60

The difficulty in developing PDE4 inhibitors with acceptable safety profiles was made

worse by side effects associated with PDE4 inhibition itself. These include nausea, emesis,

abdominal pain and diarrhea.61-63 These have been factors in the failure of rolipram,

cilomilast and piclamilast and nausea and diarrhea appear on the label of Daxas©

(roflumilast).60 The severity and frequency of side-effects are dependent on several factors;

the PDE4 subtype (with PDE4D associated with emesis), the specific PDE4 isoforms or

 

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localized populations of PDE (e.g. in the brain) inhibited and the distribution of the inhibitor

in the body.64 Additionally PDE4 inhibitors may act as dual inhibitors of PDE3 raising the

potential of cardiac safety concerns.65 The importance of selectivity in the development of

PDE inhibitors is highlighted by the lengthy path to develop the first drug targeted at PDE4

inhibition.

N

Cl Cl

HN O

O

F

F

O

RoflumilastPDE4 IC50 0.8 nM

NN O

NHN O

N N

O

O

HN

O O

SNN

SildenafilPDE5 IC50 3.5 nM

TadalafilPDE5 IC50 5 nM

OO

NH

O

H2N

AmrinonePDE3 IC50 15 M

N

NH

N

O

MilrinonePDE3 IC50 1.5 M

N

N

HN O

NHN O

ZaprinastPDE5 IC50 2.0 Mnon-selective

NN O

NHN O

pyrazolopyrimidonePDE5 IC50 0.3 M

NHN

N

N

O

HO

NH2

O

OP

O

O

HO

cGMP

N

Cl Cl

HN O

O

O

PiclamilastPDE4 IC50 1 nM

O

O

RolipramPDE4 IC50 1.5 M

HNO

N

HN

GR30040XPDE5 IC50 0.3 M

N O

OHN

-CCEPDE5 IC50 0.8 M

N

O

O

N

HN

Hydantoin Lead 2aPDE5 IC50 8 nM

N

O

O

O

A

B

C

D

Figure 8: The discovery of the drugs, milrinone (A), roflumilast (B), sildenafil (C), and

tadalafil (D), that target PDEs.

Sildenafil stands out as the first rationally designed PDE inhibitor to reach the market and

a remarkably successful drug under the name Viagra (Figure 8C). The program was initiated

by Pfizer to discover a treatment for hypertension and other cardiovascular indications in

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1985, when little was known about the role of cGMP or the target PDE5.66 The group

studied the electronic distributions of cGMP and zaprinast (known to bind PDE5 weakly)

to determine the dipole moments and the conformations of the ligands when crystalized.

This enabled the suggestion of alternative hetrocycles and decorations to the scaffold that

maintained the ligand’s ability to form core interactions. This resulted in an intermediate

pyrazolopyrimidone with a 10 fold higher inhibition of PDE5 than zaprinast. From here the

focus was on mimicking the phosphate group of cGMP to further stabilize binding, achieved

with the addition of a sulfonamide group. Despite the vector of the sildenafil sulfonamide

differing greatly from that of the cGMP phosphate in later crystal structures, sildenafil

proved 1000 fold more potent than zaprinast and much more selective for PDE5. The

clinical trials gave surprising results that led to sildenafil being labeled as a treatment for

erectile dysfunction (ED) instead of as a treatment for cardiovascular disease, though the

label was later extended to include pulmonary arterial hypertension (PAH).

The success of sildenafil on the market, lead to several me-too drugs, vardenafil, udenafil,

tadalafil and most recently avanafil. The discovery routes of vardenafil, udenafil (developed

in South Korea) and avanafil (developed in Japan) are not published. The discovery of

tadalafil progressed from ethyl β-carboline-3-carboxylate (β-CCE), which was found to

inhibit PDE5 (Figure 8D).67 In a program of medicinal chemistry exploring the chemical

space around β-CCE the intermediate GR30040X was discovered to improve the inhibition

of PDE5. This was optimized further to the hydantoin lead 2a resulting in a 50 fold

improvement in PDE5 inhibition and again to form tadalafil with a minor improvement in

PDE5 inhibition.68 The exact progression has been questioned in court by Vanderbilt

University. In their claim, which was rejected by the court, they state that a compound (8-

