Characteristics of the Protoporphyrin IX Binding Sites on Human Serum Albumin Using Molecular Docking

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Article Characteristics of the Protoporphyrin IX Binding Sites on Human Serum Albumin Using Molecular Docking

Leszek Sułkowski 1,*, Bartosz Pawełczak 2, Mariola Chudzik 2 and Małgorzata Maci ˛azek-Jurczyk˙ 2

1 Department of General and Vascular Surgery, Regional Specialist Hospital, Bialska 104/118, 42-218 Cz˛estochowa,Poland 2 Department of Physical Pharmacy, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Jagiello´nska4, 41-200 Sosnowiec, Poland; [email protected] (B.P.); [email protected] (M.C.); [email protected] (M.M.-J.) * Correspondence: [email protected]; Tel.: +48-792-244-177

Academic Editors: Josef Jampilek and Atanas G. Atanasov Received: 25 September 2016; Accepted: 2 November 2016; Published: 17 November 2016

Abstract: Human serum albumin (HSA) is the main plasma protein responsible for a distribution of drugs in the human circulatory system. The binding to HSA is one of the factors that determines both the pharmacological actions and the side effects of drugs. The derivative of heme, protoporphyrin IX (PpIX), is a hydrophobic photosensitizer widely used in photodynamic diagnosis and therapy of various malignant disorders. Using absorption and fluorescence spectroscopy, it has been demonstrated that PpIX forms complexes with HSA. Its binding sites in the tertiary structure of HSA were found in the subdomains IB and IIA. PpIX binds to HSA in one class of binding sites with the association constant of 1.68 × 105 M−1 and 2.30 × 105 M−1 for an excitation at wavelength λex = 280 nm and 295 nm, respectively. The binding interactions between HSA and PpIX have been studied by means of molecular docking simulation using the CLC Drug Discovery Workbench (CLC DDWB) computer program. PpIX creates a strong ‘sandwich-type’ complex between its highly conjugated porphine system and aromatic side chains of tryptophan and tyrosine. In summary, fluorescent studies on binding interactions between HSA and PpIX have been confirmed by the results of computer simulation.

Keywords: photodynamic diagnosis; photodynamic therapy; protoporphyrin IX; human serum albumin; UV-VIS; emission ﬂuorescence; molecular docking

1. Introduction The laser light affecting tissues can be used in the treatment of cancers and pre-cancerous lesions. An illumination initiates a photochemical reaction leading to the formation of cytotoxic compounds, which induce cell death [1,2]. Damage of the mitochondrial membrane, liposomes, Golgi apparatus, cytoplasmatic reticulum and nuclear membrane has been observed. This reaction cannot occur without a photosensitizer [3,4]. Photodynamic diagnosis (PDD) and therapy (PDT) take advantage of this phenomenon. The prevalence of the photodynamic reaction in neoplasm tissue and its surroundings is essential. PDD and PDT of cancer and pre-cancerous lesions require photosensitizers, which are activated by light [1,4]. Any particular photosensitizer cannot be highly effective in the treatment of different neoplasms. Its effectiveness depends on the chemical structure, lipo- or hydro-phobic properties, carrier and solvent, type of neoplasm, pH of intracellular and extracellular ﬂuid, concentration of photosensitizer and the type of complexes formed with carriers.

Molecules 2016, 21, 1519; doi:10.3390/molecules21111519 www.mdpi.com/journal/molecules Molecules 2016, 21, 1519 2 of 19

The porphyrin is a macrocycle composed of four pyrrole rings bridged by four sp2 hybridized carbon atoms [2,5]. Protoporphyrin IX (PpIX) is a clinically useful hydrophobic photosensitizer, which accumulatesMolecules the2016, lipid21, 1519 structures of cancer cells. PpIX is a heme metabolite [1,6,7]. PpIX is used2 of 19 in both PDD and PDT.The porphyrin is a macrocycle composed of four pyrrole rings bridged by four sp2 hybridized Sincecarbon human atoms serum[2,5]. Protoporphyrin albumin (HSA) IX (PpIX) is a component is a clinically of useful the circulatory hydrophobic system photosensitizer, [8], the understanding which of porphyrinaccumulates binding the lipid to HSA structures is crucial of cancer for PDD cells. and PpIX PDT is a successheme metabolite [5,9,10]. [1,6,7]. PpIX is used in Oneboth of PDD the and most PDT. important properties of HSA is its ability to bind and transport many endo- and exo-genic compounds,Since human serum which albumin have no(HSA) designed is a component transport of the proteins. circulatory Nevertheless, system [8], the compounds understanding having specificof transportporphyrin proteinsbinding to can HSA also is becrucial transported for PDD and by HSA.PDT success The formation [5,9,10]. of albumin-ligand complexes One of the most important properties of HSA is its ability to bind and transport many endo- and in the blood serum preserves ligands against oxidation, reduces their toxicity and improves their exo-genic compounds, which have no designed transport proteins. Nevertheless, compounds having solubility,specific and transport therefore, proteins the formationcan also be transported of the complexes by HSA. improvesThe formation the of transportation albumin-ligand ofcomplexes ligands. Preclinicalin the blood studies serum preserves are crucial ligands in order against to oxidat supportion, andreduces develop their toxicity PDD andand improves PDT. In ourtheir study, absorptionsolubility, and and fluorescence therefore, the UV formation spectroscopy of the complexes was used improves to find the PpIX transportation binding of sites ligands. in transporting protein tertiaryPreclinical structure. studies Molecularare crucial in docking order to was support applied and develop to check PDD and and confirm PDT. In our our experimental study, study.absorption The CLC and Drug fluorescence Discovery UV Workbench spectroscopy (CLC was DDWB) used to find molecular PpIX binding docking sites computer in transporting program is an integratedprotein tertiary virtual structure. environment Molecular for exploringdocking was various applied biochemical to check and processes,confirm our such experimental as binding of study. The CLC Drug Discovery Workbench (CLC DDWB) molecular docking computer program is small molecules (medicines, hormones, vitamins or minerals) to bioactive macromolecules (carrier an integrated virtual environment for exploring various biochemical processes, such as binding of proteins,small receptors molecules or (medicines, nucleic acids). hormones, Inthe vitamins study, or molecular minerals) to docking bioactive simulation macromolecules was (carrier used for the simulationproteins, of bindingreceptors interactions or nucleic acids). between In the the study, macromolecule molecular docking of HSA simulation and the moleculewas used for of PpIX.the simulation of binding interactions between the macromolecule of HSA and the molecule of PpIX. 2. Results and Discussion 2. Results and Discussion 2.1. Formation of the PpIX-HSA Complex 2.1. Formation of the PpIX-HSA Complex The absorption spectra of HSA (I), PpIX (II), as well as the PpIX-HSA system (III) are shown in Figure1The. Difference absorption spectra spectra of (III HSA− (I),I and PpIX III (II),− asII) well were as the also PpIX-HSA recorded. system The (III) absorption are shown spectrum in (I + II)Figure that arose1. Difference by the spectra addition (III of− I spectraand III − of II) PpIX were andalso recorded. HSA does The not absorption overlap spectrum the spectrum (I + II) of the that arose by the addition of spectra of PpIX and HSA does not overlap the spectrum of the complex complex (III). Similarly, difference spectra (III − I) and (III − II) do not overlap spectra (II) and (I), (III). Similarly, difference spectra (III − I) and (III − II) do not overlap spectra (II) and (I), respectively. respectively.A new quality A new in quality the PpIX-HSA in the PpIX-HSA system appeared. system This appeared. suggests This that suggestsa complex that between a complex HSA and between HSA andPpIX PpIX may be may formed. be formed.

