Penetrating the Blood-Brain Barrier with New Peptide–Porphyrin Conjugates Having anti-HIV Activity

Passing through the blood-brain barrier (BBB) to treat neurological conditions is one of the main hurdles in modern medicine. Many drugs with promising in vitro profiles become ineffective in vivo due to BBB restrictive permeability. In particular, this includes drugs such as antiviral porphyrins, with the ability to fight brain-resident viruses causing diseases such as HIV-associated neurocognitive disorders (HAND). In the last two decades, BBB shuttles, particularly peptide-based ones, have shown promise in carrying various payloads across the BBB. Thus, peptide–drug conjugates (PDCs) formed by covalent attachment of a BBB peptide shuttle and an antiviral drug may become key therapeutic tools in treating neurological disorders of viral origin. In this study, we have used various approaches (guanidinium, phosphonium, and carbodiimide-based couplings) for on-resin synthesis of new peptide–porphyrin conjugates (PPCs) with BBB-crossing and potential antiviral activity. After careful fine-tuning of the synthetic chemistry, DIC/oxyma has emerged as a preferred method, by which 14 different PPCs have been made and satisfactorily characterized. The PPCs are prepared by coupling a porphyrin carboxyl group to an amino group (either N-terminal or a Lys side chain) of the peptide shuttle and show effective in vitro BBB translocation ability, low cytotoxicity toward mouse brain endothelial cells, and low hemolytic activity. Three of the PPCs, MP-P5, P4-MP, and P4-L-MP, effectively inhibiting HIV infectivity in vitro, stand out as most promising. Their efficacy against other brain-targeting viruses (Dengue, Zika, and SARS-CoV-2) is currently under evaluation, with preliminary results confirming that PPCs are a promising strategy to treat viral brain infections.

Scheme 1. Schematic representation of on-resin synthesis of P4-PP. The formation of two putative diastereomers is showed through conjugation of -COOH groups of PP with Lys sidechain NH 2 group of P4 peptide.  Figure S1. Raw TIC-MS spectra of all 14 synthesized PPCs. The order of presented spectra is identical with the order of the conjugate in Table S1.

LC ANALYSIS
The formation of the putative diastereomeric species (see scheme S1) was evaluated though LC analysis using various ion-pair reagents and detection wavelengths. The conjugates (P2-MP, P2-PP and P4-MP) were dissolved in 25% MeCN/H 2 O at final concentration of 1 mg/mL. Each conjugate solution (5 µL injected) was analyzed by a linear 25%-70% MeCN (into 0.1% TFA, or 0.1% HFBA, in H 2 O) gradient over 45 min using PDA detection. In all tested cases, only one elution peak corresponding to the synthesized conjugate, was observed.     Table S2; their 1 H chemical shifts can be measured at the vertical y-axis). Cross-peaks corresponding to the same amino acid residue are linked by vertical lines. The cross-peaks belonging to the N  H 3 amino group of K6 side chain and to the N  H guanidinium proton of the R7 side chains are also labelled. (B) Region showing the crosspeaks for the pair of N ' H 2 amide protons of the two glutamine residues (Q2 and Q3; see Table  S2), which are symmetrical relative to the diagonal.  Table S2) and the amino acid they belong to. 1 H and 13 C chemical shifts can be measured at the horizontal and vertical axis, respectively.