(4-hydroxy phenylthio)-IBMX) in a research proposal they provided to the Glaxo France

laboratory was used to progress the discovery of tadalafil.69

 

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Figure 9: The fold inhibitions of PDE1, PDE6 and PDE11 relative to the inhibition of

PDE5 for drugs that target PDE5.70, 71

All drugs targeting PDEs developed so far were conceived before the first PDE crystal

structures were published. This precludes the use of structure-based drug design methods

to support the development of compounds selective for one particular PDE family. As has

been discussed in relation to PDE4, selectivity is a key aspect of drugs targeting PDEs as

the unintentional inhibition of off target PDEs can lead to significant side effects. This is

perhaps most visible in the various drugs targeting PDE5 which show some correlation

between PDE selectivity and side effects (Figure 9). Inhibition of PDE1 is associated with

cardiac events and flushing, side-effects most common to sildenafil, though these effects

may also result from PDE5 inhibition.72 Inhibition of PDE6 (found in the retina) can lead

to visual disturbances, a prominent side effect of sildenafil and udenafil. Inhibition of

PDE11 is associated with myalgia and back pain, side effects most prominent for tadalafil.72

Developing PDE inhibitors into drugs today means being aware of the selectivity profiles

of compounds across a panel of PDEs in order to identify the desired selectivity and

structural biology plays a key role in that process.

1.4 PDE Crystal structure analysis

The 168 PDE crystal structures available in the PDB are a valuable resource for the

discovery of novel PDE inhibitors. An extensive analysis of the PDE crystal structures is

1

10

100

1000

10000

sildenafil vardenafil udenafil tadalafil avanafil

Fold PDE5

 inhibition

PDE1 PDE6 PDE11

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presented in Chapter 2, in which IFPs were used to assess the interactions formed between

ligands, metals, water molecules and the PDE binding pocket in each of the PDE crystal

structures. Such analyses provide medicinal chemists with an overview of complex

structural data in a practical format allowing them to quickly compare multiple PDE crystal

structures.

The effective visualization of the information from a PDE-ligand complex can help the

interpretation and comparison of multiple PDE crystal structures. Retaining information

about the complex when viewing just the ligand is a way to compare many crystal structures

in an efficient manner, as shown in Figure 10. Here the colors of the pocket regions close

to atoms are projected onto the atoms in a figure of the ligand structure. In this way a single

figure of the ligand can be used to convey information about the binding mode of the ligand

in the binding site and the degree to which each atom of the ligand interacts with regions of

the pocket. The color coded ligand figures are generated by applying a distance dependent

translucence in relation to each of the regions for each atom of the ligand and then

overlaying and averaging the resulting figures.

Figure 10: Visualizing occupation of pocket regions in single ligand figures using the color

coding of the PDE pocket. A) The pocket color coding used to color ligand atoms. B) The

two enantiomers of rolipram bound to PDE4B (PDB code 1XN0). C) The conformation of

piclamilast as bound to PDE4B (PDB code 1XM4). D) The conformation of roflumilast as

bound to PDE4B (PDB code 1XMU).

The comparison of the shapes and pharmacophore points of PDE binding pockets can also

provide information on the similarity between PDEs. A new method was devised for this

task. The Pymol plugin, Castp was used to generate binding sites from each published PDE

crystal structure using a standardized algorithm (Figure 11A).73 The binding sites were then

overlaid using the software application ROCS to score the similarity between PDE binding

 

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pockets.74 ROCS was developed to perform 3D pharmacophore searches on small

molecules, applying it to binding sites was a novel use. The package uses a combination of

shape overlap and pharmacophore overlap to optimize and rank the overlap of structures

and generates a set of coordinates for the best fit. Averaging the scores across the PDE

subtypes provided the input for the table shown in Figure 11B. This can be used to identify

the most likely off targets when targeting a particular PDE subtype, or to identify similar

PDEs when selecting compounds to test on a novel PDE subtype for which few inhibitors

are known. The same process can be applied across protein classes to identify potential off-

target binding sites and similar binding sites for which ligands may prove valuable starting

points and against which drugs may potentially be repurposed. Looking for example at the

TbrPDEB1 and lmjPDEB1 parasite PDEs, the most similar binding pockets are indicated

to be PDE4B and PDE4D, a finding supported by sequence similarity, analysis of the

binding sites and the activities of TbrPDEB1 inhibitors across the complete set of human

PDEs.35, 75

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Figure 11: A) The process followed to compare the binding sites of PDE crystal structures.