Figure 1. Difference spectra of protoporphyrin IX (PpIX) (5 × 10−6 M)-human serum albumin (HSA) Figure 1. Difference spectra of protoporphyrin IX (PpIX) (5 × 10−6 M)-human serum albumin (HSA) (1 × 10−6 M) systems. (1 × 10−6 M) systems. To obtain evidence of the complex formation between HSA and PpIX, fluorescence analysis Towas obtain conducted. evidence of the complex formation between HSA and PpIX, ﬂuorescence analysis was conducted. Molecules 2016, 21, 1519 3 of 19

TheMolecules fluorescence 2016, 21, 1519 of HSA excited at both wavelength λex = 280 nm and 295 nm was quenched3 of 19 by PpIXMolecules (Figure 2016, 221)., 1519 The fluorescence of HSA excited at both λex = 280 nm and 295 nm decreased3 of 19 in The fluorescence of HSA excited at both wavelength λex = 280 nm and 295 nm was quenched by the presence of PpIX (Figure2). A hypsochromic shift of the HSA maximum emission fluorescence PpIX (Figure 2). The fluorescence of HSA excited at both λex = 280 nm and 295 nm decreased in the λ The fluorescence of HSA excited at both wavelength λex = 280 nm and 295 nm was quenched by at expresence= 334 andof PpIX 340 (Figure nm by 2). 7 A and hypsochromic 5 nm, respectively, shift of the is HSA observed. maximum The emission quenching fluorescence effect of at PpIX PpIX (Figure 2). The fluorescence of HSA excited at both λex = 280 nm and 295 nm decreased in the on theλex fluorescence= 334 and 340 ofnm HSA by 7 increasesand 5 nm, respectively, with increasing is observed. concentration The quenching of the effect ligand. of PpIX The on quenching the presence of PpIX (Figure 2). A hypsochromic shift of the HSA maximum emission fluorescence at of thefluorescence protein fluorescence of HSA increases may with be anincreasing effect ofconcentration energy transfer of the ligand. between The tryptophan quenching of (Trp) the and λex = 334 and 340 nm by 7 and 5 nm, respectively, is observed. The quenching effect of PpIX on the tyrosineprotein (Tyr) fluorescence fluorophores may be in an the effect HSA of andenergy chromophores transfer between in tryptophan the PpIX. (Trp) That and may tyrosine occur (Tyr) when the fluorescence of HSA increases with increasing concentration of the ligand. The quenching of the fluorophores in the HSA and chromophores in the PpIX. That may occur when the donor-acceptor donor-acceptorprotein fluorescence distance may does be notan effect exceed of energy 10 nm transfer [11]. Thisbetween confirms tryptophan the formation(Trp) and tyrosine of the (Tyr) complex distance does not exceed 10 nm [11]. This confirms the formation of the complex PpIX-HSA. A PpIX-HSA.fluorophores A blue-shift in the HSA of the and emission chromophores fluorescence in the PpIX. maximum That may is caused occur when by conformational the donor-acceptor changes blue-shift of the emission fluorescence maximum is caused by conformational changes in the HSA, in thedistance HSA, whichdoes not involve exceed the10 nm indole [11]. ringThis locatedconfirms withinthe formation the hydrophobic of the complex pocket PpIX-HSA. in the serumA which involve the indole ring located within the hydrophobic pocket in the serum albumin molecule. albuminblue-shift molecule. of the The emission environment fluorescence of the maximum fluorophore is caused thus by becomes conformational less polar. changes The effectin the ofHSA, solvent The environment of the fluorophore thus becomes less polar. The effect of solvent polarity on serum polaritywhich on involve serum the albumin indole ring fluorescence located within has beenthe hydrophobic described pocket previously in the [serum12]. The albumin blue-shift molecule. of HSA albumin fluorescence has been described previously [12]. The blue-shift of HSA emission fluorescence emissionThe environment fluorescence of excited the fluorophore at λ = 295thus nm becomes can be less attributed polar. The to effect the significant of solvent polarity alteration on serum within the excited at λex = 295 nm can beex attributed to the significant alteration within the fluorophore’s albumin fluorescence has been described previously [12]. The blue-shift of HSA emission fluorescence fluorophore’senvironment, environment, demonstrating demonstrating the exposure theof Trp214 exposure to the of solvent. Trp214 to the solvent. excited at λex = 295 nm can be attributed to the significant alteration within the fluorophore’s environment, demonstrating the exposure of Trp214 to the solvent.

Figure2. Quenching of HSA fluorescence by PpIX obtained for 280 nm (▬■▬) and 295 nm (▬●▬) Figure 2. Quenching of HSA fluorescence by PpIX obtained for 280 nm (– –) and 295 nm (–•–) −6 excitation wavelength. In the inserts: fluorescence emission spectra of HSA at concentration 1 × 10 − excitationFigure2. wavelength. Quenching In of the HSA inserts: fluorescence fluorescence by PpIX emission obtained spectra for 280 of HSAnm (▬ at■ concentration▬) and 295 nm 1(▬×●10▬) 6 M M (I) and HSA in the presence of increasing PpIX concentration in range from 1 × 10−5 M to 3 × 10−4 M excitation wavelength. In the inserts: fluorescence emission spectra of HSA at ×concentration−5 1 × 10−6− 4 (I) and(II, HSA III, IV, in V, the VI, presence VII, VIII) of excited increasing at 280 PpIXnm (Insert concentration 1) and 295 innm range (Insert from 2). 1 10 M to 3 10 M −5 −4 (II, III,M IV, (I) V,and VI, HSA VII, in VIII) the presence excited of at increasing 280 nm (Insert PpIX concentration 1) and 295 nm in (Insertrange from 2). 1 × 10 M to 3 × 10 M (II, III, IV, V, VI, VII, VIII) excited at 280 nm (Insert 1) and 295 nm (Insert 2). The presence of HSA causes the increase of PpIX fluorescence excited at both λex = 280 nm and 295 nm (Figure 3). The presenceThe presence of HSA of HSA causes causes the the increase increase of of PpIXPpIX fluorescencefluorescence excited excited at atboth both λex λ=ex 280= nm 280 and nm and 295 nm295 (Figure nm (Figure3). 3).

(A) (A)

Figure 3. Cont.