MASS SPECTROMETRY
We were able to assign the masses of some side-reaction products formed during the conjugation reactions with various activation strategies. For example, in PyBOP/DIPEA activation, the peaks at 10.6 min (m/z 1444.8, Figure 2a) and 10.5 min (m/z 1816.9, Figure 2d), have respectively an m/z increase of +54 Da relative to the target conjugate. By performing tandem MS of this product and assigning the a-series of ions (data not shown), we could identify that the modification occurred on the porphyrin section of the molecule (Table S3), though further detailed characterization was not pursued. Similarly, in the case of HATU/DIPEA activation, the side product that co-elute within the conjugate peak was assigned with a mass increase of +27 Da, compared to the mass of the corresponding conjugate. This product was also present in all tested activation protocols, but in much lower extent. Based on the MS/MS spectra the modification of +27 Da can also be related with the porphyrin molecule (Table S3) (Table S3, mass increase of +824 Da and +1201 Da). Furthermore, some of the formed side products were related with the peptide part of conjugate. For example, when P3 was used in the conjugation reaction there is appearance of one additional peak with mass increase of +32 Da that elutes immediately before the conjugate peak (m/z 1422.8, Figure 2a-c). This peak probably corresponds to oxidation of the Trp residue, that is most susceptible residue for oxidation in the sequence, as the mass difference of +32 Da can be attributed to one of the well-characterized Trp-oxidation products. [1][2][3][4][5] Tandem mass spectra of this oxidized product revealed the full a-and b-series of ions with no detectable peaks corresponding to oxidized product. In contrary, the y-series of ions displayed peaks with +32 Da mass increase confirming that modification occurred at the corresponding Trp residue (data not shown).
In all of them *relative to the mass of the conjugate Figure S6. DIPEA relevance on conversion rate with MPIX hydrochloride as porphyrin coupling partner. Bars refer to peptide (grey) and conjugate (black) integrated areas in the HPLC chromatograms of the reaction crudes.

TZM-bl Cell Viability Studies
The cytotoxic effects of each PPC on TZM-bl cell cultures were assessed to ensure that HIV inhibition was a result of the compounds effect on the virus and not on the host cells used in the assay. A resazurin reduction fluorometric assay was performed. Resazurin, the active compound in alamarBlue ® , is a blue dye that can be reduced to a pink fluorescent intermediate, resorufin, as a result of cell metabolic activity. 6 TZM-bl cells were seeded at 2x10 4 cells/well in tissue culture-treated 96-well microplates and incubated for 24 h. Cells were then incubated with serial dilutions (2fold) of single peptide and porphyrins, or PPC, for 3 h, after which the mixture was replaced with fresh complete medium. Untreated cells (in the absence of peptideporphyrin combinations) were used as a control. After 42 h, media was replaced with alamarBlue ® reagent and incubated for an additional 3 h. Resazurin reduction was quantified by resorufin fluorescent emission intensity, measured in an Infinite M200 microplate reader. Fluorescence emission intensity was collected at 590 nm, using a fixed λ exc of 560 nm. Excitation and emission slits were 9 and 20 nm, respectively. Cell viability was calculated through the following formalism: (1) where I EI corresponds to the resorufin fluorescence emission intensity in the presence of EIs or combination, I control to the fluorescence emission intensity in the absence of EIs and I background to the background fluorescence emission from the non-reduced alamarBlue ® reagent.

PPCs internalization into bEnd.3 cells
bEnd.3 cells were seeded 50000 cells/500 μL in 24-well flat-bottomed plates (Corning, New York, USA) for 24h. Then, medium was removed and cells were washed twice with PBS and once with medium, and incubated for 24 h with different PPCs, at a final concentration of 10 μM in a humidified atmosphere of 5% CO 2 at 37 °C. Finally, cells were collected and washed twice with PBS. The mean fluorescence intensity of 10000 cells was analyzed with BD LSRFortessa X-20 flow cytometer (BD Biosciences, San José, USA) -Laser Violet (405 nm) and detector BV650 (670/30 nm). All experiments were performed in duplicates in three independent days.
In order to elucidate the internalization potential of the PPC, an internalization assay was performed. bEnd.3 cells incubated with 10 µM PPC or porphyrins for 24 h, and internalization was assessed using flow cytometry. Assuming that the fluorescence quantum yield of the PPC is similar to that of porphyrins, we used the cells mean fluorescence intensity (MFI) to quantity PPC internalization. The results ( Figure S8) show that the peptide conjugation enhances porphyrin delivery into cells. Figure S8 -PPCs internalization in bEnd.3 cells. The cells were incubated with 10 µM PPC or porphyrins for 24 h, and internalization was assessed using flow cytometry. Cells mean fluorescence intensity (MFI) was used to quantify PPCs internalization.