In the first step the binding site is extracted by Castp and in the second the binding sites are

compared by ROCs. B)A ROCS analysis of the binding sites extracted from PDE crystal

structures showing the highest sum of shape and color (pharmacophore similarity) between

crystal structures of any two PDE subtypes. The cells are colored as a heat map with green

indicating high similarity and red indicating lack of similarity.

1.5 PDEs as targets for the treatment of neglected tropical diseases

The catalytic domains of both human and parasite PDEs share a high degree of structural

similarity (Figure 12A). This similarity enriches the chances of identifying novel parasite

PDE inhibitors when screening collections of human PDE inhibitors and has led to the

discovery of a number of early hit molecules in parasite PDE drug discovery efforts.32, 36, 76,

77 However, compounds are needed that are selective for parasite PDEs, so once an inhibitor

of a parasite PDE is found it must be optimized to achieve selectivity against the human

PDE with the greatest similarity. In the case of TbrPDEB1 and LmjPDEB1 it is the human

PDE4 that shows the greatest similarity structurally and pharmacologically.

Achieving selectivity requires ligands bound to parasite PDEs to form interactions with

residues that differ from the residues at the same position the human PDE or that occupy

space that is not accessible in the human PDE. Subtle factors such as protein flexibility and

water network disruption also play a role, but are hard to target in ligand design. In the case

of TbrPDEB1 and human PDE4, the key interactions between PDE4 inhibitors bound to

PDE4 and TbrPDEB1 are conserved. In order to develop TbrPDEB1 selective compounds,

they need to interact with an unconserved region of the pocket. The parasite specific P-

pocket is a region that differs between parasite and human PDEs and has been the focus for

developing selective parasite PDE inhibitors (Figure 12B-E).24, 35, 77 The P-pocket is very

similar in TbrPDEB1 and LmjPDEB1, in TcrPDEC the position and size differ and in

PDE4, like other human PDEs the P-pocket is not present.

 

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Figure 12: A) A superposition of crystal structures of TbrPDEB1 (blue, 4I1535), LmjPDEB1

(green, 2R8Q77) with IBMX bound (blue), TcrPDEC (purple, 3V9437) with WYQ bound

(pink) and PDE4B (yellow, 1XM478) with piclamilast bound (green). Close ups of the

binding pocket are shown in B (TbrPDB1), C (LmjPDEB1), D (TcrPDEC) and E (PDE4B)

with the P-pocket indicated in parasite PDEs.

HAT is being targeted through inhibition of TbrPDEB1 and TbrPDEB2 simultaneously.

This has been shown to result in a halt of cell proliferation, lysis of the cells and in vivo

clearance of the parasites from infected mice in a series of validation studies.32, 34, 75 The

sequence identity when TbrPDEB1 and TbrPDEB2 are aligned is 75% and no residues on

the surface of the binding site differ (Figure 13) coupled with the correlation in the

inhibition of these enzymes, this allows efforts to be focused on just TbrPDEB1. 32, 33, 35, 36

Figure 13: A superposition a TbrPDEB1 crystal structure (blue, 4I15) and a homology

model of TbrPDEB2 (red) showing the residues that differ as sticks.

 

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The exact roles of cAMP in the Trypanosoma brucei life cycle is still uncertain.39 It plays a

role in differentiation of trypanosomes through the activation of protein kinase A (PKA).79

The concentration of cAMP also impacts cytokinesis during cell division. When TbrPDEB1

and TbrPDEB2 are inhibited or inactivated through RNAi, the internal cAMP concentration

increases dramatically.34 Cells appear phenotypically affected as cytokinesis begins and

multiple nuclei and kinetoplasts appear, a process that eventually leads to cell lysis.32-34

Targeting PDEs may also be applicable to treat other neglected tropical diseases.