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(B)

(C)

(D)

−5 Figure 3. The increase of PpIX (I, 5 × 10 − M)5 fluorescence excited at λex = 280 nm (A) and 295 nm (B) in the Figure 3. The increase of PpIX (I, 5 × 10 M) fluorescence excited at λex = 280 nm (A) and 295 nm (B) presence of HSA (II, 2 × 10−7 M; III, 3 × 10−7 M; IV, 4 × 10−7 M; V, 5 × 10−7 M; VI, 7.5 × 10−7 M; VII, 1 × 10−6 M; in the presence of HSA (II, 2 × 10−7 M; III, 3 × 10−7 M; IV, 4 × 10−7 M; V, 5 × 10−7 M; VI, 7.5 × 10−7 M; VIII, 1.25 × 10−6 M; IX, 2 × 10−6 M; X, 2.5 × 10−6 M; XI, 4 × 10−6 M; XII, 5 × 10−6 M; XIII, 9 × 10−6 M). Quenching VII, 1 × 10−6 M; VIII, 1.25 × 10−6 M; IX, 2 × 10−6 M; X, 2.5 × 10−6 M; XI, 4 × 10−6 M; XII, 5 × 10−6 M; of HSA (I, 7.5 × 10−7 M) fluorescence excited at λex = 280 nm (C) and 295 nm (D) in the presence of PpIX (II, −6 −7 XIII, 91 × 1010−5 M; M).III, 3 Quenching × 10−5 M; IV, of5 × HSA 10−5 M; (I, V, 7.5 9 × 1010−5 M;M) VI, fluorescence1 × 10−4 M; VII,excited 1.5 × 10− at4 M;λ exVIII,=280 3 × 10 nm−4 M). (C) and 295 nm (D) in the presence of PpIX (II, 1 × 10−5 M; III, 3 × 10−5 M; IV, 5 × 10−5 M; V, 9 × 10−5 M; VI, 1 × 10Both−4 M; phenomena, VII, 1.5 × 10 i.e.,−4 quenchingM; VIII, 3 ×of 10HSA−4 M).fluorescence by PpIX (Figures 2 and 3) and an increase of PpIX fluorescence in the presence of HSA (Figure 3), indicate the possibility of an interaction Bothbetween phenomena, HSA and PpIX i.e., quenchingin the binding of HSAsites where fluorescence the Trp and by PpIXTyr residues (Figures are2 and located,3) and i.e., an IB, increase IIA of PpIXand fluorescenceIIIA [13,14]. in the presence of HSA (Figure3), indicate the possibility of an interaction between2.2. Association HSA and PpIXand Stern–Volmer in the binding Constants sites where the Trp and Tyr residues are located, i.e., IB, IIA and IIIA [13,14]. In order to estimate the stability and the strength of the binding of a photosensitizer with protein, 2.2. Associationthe association and (K Stern–Volmera) and Stern–Volmer Constants (KS-V) constants were determined (Figures 4 and 5, respectively). The shape of the plot (Figure 4) points to the presence of one class of binding sites for PpIX molecules Inin orderthe HSA to estimatetertiary structure. the stability The andStern–Volmer the strength constant of the allows binding estimating of a photosensitizer how close the with protein protein, the association (Ka) and Stern–Volmer (KS-V) constants were determined (Figures4 and5, respectively). The shape of the plot (Figure4) points to the presence of one class of binding sites for PpIX molecules in the HSA tertiary structure. The Stern–Volmer constant allows estimating how close the protein fluorophore PpIX is located. Both Ka and KS-V (Table1) are of the same order. This may suggest that Molecules 2016, 21, 1519 5 of 19 Molecules 2016, 21, 1519 5 of 19

fluorophore PpIX is located. Both Ka and KS-V (Table 1) are of the same order. This may suggest that Molecules 2016, 21, 1519 5 of 19 withinwithin one one class class of bindingof binding sites sites on on the the HSA HSA tertiarytertiary structure, structure, PpIX PpIX forms forms a stable a stable complex complex in the in the subdomain IIA where Trp 214 is located and/or in other sites (IB, IIIA) containing Tyr residues. fluorophoresubdomain IIA PpIX where is located. Trp 214 Both is located Ka and and/or KS-V (Table in other 1) are sites of (IB,the IIIA)same containingorder. This Tyr may residues. suggest that within one class of binding sites on the HSA tertiary structure, PpIX forms a stable complex in the subdomain IIA where Trp 214 is located and/or in other sites (IB, IIIA) containing Tyr residues.

Figure4. Scatchard dependence for the PpIX-HSA complex excited at 280 nm (■) (y = −23.317x + Figure 4. Scatchard dependence for the PpIX-HSA complex excited at 280 nm ()(y = −23.317x + 23.663) and 295 nm (●) (y = −18.44x + 18.999). Data determined from emission fluorescence spectra. 23.663) and 295 nm (•)(y = −18.44x + 18.999). Data determined from emission fluorescence spectra. In the insert 1: fluorescence emission spectra of HSA at concentration 7.5 × 10−7 M (I) and HSA in the In theFigure4. insert Scatchard 1: fluorescence dependence emission for the spectra PpIX-HSA of HSA complex at concentration excited at 280 7.5 nm× 10 (■−)7 (My = (I) −23.317 and HSAx + in presence of increasing PpIX concentration in range from 1 × 10−5 M to 3 × 10−4 M (II, III, IV, V, VI, VII, 23.663) and 295 nm (●) (y = −18.44x + 18.999). Data determined from −emission5 fluorescence−4 spectra. the presence of increasing PpIX concentration in range from 1 × 10 M to 3 × 10 M (II, III,−6 IV, V, InVIII) the excited insert at1: fluorescence280 nm. In the emission insert 2: spectrafluorescenc of HSAe emission at concentration spectra of HSA7.5 × at10 concentration−7 M (I) and HSA 1 × 10in the M VI, VII, VIII) excited at 280 nm. In the insert 2: fluorescence emission spectra−5 of HSA at concentration−4 presence(I) and HSA of increasingin the presence PpIX of co increasingncentration PpIX in rangeconcentration from 1 ×in 10 range−5 M tofrom 3 × 1 10 × −104 M M(II, to III, 3 ×IV, 10 V, M VI, (II, VII, III, 1 × 10−6 M (I) and HSA in the presence of increasing PpIX concentration in range from 1 × 10−5 M to VIII)IV, V, excited VI, VII, at VIII) 280 nm.excited In the at 295 insert nm. 2: [Lb], fluorescenc bound eli emissiongand concentration; spectra of HSA [Lf], atfree concentration ligand concentration. 1 × 10−6 M 3 × 10−4 M (II, III, IV, V, VI, VII, VIII) excited at 295 nm. [Lb], bound ligand concentration; [Lf], free (I) and HSA in the presence of increasing PpIX concentration in range from 1 × 10−5 M to 3 × 10−4 M (II, III, ligand concentration. IV, V, VI, VII, VIII) excited at 295 nm. [Lb], bound ligand concentration; [Lf], free ligand concentration.

Figure5. Stern-Volmer dependence for the PpIX-HSA complex excited at 280 nm (■) (y = 0.0434x + 0.9971) and 295 nm (●) (y = 0.0571x + 0.9551). Data determined from emission fluorescence spectra. In the insert 1: fluorescence emission spectra of HSA at concentration 7.5 × 10−7 M (I) and HSA in the Figure5. Stern-Volmer dependence for the PpIX-HSA complex excited at 280 nm (■) (y = 0.0434x + Figurepresence 5. Stern-Volmer of increasing dependence PpIX concentration for the in PpIX-HSA range from complex 1 × 10−5 M excited to 3 × 10 at−4 280M (II, nm III, ( IV,)( V,y =VI, 0.0434 VII, x + 0.9971) and 295 nm (●) (y = 0.0571x + 0.9551). Data determined from emission fluorescence spectra. 0.9971) and 295 nm (•)(y = 0.0571x + 0.9551). Data determined from emission fluorescence spectra.−6 VIII) excited at 280 nm. In the insert 2: fluorescence emission spectra of HSA at−7 concentration 1 × 10 In the insert 1: fluorescence emission spectra of HSA at concentration 7.5 × 10 M (I)−7 and HSA in the In the insert 1: fluorescence emission spectra of HSA at concentration 7.5 × 10 −5M (I) and− HSA4 in presenceM (I) and of HSA increasing in the presence PpIX concentration of increasing in PpIXrange concentration from 1 × 10−5 Min rangeto 3 × 10from−4 M 1 (II,× 10 III, MIV, to V, 3×10 VI, VII, M the presence of increasing PpIX concentration in range from 1 × 10−5 M to 3 × 10−4 M (II, III, IV, V, VIII)(II, III, excited IV, V, atVI, 280 VII, nm. VIII) In excitedthe insert at 2:295 fluorescen nm. ce emission spectra of HSA at concentration 1 × 10−6 VI, VII,M (I) VIII) and excitedHSA in the at 280 presence nm. In of the increasing insert 2: PpIX fluorescence concentration emission in range spectra from 1 of × HSA10−5 M at to concentration 3×10−4 M −6 −5 1 × (II,10 III,M IV, (I) V, and VI, HSAVII, VIII) in the excited presence at 295 of nm. increasing PpIX concentration in range from 1 × 10 M to 3×10−4 M (II, III, IV, V, VI, VII, VIII) excited at 295 nm.

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Table 1. Association constants (Ka), number of binding sites (n) and Stern–Volmer constants (KS-V) for Tablethe PpIX-HSA 1. Association complex. constants (Ka), number of binding sites (n) and Stern–Volmer constants (KS-V) for the PpIX-HSA complex.

λex = 280 nm λex = 295 nm λex = 280 nm λex = 295 nm K = 1.68 × 105 M−1 K = 2.30 × 105 M−1 aKa = 1.68 × 105 M−1 Ka = 2.30a × 105 M−1 PpIX-HSA n = 1 n = 1 PpIX-HSA n = 1 n = 1 K = 1.78 × 105 M−1 K = 1.93 × 105 M−1 S-VKS-V = 1.78 × 105 M−1 KS-V =S-V 1.93 × 105 M−1

Spatial structurestructure ofof the the PpIX PpIX molecule molecule bound bound to HSA to HSA may may have have an effect an oneffect the on strength the strength of binding of bindingand the distanceand the distance between between the ligand the and liga thend ﬂuorophore and the fluorophore of the protein. of theπ -protein.π interactions π-π interactions leading to “sandwich-type”leading to “sandwich-type” structures shouldstructures be takenshould into be accounttaken into [15 account]. [15].

2.3. Molecular Molecular Properties of PpIX The chemical 2D and 3D structure of PpIX with minimum energy conformation and its predicted physico-chemical properties properties are are shown shown in in Figures 66 andand7 .7. PpIX PpIX is is a highlya highly pharmacologically-active pharmacologically-active heterocyclic organic organic compound compound composed composed of of four four conjugated conjugated pyrrole pyrrole rings rings (A, (A, B, B, C and D) connected via four methane methane bridges. bridges. The The macrocycle macrocycle system system of of PpIX PpIX is issubstituted substituted by by four four methyl methyl groups groups at positionsat positions C(3), C(3), C(7), C(7), C(12), C(12), C(17), C(17), two twovinyl vinyl groups groups at positions at positions C(8), C(8), C(13) C(13) and two and propionic two propionic acid groupsacid groups at positions at positions C(2), C(18). C(2), The C(18). pro-tropic The pro-tropic tautomerism tautomerism in macrocyclic in macrocyclic porphine porphinering at positions ring at N(21)H/NoH,positions N(21)H/NoH, N(22)H/NoH, N(22)H/NoH, N(23)H/NoH N(23)H/NoHand N(24)H/NoH and has N(24)H/NoH been the subject has been of several the subject studies of [16–19].several studiesComputer [16 analysis–19]. Computer of the PpIX analysis structure of the sugg PpIXested structure that this suggestedcompound that may this exist compound in equilibrium may asexist four in tautomeric equilibrium forms as four N(21)H-N(22)H tautomeric forms ↔ N(22)H-N(23)H N(21)H-N(22)H ↔↔ N(23)H-N(24)HN(22)H-N(23)H ↔↔ N(22)H-N(24)H;N(23)H-N(24)H however,↔ N(22)H-N(24)H; in solutions however, at physiological in solutions pH, the at major physiological tautomer of pH, PpIX the is majorN(22)H-N(23)H tautomer (about of PpIX 49% is distribution)N(22)H-N(23)H (Figure (about 6). 49%PpIX distribution) is a weak acidic (Figure compou6). PpIXnd, iswhich a weak dissociates acidic compound, in solutions which at pH dissociates 7.4. PpIX mainlyin solutions occurs at as pH an 7.4. anionic PpIX form mainly (about occurs 99.65% as an in anionic a mixture form of (aboutneutral 99.65% and ionic in a forms) mixture (Figure of neutral 7A). Additionally,and ionic forms) the (FigurePpIX molecule7A). Additionally, is distinguished the PpIX by molecule a high potential is distinguished of the formation by a high of potential hydrogen of bondsthe formation and hydrophobic of hydrogen π-π bonds stacking and interactions hydrophobic (Figureπ-π stacking 7B). interactions (Figure7B).

Figure 6. Chemical structurestructureof of the the dominant dominant PpIX PpIX tautomer tautomer in aqueousin aqueous solution solution at pH at pH 7.4. 7.4. Atoms Atoms and andrings rings are numberedare numbered in order in order of the of docking the docking results’ results’ interpretation. interpretation.

MoleculesMolecules2016 2016, 21,, 21 1519, 1519 7 of7 19 of 19

(A)

(B)

FigureFigure 7. 7.Three-dimensional Three-dimensional structure of of the the PpIX PpIX molecu moleculele in the in energy the energy optimized optimized conformation conformation used in molecular docking. (A) Interpolated charges; (B) hydrogen bond donors and acceptors. Models are used in molecular docking. (A) Interpolated charges; (B) hydrogen bond donors and acceptors. Models shown in the ball and stick representation with the van der Waals surface (atom surfaces are scaled are shown in the ball and stick representation with the van der Waals surface (atom surfaces are scaled to 100% of the van der Waals radii). For the sake of clarity, only polar hydrogen atoms are displayed. to 100% of the van der Waals radii). For the sake of clarity, only polar hydrogen atoms are displayed. 2.4. Visualization of the PpIX Binding Sites in the HSA Molecule 2.4. Visualization of the PpIX Binding Sites in the HSA Molecule The HSA primary structure is a sequence of 585 amino acids, which form one polypeptide chain stabilizedThe HSA by primary 17 disulfide structure bridges is a[14]. sequence The secondary of 585 amino structure acids, of which HSA formbased one on polypeptidethe X-ray chaincrystallographic stabilized by analysis 17 disulﬁde shows bridges that the [HSA14]. Thepolypeptide secondary chain structure forms about of HSA thirty based α-helices, on the many X-ray crystallographic analysis shows that the HSA polypeptide chain forms about thirty α-helices, many