Leishmaniasis is a disease found across the intertropical and temperate regions of the world

where it is caused by infection by Leishmania species (Leishmania major, Leishmania

infantum, Leishmania donovani, Leishmania mexicana or Leishmania braziliensis) that are

spread by the phlebotomine sandfly. As in the case of trypanosomes, it is the PDEB family

which is has been most studied as a drug target. However in the case of Leishmanial PDEB1

and PDEB2 the targets have not been validated and it remains uncertain whether they are

essential.80 A particular complication in assessing the sensitivity of Leishmania species to

PDE inhibition is that the parasites can be present in either promastigote or amastigote forms

and the amastigotes are found within human cells. Assays have relied on visual inspection

of human cells for the content of amastigotes, or high throughput yet poorly predictive

promastigote screening, though the first biochemical assays have recently been developed

within the T4-302 project.81, 82

Chagas disease is found in South America where it is spread by the faeces of triatomine

bugs infected with Trypanosoma cruzi. In the case of Trypanosoma cruzi inhibition of

PDEC has been validated as a means of controlling the parasite.23 The crystal structures of

Trypanosoma cruzi PDEC show a binding site ,which diverges significantly from those of

the PDEB families in Trypanosoma brucei or Leishmania species.

Malaria is no longer considered a neglected tropical disease and significant and ongoing

efforts are being made to control the spread of malaria.83 Nevertheless, continued drug

discovery efforts are prudent given the rate at which resistance to antimalarial drugs has

grown in the past.84 Malaria is caused by infection with one of the Plasmodium species

(Plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, amongst others)

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following a bite by an infected female Anopheles mosquito. Although Plasmodium PDEs

are yet to be validated as drug targets for the control of malaria, there are indications that

PDE inhibitors could function as antiplasmodials.22, 85

Figure 14: A phylogenetic tree showing the evolutionary relationships between known

human and parasite PDEs and a series of putative parasite PDEs.

Looking beyond these four diseases there are other neglected tropical diseases caused by

parasites where research on PDEs is still in its infancy. In order to identify PDE sequences

in these parasites a series of BLAST searches were performed starting from each human

PDE canonical sequence and each verified parasite PDE. The results were filtered to

identify sequences derived from the following genera, Trypanosoma, Leishmania,

Plasmodium, Schistosoma, Brugia, Wuchereria, Loa, Onchocerca and Wolbachia. The

 

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remaining sequences were further filtered on the presence of residues known to play key

roles in the function of PDEs. The remaining sequences were realigned and the phylogenic

tree shown in Figure 14 was generated. Two genera were not present in the results,

Onchocerca and Wolbachia. The putative PDEs of Schistosoma, Brugia, Wuchereria and

Loa were found to show greater similarity to human PDEs than to those of Trypanosoma,

Leishmania and Plasmodium. This suggests that screening human PDE inhibitors against

Schistosoma, Brugia, Wuchereria and Loa could prove to be an excellent starting point to

validate these PDEs as potential drug targets.

1.6 Research Aim

The aim of the research presented in this thesis was to discover novel selective inhibitors of

TbrPDEB1 with the potential for development into drugs to treat HAT. In support of that

aim, improving the structural understanding of PDEs was set as a priority in order to allow

rational drug design and support future PDE drug discovery efforts.

1.7 PDE Drug Discovery

During the research presented in this thesis a diverse range of drug-discovery methods were

deployed with the aim of discovering parasite PDE inhibitors. Several key drug-discovery

resources were available at the start of the project: the publicly available crystal structures

of many human PDEs and the parasite PDE LmjPDEB1, a proprietary lead compound series

that potently inhibits parasite PDEB subtypes, a screening assay for parasite and human

PDE inhibition and a phenotypic parasite screening assay. Over the course of the project

crystal structures of TbrPDEB1 became available opening the door to further drug-

discovery methods.

In Chapter 3 TbrPDEB1 inhibitors were found using fragment-based drug design (FBDD).

In FBDD, molecules that satisfy the definition of a fragment, e.g. molecules with 20 or

fewer heavy atoms, are identified as starting points for further optimization. These initial

fragments can be identified by fragment screening using computational screening,

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pharmacological screening, or, as in this case, from literature. Fragments tend to show weak

activity. However, when corrected by the number of atoms, the ligand efficiency (LE) of

fragments can be high. Maintaining a high LE while growing fragments is a key challenge

in FBDD. Structural knowledge of the ligand binding site and the binding mode of the

fragment provide the means to rationally grow the fragment while trying to keep the LE

high. In the case of targeting TbrPDEB1, a homology model was created of TbrPDB1 using

the LmjPDEB1 crystal structure as a reference. This homology model was used to predict

the binding modes of the fragments and intermediates during the fragment growing process.