Molecules 2016, 21, 1519 8 of 19

Molecules 2016, 21, 1519 8 of 19 coiled-coils and none β-sheets [20]. The tertiary HSA structure consists of three homologous domainscoiled-coils (I, and II and none III) β-sheets formed [20]. as The the heart-shapedtertiary HSA structure conformation consists [21 of]. three Each homologous domain is domains comprised (I, ofII and two III) subdomains formed as the (A andheart-shaped B) [14]. Each conformation subdomain [21]. A Each is composed domain is of comprised six (h1–h6) of twoα-helices, subdomains while the(A and subdomain B) [14]. Each B of subdomain four (h1–h4) Aα is-helices composed [20]. of HSA six (h1–h6) contains α-helices, one tryptophan while the residue subdomain (Trp B 214) of locatedfour (h1–h4) deeply α-helices in its hydrophobic [20]. HSA matrixcontains in Subdomainone tryptophan IIA [ 14residue]. Sudlow (Trp [ 13214)] classified located twodeeply principal in its drug-bindinghydrophobic matrix sites in in HSA Subdomain molecule IIA located [14]. inSudlow Subdomains [13] classified IIA (Sudlow’s two principal Site 1) anddrug-binding IIIA (Sudlow’s sites Sitein HSA 2). molecule located in Subdomains IIA (Sudlow’s Site 1) and IIIA (Sudlow’s Site 2). As an introduction to molecular modelling, seven possible binding sites, which represent the free space inside the HSA structure able toto interactinteract withwith variousvarious ligands,ligands, havehave beenbeen identified.identified. The ligand binding sites of HSA detected in this study are shownshown in FigureFigure8 8.. AmongAmong allall ofof thethe detected detected ligandligand binding sites of has, the lowest interaction energy results for docked PpIX were found only for Site 1 (hemin site [[22])22]) andand SiteSite 22 (Sudlow’s(Sudlow’s SiteSite 11 [[13]13])) (Figure(Figure8 8).). TheseThese sitessites werewere selectedselected asas proposedproposed binding sites for PpIX. The localization and detaileddetailed characterization of the detected ligand binding sites and the energy of their interactions with PpIX are presented in Table2 2.. TheThe firstfirst sitesite (Site(Site 1)1) isis thethe largest one and located deeply in the hydrophobic matrixmatrix of Subdomain IB (Figure9 9A).TheA).The secondsecond sitesite is found deeply in the hydrophobic pocket ofof SubdomainSubdomain IIAIIA insideinside thethe proteinprotein corecore (Figure(Figure9 9B).B).

Figure 8.8. Macromolecule of HSA (PDB 1AO6) complexedcomplexed withwith thethe PpIXPpIX molecules.molecules. The protein backbone is shown schematically as a ribbon. Each subdomain of HSA is marked according to the model proposed by Carter and Ho [[14].14]. The best docking results (number 1 and 2) are shown in the CPK representation.representation. OtherOther docking docking results results are are shown show inn thein the ball ball and and stick stick representation. representation. For theFor sake the ofsake clarity, of clarity, only polaronly polar hydrogen hydrogen atoms atoms are displayed. are displayed.

Molecules 2016, 21, 1519 9 of 19

Table 2. Docking results for PpIX molecule inside HSA (1AO6.pdb) showing detailed analysis of the ligand binding sites, protein-ligand interaction energy and major residues involved in hydrogen bonding and hydrophobic interactions. Computational data obtained using the CLC DDWB program [23] and BIOVIA DS Visualizer software [24].

Binding Sites in HSA PpIX-HSA Complexes Volume Radius Energy No. of Site Location Domain Subdomain α-Helix Common Interacting Residues with PpIX (Å3) (Å) (a.u.) a H-bonds h1, h2, Arg 117, Tyr 138, Ile 142, His 146, Phe 149, Phe 1 787.46 15 Core I B −73.55 4 (6) b h3, h4 157, Tyr 161, Arg 186 I B h4 Ala 191, Ala 194, Lys 195, Leu 198, Trp 214, Arg II A h1, h2 2 420.35 13 Core −70.34 3 (5) b 218, Val 343, Lys 436, Lys 444, Pro 447, Cys 448, B h3 Asp 451, Tyr 452, Val 455 III A h3, h4 II A h1, h2 3 165.38 11 Surface B h2, h3 −11.42 0 (2) b Phe 206, Arg 209, Ala 210, Lys 351 III A h5, h6 A h2, h3 4 146.94 10 Surface I −48.25 2 (4) b Glu 17, Ala 21, Ala 158, Lys 159, Lys 162 B h2, h3 A h1, h2 Leu 394, Leu 398, Ala 406, Arg 410, Lys 413, Thr 5 65.02 10 Surface III −44.90 2 (4) b B h2, h3 540, Lys 541 Pro 421, His 510, Thr 506, Phe 509, His 510, Lys 6 52.22 10 Surface III B h1, h2 −33.56 1 (3) b 524, Ala 528 7 52.22 10 Surface III A h2, h5, h6 −49.05 2 (4) b Met 87, Gln 32, Gln 33, Lys 466 a The unit of protein-ligand interaction energy is presented in arbitrary units (a.u.); the lower the value, the better the binding afﬁnity; b including intramolecular H-bonds. Molecules 2016, 21, 1519 10 of 19 Molecules 2016, 21, 1519 10 of 19

(A)

(B)

FigureFigure 9. Superposition 9. Superposition of of the the PpIX PpIX molecule molecule inside inside the thehemin hemin sitesite (A) and Sudlow’s Sudlow’s Site Site 1 1(B (B) )of of the the HSAHSA macromolecule. macromolecule. The The geometry geometry of of the the bindingbinding sitessites is colored according according to to its its hydrophobicity. hydrophobicity. The ligand is shown in the ball and stick representation. Aromatic amino acids are shown in the thin The ligand is shown in the ball and stick representation. Aromatic amino acids are shown in the thin stick representation. For the sake of clarity, only polar hydrogen atoms are displayed. stick representation. For the sake of clarity, only polar hydrogen atoms are displayed. 2.5. Binding Site Interactions between PpIX and HSA Amino Acids 2.5. Binding Site Interactions between PpIX and HSA Amino Acids The results of computer simulation showed that there are many common interaction residues submittedThe results for the of computerbinding of simulationPpIX into Sites showed 1 and 2 that (Figures there 10 are and many 11, Table common 3). There interaction was a combined residues submittedhydrogen for bonding the binding and of hydrophobic PpIX into Sites interaction 1 and 2 (Figurespattern observed10 and 11 upon, Table docking3). There of was the aPpIX combined into hydrogenSites 1 and bonding 2 of HSA. and hydrophobicThe docked ligand interaction manifest patterned multicenter observed hydrophobic upon docking interaction of the PpIXbetween into Sitesthe 1 andhighly 2 ofconjugated HSA. The system docked of ligandPpIX and manifested the side ch multicenterains of amino hydrophobic acids located interaction inside Site between1 (Tyr 138, the highlyIle 142, conjugated His 146, Phe system 149, ofPhe PpIX 157, andArg 186 the and side Lys chains 190) ofand amino Site 2 acids(Ala 191, located Ala 194, inside Lys Site 195, 1 Leu (Tyr 198, 138, Ile 142,Trp 214, His Arg 146, 218, Phe Val 149, 343, Phe Lys 157, 436, Arg Lys 186 444, and Pro Lys 447, 190) Cys and 448, SiteAsp 2451, (Ala Tyr 191, 452 Ala and 194, Val Lys455) 195, (Figure Leu 10, 198, TrpTable 214, Arg3). PpIX-HSA 218, Val 343,complexes Lys 436, are Lys stabilized 444, Pro by 447,π-π Cysstacking 448, interactions Asp 451, Tyr with 452 aromatic and Val amino 455) acids (Figure into 10 , TableSites3). 1 PpIX-HSA (Tyr 138, Phe complexes 149, Phe 157) are stabilizedand 2 (Trp by214,π Ty-πr stacking452). Furthermore, interactions hydrog withen aromatic bonding interactions amino acids intoplay Sites an 1 important (Tyr 138, role Phe in 149, the Phebinding 157) of and PpIX. 2 (Trp The 214,most Tyr important 452). Furthermore, amino acid residues hydrogen involved bonding in interactionshydrogen playbonding an important interactions role are in Arg117 the binding (Site 1) of, Tyr PpIX. 161 The(Site most1) and important Lys 444 (Site amino 2). In acid summary, residues involvedthese residues in hydrogen that show bonding close interactions interactions are with Arg117 the PpIX (Site molecule 1), Tyr 161 inside (Site Site 1) and 1 and Lys Site 444 2 (Site may 2). In summary,appear to thesebe the residues key contributors that show to close the high interactions binding withaffinity. the Even PpIX though molecule internal inside stabilization Site 1 and Site by 2 mayintramolecular appear to be hydrogen the key contributors bonds was observed to the high in the binding ligand afﬁnity. structure Even after though docking internal simulation stabilization for both binding Sites 1 and 2, the docking results suggest that PpIX has a good binding affinity for the HSA. by intramolecular hydrogen bonds was observed in the ligand structure after docking simulation for both binding Sites 1 and 2, the docking results suggest that PpIX has a good binding afﬁnity for the HSA.

Molecules 2016, 21, 1519 11 of 19

Table 3. Key protein-ligand contacts of bound PpIX to the ligand binding Sites 1 and 2 inside HSA (1AO6.pdb). Computational data obtained using the CLC DDWB program [23] and BIOVIA DS Visualizer software [24].

Hydrogen Bonding Interactions Site 1 Site 2 PDB atom name Ligand atom Type of contact Distance (Å) PDB atom name Ligand atom Type of contact Distance (Å) (D) Arg117:HN (bb) (A) PpIX:O(pp) Conventional 2.02 * (D) Lys444:HZ1 (sc) (A) PpIX:O (pp) Conventional 2.01 * (D) Arg117:HE (sc) (A) PpIX:O (pp) Conventional 2.77 * (D) Lys444:HZ1 (sc) (A) PpIX:O (pp) Conventional 2.25 * (D) Tyr161:HH (sc) (A) PpIX:O (pp) Conventional 2.00 * (D) Lys444:HZ2 (sc) (A) PpIX:O (pp) Conventional 2.58 * π-Donor (D) PpIX:NH (r) Conventional (D) Arg186:HE (sc) (A) PpIX: (r) 3.01 * 2.21 * Non classical (A) PpIX:N (r) Intramolecular (D) PpIX:NH (r) Conventional (D) PpIX:NH (r) Conventional 2.21 * 2.24 * (A) PpIX:N (r) Intramolecular (A) PpIX:N (r) Intramolecular (D) PpIX:NH (r) Conventional 2.24 * (A) PpIX:N (r) Intramolecular Hydrophobic Interactions Site 1 Site 2 PDB atom name Ligand atom Type of contact Distance (Å) PDB atom name Ligand atom Type of contact Distance (Å) Tyr138 (sc) PpIX:C(v) π-Alkyl 5.05 Ala191 (sc) PpIX:C (v) Alkyl 4.09 Ile142 (sc) PpIX:C (m) Alkyl 4.52 Ala191 (sc) PpIX:C (m) Alkyl 4.10 Ile142 (sc) PpIX:C (v) Alkyl 4.57 Ala194 (sc) PpIX:C (v) Alkyl 3.90 Ile142:CD1 (sc) PpIX (pr) π-σ 3.15 Lys195 (sc) PpIX:C (v) Alkyl 4.00 His146 (sc) PpIX:C( m) π-σ 3.62 Lys195 (sc) PpIX (pr) π-Alkyl 4.88 His146 (sc) PpIX:C (v) π-Alkyl 5.10 Leu198 (sc) PpIX:C (m) Alkyl 5.14 Phe149 (sc) PpIX:C (m) π-Alkyl 4.83 Trp214 (sc) PpIX:C (v) π-Alkyl 4.49 Phe157 (sc) PpIX:C (v) π-Alkyl 4.50 Trp214 (sc) PpIX:C (v) π-Alkyl 4.61 Arg186 (sc) PpIX (chms) π-Alkyl 3.11 Arg218 (sc) PpIX:C (v) Alkyl 4.55 Arg186 (sc) PpIX (pr) π-Alkyl 3.46 Val343 (sc) PpIX:C (v) Alkyl 3.49 Arg186 (sc) PpIX (pr) π-Alkyl 4.35 Lys436 (sc) PpIX:C (m) Alkyl 5.31 Arg186 (sc) PpIX (pr) π-Alkyl 4.72 Lys436 (sc) PpIX:C (m) Alkyl 3.79 Arg186 (sc) PpIX (pr) π-Alkyl 5.42 Lys436 (sc) PpIX (pr) π-Alkyl 5.27 Molecules 2016, 21, 1519 12 of 19

Table 3. Cont.

Hydrophobic Interactions Site 1 Site 2 PDB atom name Ligand atom Type of contact Distance (Å) PDB atom name Ligand atom Type of contact Distance (Å) Pro447 (sc) PpIX (pr) π-Alkyl 5.21 Pro447:C,O (bb) Amide Lys190 (sc) PpIX:C (v) Alkyl 4.43 PpIX (pr) 3.94 Cys448:N (bb) π-Stacked Lys190 (sc) PpIX:C (m) Alkyl 4.72 Cys448 (sc) PpIX (pr) π-Alkyl 5.32 Lys190:CD1 (sc) PpIX (pr) π-σ 3.94 Cys448 (sc) PpIX (chms) π-Alkyl 5.33 Asp451:C,O (bb) Amide Lys190 (sc) PpIX (pr) π-Alkyl 5.27 PpIX (pr) 4.47 Tyr452:N (bb) π-Stacked Asp451:C,O (bb) Amide PpIX (chms) 5.66 Tyr452:N (bb) π-Stacked Tyr452 (sc) PpIX (pr) π-π T-shaped 4.96 Tyr452 (sc) PpIX (m) π-Alkyl 4.59 Tyr452 (sc) PpIX (v) π-Alkyl 5.48 Val455 (sc) PpIX:C (m) Alkyl 4.56 Val455 (sc) PpIX:C (v) Alkyl 3.97 Val455 (sc) PpIX (pr) π-Alkyl 5.43 Miscellaneous Interactions Unfavorable Interactions Site 2 Site 2 PDB atom name Ligand atom Type of contact Distance (Å) PDB atom name Ligand atom Type of contact Distance (Å) Cys448:SG (sc) PpIX (pr) π-Sulfur 3.76 Lys436:HZ2 (sc) PpIX:C (m) Steric Bumps 1.04 Lys436:HZ3 (sc) PpIX:H (m) Steric Bumps 1.34 Lys436:NZ (sc) PpIX:H (m) Steric Bumps 1.37 Lys436:HZ2 (sc) PpIX:C (m) Steric Bumps 1.64 Lys436:NZ (sc) PpIX:C (m) Steric Bumps 2.20 * Strong H-bond (max distance = 3.4 Å), weak H-bond (max distance = 3.8 Å); (A), H-bond acceptor; (D), H-bond donor; (sc), side chain functional group of the amino acid; (bb), backbone; (pp), propionic acid group; (v), vinyl group; (m), methyl group; (pr), pyrrole ring; (chms), conjugated heterocyclic macrocycle system. Molecules 2016, 21, 1519 13 of 19 Molecules 2016, 21, 1519 13 of 19