In Chapter 4 virtual screening was used to identify novel TbrPDEB1 inhibitors using the

first TbrPDEB1 crystal structure to be published. Virtual screening is the process of

identifying active ligands from a database of molecular structures. The identification can

proceed in a ligand-based fashion, through the comparison of molecules in the database to

a reference molecule, or a pharmacophore model. A pharmacophore model abstracts several

positions in space using the conformations of one or more reference molecules. A sphere at

each isolated position describes one or more molecular features and rules regarding which

spheres and features must be matched by conformations of ligands in the database to be

considered hits. If a protein structure is available, structure based virtual screening may be

used to identify hits from a database. In this case binding modes of the molecules in the

database are predicted using docking software and one or more scoring functions are used

to identify hits. In the virtual screening method presented in Chapter 4, a database of

commercially available molecules was filtered and then docked into an unliganded

TbrPDEB1 crystal structure. The interactions between each docked pose and the protein

were calculated in the form of interaction fingerprints (IFPs) and a score was generated by

comparing the IFPs to the IFP of a reference compound. Ranking combined the docking

scores and IFP similarity scores with selected references, to identify hit compounds.

In Chapter 5 ligands were discovered using structure guided design with multiple liganded

TbrPDEB1 crystal structures and scaffold merging. In structure guided design, crystal

structures of intermediate compounds guide the discovery of target compounds, often in an

iterative process. In scaffold merging, substructures from multiple ligands are brought

together to form a new molecule. Once crystal structures showing the binding modes of

 

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potent but non-selective TbrPDEB1 inhibitors described in Chapter 4 were available, it

became clear these were not targeting a parasite specific sub-pocket, dubbed the P-pocket.

Through the analysis of all published PDE crystal structures, scaffolds were identified to

merge with those of the potent non-selective TbrPDEB1 inhibitors, with the aim of

rigidifying the ligands and forcing occupation of P-pocket. The resulting inhibitors proved

to be both potent and selective inhibitors of TbrPDEB1.

1.8 Project T4-302

The research presented in this thesis has taken place within the context of a consortium

financed mainly by Top Institute Pharma (TI Pharma). The aims of the consortium were, to

validate the use of parasite-specific PDE inhibitors as therapeutic agents in Leishmania and

Trypanosoma infections; develop suitable medicinal chemistry leads targeting PDEs as

drug candidates in these and other neglected tropical diseases (NTDs); and to provide a new

platform for TI Pharma to exploit rational mechanism-based approaches to NTD discovery.

Resources to achieve these aims were provided by each of the consortium members.

32  

Figure 15: An overview of the consortium members and their contributions to project T4-

302. The aim of this project was to identify phosphodiesterase inhibitors with the potential

to be developed into drugs, like Viagra © shown at the center.

The consortium contained seven members, TI Phama, University of Bern, Nycomed,

Mercachem, VU University Amsterdam, IOTA Pharmaceuticals, DNDi and the Royal

Tropical Institute (Figure 15). TI Pharma coordinated the public-private partnership in

which private contributions were matched by public contributions. The University of Bern

is where phosphodiesterases (PDEs) were first found to be essential for parasite

proliferation in Trypanosoma brucei and Leishmania major and they provided phenotypic

screening against the parasites. Nycomed (now part of Takeda), provided access to a series

of Trypanosoma brucei phosphodiesterase B1 (TbrPDEB1) inhibitors discovered through

 

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high throughput screening, along with pharmacological screening against parasite and

human PDEs. Mercachem provided novel PDE inhibitor synthesis contributions and

support from experienced medicinal chemists. VU University Amsterdam provided novel

PDE inhibitor computational design and synthesis contributions and novel pharmacology

technique research. IOTA provided access to a fragment library for phenotypic and

enzymatic screening and performed biochemical and biophysical screening. DNDi provided

phenotypic screening and basic toxicology screening of project compounds. The Royal

Tropical Institute in Amsterdam developed novel screening methods to identify parasite

infections and provided phenotypic screening against parasites.

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