Molecules 2016, 21, 1519 13 of 19

(A) (A)

(B) (B) FigureFigure 10. 10.Hydrogen Hydrogen bondsbonds and and miscellaneous miscellaneous (sulfur) (sulfur) interactions interactions between between the residues the residues of HSA of and HSA Figurethe molecules 10. Hydrogen of PpIX bonds in the and hemin miscellaneous site (A) and (sulfur) Sudlow’s interactions Site 1 ( Bbetween). Interactions the residues between of HSA ligand and and and the molecules of PpIX in the hemin site (A) and Sudlow’s Site 1 (B). Interactions between ligand theamino molecules acids are of PpIXvisualized in the heminin dashed site (linesA) and and Sudlow’s colored Site according 1 (B). Interactions to their type between (conventional ligand and and and amino acids are visualized in dashed lines and colored according to their type (conventional and aminonon-classical acids are H-bonds visualized are greenin dashed and olive;lines andthe miscellaneouscolored according interaction to their is type yellow). (conventional Ligands andand are non-classicalnon-classical H-bonds H-bonds areare greengreen and olive; olive; the the miscellaneous miscellaneous interaction interaction is yellow). is yellow). Ligands Ligands and are and are shown in the ball and stick representation, respectively. shownshown in in the the ball ball and and stick stick representation,representation, respectively. respectively.

(A(A) )

Figure 11. Cont.

Molecules 2016, 21, 1519 14 of 19 Molecules 2016, 21, 1519 14 of 19 Molecules 2016, 21, 1519 14 of 19

(B) (B) Figure 11. Hydrophobic interactions between the molecules of PpIX and the residues of HSA around Figure 11. Hydrophobic interactions between the molecules of PpIX and the residues of HSA around Figurethe hemin 11. Hydrophobic site (A) and Sudlow’s interactions Site between1 (B). Ligands the molecule and residuess of PpIXare sh andown thein the residues ball and of stick HSA and around the hemin site (A) and Sudlow’s Site 1 (B). Ligands and residues are shown in the ball and stick and thethin hemin stick site representation, (A) and Sudlow’s respectively. Site 1 (B). Ligands and residues are shown in the ball and stick and thinthin stick stick representation, representation, respectively. respectively. As a result of the interactions of the PpIX molecule with the HSA, amino acid residue changes inAs Asthe aa spatial resultresult ofconformationof thethe interactions interactions of the ofligandof thethe PpIX PpIXappear molecule molecule. The comparison withwith thethe of HSA,HSA, the aminomolecularamino acidacid PpIX residueresidue structure changeschanges inin thethebefore spatialspatial molecular conformationconformation docking with ofof thethe that ligandligand isolated appear.appear from the. TheThe PpIX-HSA comparisoncomparison complex ofof thethe after molecularmolecular docking is PpIXPpIX shown structurestructure in beforebeforeFigure molecularmolecular 12. The changes dockingdocking in withwiththe PpIX thatthat spatial isolatedisolated structure fromfrom the concernthe PpIX-HSAPpIX-HSA setting complexincomplex 3D the vinyl afterafter group dockingdocking in C(3) isis shownshown and inin FigureFigureC(8) 12 12.positions,. TheThe changeschanges as well inin as thethe propionic PpIXPpIX spatialspatial acid residues structurestructure in concernC(13)concern and setting settingC(17). inDue in 3D3D to the thethe vinylvinylformation groupgroup of instrongin C(3)C(3) andand C(8)C(8)hydrogen positions,positions, bonds asas wellwell with as asArg propionicpropionic 117 and acidTyracid 161 residuesresidues (Site 1) inandin C(13)C(13) Lys and444and (Site C(17).C(17). 2), DuetheDue most toto the thesignificant formationformation changes ofof strongstrong in PpIX conformation are observed. hydrogenhydrogen bonds bonds with with Arg Arg 117 117 and and Tyr Tyr 161 161 (Site (Site 1) 1) and and Lys Lys 444 444 (Site (Site 2), 2), the the most most signiﬁcant significant changes changes in PpIXin PpIX conformation conformation are are observed. observed.

(A)

Figure 12. Cont.

Molecules 2016, 21, 1519 15 of 19 Molecules 2016, 21, 1519 15 of 19

(B)

(C)

FigureFigure 12.12. TheThe differences differences in molecular in molecular conformations conformations of PpIX of before PpIX and before after and computational after computational simulation. simulation.(A) Initial conformation (A) Initial conformation of PpIX; (B) conformation of PpIX; (B )of conformation PpIX bound in of the PpIX hemin bound site; ( inC) theconformation hemin site; of (CPpIX) conformation bound in Sudlow’s of PpIX Site bound 1. Molecules in Sudlow’s are Siteshown 1. Moleculesin the ball areand shown stick representation in the ball and of sticktheir representationaromatic forms. of Intramolecular their aromatic forms.hydrogen Intramolecular bonds are highlighted hydrogen with bonds dashed are highlighted lines (1 Å = with 0.1 nm). dashed For linesthe sake (1 Å of = 0.1clarity, nm). only For thepola saker hydrogen of clarity, atoms only are polar displayed. hydrogen atoms are displayed.

3. Materials and Methods 3. Materials and Methods

3.1.3.1. Chemicals Chemicals HSA,HSA, Fraction Fraction V,V, crystallized crystallized and and lyophilized, lyophilized, waswas obtained obtained fromfromICN ICNBiomedicals Biomedicals Inc.,Inc., Aurora,Aurora, OH,OH, USA. USA. PpIXPpIX (3,7,12,17-tetramethyl-8,13-diwvnyl-2,18-porﬁnodipropionic(3,7,12,17-tetramethyl-8,13-diwvnyl-2,18-porfinodipropionic acid)acid) waswas obtainedobtained fromfrom AldrichAldrich Chem. Chem. Co., Co., Milwaukee, Milwaukee, WI, WI, USA. USA.

3.2.3.2. Absorption Absorption UV-VIS UV-VIS AnalysisAnalysis TheThe UV-VIS UV-VIS spectra spectra were were recorded recorded onon aa spectrophotometerspectrophotometer (Jasco(Jasco InternationalInternational Co.,Co., Ltd.,Ltd., Tokyo,Tokyo, Japan),Japan), thethe Jasco V-530. V-530. The The wavelength wavelength accuracy accuracy was was ±1 nm;±1 nm;the photometric the photometric accuracy accuracy was ±0.002 was Abs. at 0.5 Abs. All PpIX and HSA samples were prepared in sodium phosphate buffer (pH 7.4). The UV-VIS spectra of the PpIX-HSA system were recorded for two systems: the first containing a constant PpIX concentration and the second containing a constant HSA concentration. The

Molecules 2016, 21, 1519 16 of 19

±0.002 Abs. at 0.5 Abs. All PpIX and HSA samples were prepared in sodium phosphate buffer (pH 7.4). The UV-VIS spectra of the PpIX-HSA system were recorded for two systems: the ﬁrst containing a constant PpIX concentration and the second containing a constant HSA concentration. The differences of the UV-VIS spectra of the PpIX-HSA system were recorded, as well. The intensity and shape of all of the recorded spectra were corrected for the absorption of the buffer using software (Spectra Manager version 1.55.00, Jasco International Co., Ltd., Tokyo, Japan) supplied by Jasco.

3.3. Fluorescence Analysis Emission fluorescence spectra were recorded with 1 cm × 1 cm × 4 cm quartz cells on a KONTRON SFM-25 Instrument AG (Kontron AG, Zurich, Switzerland), 30 min after sample preparation at 25 ◦C. Correcting the error of the apparatus for wavelength and relative fluorescence was equal to λ = ±1 nm, RF = ±0.01, respectively. For excitation wavelength λex = 280 nm, 295 nm and 405 nm, the scan range was 280–400 nm, 295–400 nm and 550–750 nm, respectively. By using fluorescence data, the quenching curves, i.e., (f ([PPIX]:[HSA]) = F/F0), of HSA in the presence of PpIX have been plotted (F0, fluorescence of HSA in the absence of PpIX; F, fluorescence of HSA in the presence of PpIX). The quenching of HSA fluorescence caused by PpIX was used to determine [PpIXb] from: ∆F [PpIXb] = · [HSA]t ∆Fmax where ∆F = F0 − F; [HSA]t the total concentration of HSA; ∆Fmax the maximal fluorescence decrease (to determine that the ∆Fmax afixed concentration of HSA was titrated with increasing concentration of PpIX); 1 is an intersect of the ordinate axis on the plot 1 versus 1 . ∆Fmax ∆F [PpIXtotal] The concentration of unbound (free) PpIX was obtained from: h i PpIX f = [PpIXtotal] − [PpIXb]

3.4. Association Constants and Quenching Constants The association constants of PpIX to HSA were estimated by the method described by Hiratsuka [25]. The Stern–Volmer constants were calculated according to the equation [11]:

RFo/RF = 1 + KS-V × [Q] where RFo and RF are the ﬂuorescence intensities in the absence and presence of the quencher, respectively; KS-V is the collisional quenching constant; [Q] is the quencher (PpIX) in the complex HSA-PpIX.

3.5. Computational Simulation The docking experiment was performed using the CLC Drug Discovery Workbench (CLC DDWB) computer program [23]. Docking results were graphically elaborated using the BIOVIA Discovery Studio Visualizer (DS) software [24]. The crystal structure of HSA (Chains A and B) required for computer simulation was downloaded from the Protein Data Bank (PDB) database [26] with 4-letter PDB accession 1AO6 and imported to the CLC DDWB computer program. The downloaded protein structure was obtained by the X-ray diffraction method with a resolution of 2.5 Å by Sugio et al. [20]. The loaded ﬁle 1AO6.pdb contains two single, unglycosylated and ligand-free polypeptide chains, A and B. The chain A was chosen to be a target protein structure. Before the docking experiment, the chain B and all crystallographic water molecules were removed from protein ﬁle 1AO6.pdb. Then, the ionizable amino acid residues of the protein’s Chain A were set to their protonation states, which exist in physiological conditions at pH 7.4. Molecules 2016, 21, 1519 17 of 19

Amino acids with positive electrically-charged side chains such as Arg (PDB atom: NH1, NH2 and NE), His (PDB atom: ND1) and Lys (PDB atom: NZ) were protonated, while those of amino acids with negative electrically-charged side chains, such as Asp (PDB atom: OD1 and OD2) and Glu (PDB atom: OE1 and OE2), were deprotonated. The two-dimensional (2D) structure of PpIX was built using the ChemDraw Ultra [27] program and converted to the three-dimensional (3D) representation by the use of the Chem3D Ultra [27] program. Subsequently, the overall geometry optimizations and partial atomic charge distribution calculations of the ligands were performed with the same program using the Austin Model 1 (AM1) semi-empirical molecular orbital method [28]. The CLC DDWB program and Marvin Sketch [29] software were used for characterizing the molecular properties of PpIX, including molecular mass, number of rotatable bonds, XlogP, hydrogen bond acceptors and donors, van der Waals surface, water solubility, dissociation constant (pK), major microspecies and major tautomeric forms at pH 7.4.

3.6. Molecular Docking Procedure Molecular docking simulation consists of four steps: finding binding sites, docking ligand to the binding sites, virtual screening and, finally, visualization of the docking results. Initially, potential ligand-binding sites in the HSA structure were automatically detected by the Find Binding Pockets algorithm implemented in CLC DDWB. Then, the detected binding sites were classified according to their molar volumes (all sites with a volume less than 50 Å3 were discarded) and analyzed by selecting neighboring amino acids within a 10–15 Å radius from the center of each binding site. In the second step, the PpIX molecule was fitted separately into each detected ligand-binding site of HSA by the CLC DDWB search algorithm with standard docking simulation settings (No. of iterations for each ligand = 100; number of docking results returned for each ligand = 1). In this step, the rotation around flexible bonds was changing the conformation of the ligand. During the experiment, the binding energy for each obtained complex was automatically scored by the PLANTSPLP scoring function used in the CLC DDWB program and presented in arbitrary units (a.u.) [30]. Finally, all solutions of protein-ligand complexes were ranked according to their docked energy (virtual screening). A very negative score corresponds to a strong binding, while a less negative or even a positive score corresponds to a weak or non-existing binding. In the last step, the complexes with the best score were visualized as the final docking results.

4. Conclusions The binding of medicines to plasma proteins determines their pharmacological actions and side effects. A significant role in the drug carriage is played by HSA. In this work, spectroscopic and molecular docking studies were applied in order to identify the likely PpIX binding sites of HSA and to explain the mechanism of binding the PpIX to this carrier protein. The formation of the complex between PpIX and HSA was confirmed by the analysis of the difference of the UV-VIS spectra and spectrofluorescence quenching analysis. Based on the absorption analysis, it was concluded that PpIX may form complexes with HSA in human plasma. Additionally, the fluorescence analysis results show that PpIX is able to interact with both Trp and Tyr residues. These results suggest that PpIX most likely binds to HSA near Subdomains IIA (where Trp 214 and Tyr 263 are located). Furthermore, the binding site of PpIX on HSA may be located near Subdomains IB (Tyr 138, Tyr 140, Tyr 148, Tyr 150, Tyr 161), IIB (Tyr 332, Tyr 334, Tyr 341) and/or IIIA (Tyr 401, Tyr 411, Tyr 452). The spectroscopic studies were confirmed by the results of computer simulation. The docking results clearly indicate Subdomains IB (hemin binding site) and IIA (Sudlow’s Site 1) as the major binding sites for PpIX on the HSA molecule. Most likely, PpIX forms a strong “sandwich-type” complex between its highly conjugated porphine system and aromatic side chains of Trp and Tyr. Finally, nine amino acid residues (Arg 117, Tyr 138, Ile 142, His 146, Phe 149, Phe 157, Tyr 161, Arg 186 and Lys 190) involved in the binding with PpIX in Site 1 of HSA are the same as those for Molecules 2016, 21, 1519 18 of 19 heme, which was described by Wardell et al. [22]. This reveals that PpIX and heme may interact in Subdomain IIA of the tertiary structure of HAS.

Acknowledgments: This work was supported by Grants KNW-2-O15/N/6/K and KNW-1-034/K/6/0 from Medical University of Silesia, Katowice, Poland. Calculations have been carried out using resources provided by Wroclaw Centre for Networking and Supercomputing (http://wcss.pl), Grant No. 382. Author Contributions: L.S. and B.P.: conception and design of the study. L.S. and B.P.: analysis and interpretation of the results. L.S., B.P. and M.C.: preparation of the manuscript. M.M.-J.: critical revision. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Sample Availability: Samples of the compound PpIX and PpIX-HSA complexes are available (in Mol Files) from the authors.

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