98 99 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 Synchrotron TeraHertz spectroscopy and neutron scattering pave theway to understanding chemoresistance Related publication: Batista de Carvalho, L. A. E., Mamede, A. P., Batista de Carvalho, A. L. M., Marques, J., Cinque, G., Rudić, S., &Marques, M. P. M. Metallodrug-protein interaction probed by synchrotron terahertz and neutron scattering spectroscopy. Biophysical Journal , 120 3070–3078 (2021). DOI : 10.1016/j.bpj.2021.06.012 Publication keywords: Anticancer drugs; Human serumalbumin; Metallothionein; SynchrotronTHz spectroscopy; Inelastic neutron scattering spectroscopy; Fourier transform infrared spectroscopy M etallodrugs are pharmaceutical drugs that contain metals such as silver or platinum. The low bioavailability and toxicity of metallodrugs, and the development of resistance to them, are still severe handicaps in cancer treatment. These factors drastically restrict chemotherapy success and affectmillions of patients. To overcome these drawbacks, we must thoroughly understand the mechanisms underlying drug transport and resistance, which control the agent’s biodistribution and availability at its pharmacological target. Intracellular inactivation of the drug via coordination to biomolecules apart fromthe target (DNA) is one suchmechanism involvingmetallothioneins (MTs). The Multimode InfraRed Imaging and Microspectroscopy (MIRIAM) beamline B22 has been established as an invaluable tool to monitor biomolecular binding and conformational rearrangements, and researchers used a combination of coherent synchrotron-radiation TeraHertz spectroscopy and inelastic neutron scattering to investigate two platinum (Pt)- and palladium (Pd)-polyamine agents that have yielded promising results toward some types of human cancers. Their results revealedan impact of thePt- andPd-agents onprotein structure, conformational behaviour andoverall flexibility, similar to former observations on the effect of this type ofmetallodrugs on DNA. These findings are expected to contribute to a better understanding of the drug’s mode of action, particularly of the inactivation processes associated with drug-protein linkage prior to DNA binding that underlie resistance and toxicity. This will assist in the design of improved platinum- and palladium-derived anticancer agents with higher bioavailability at the target, lower acquired resistance and decreased adverse side effects, thus enhancing chemotherapy effectiveness. Cancer is still a major public health problem worldwide. Numerous cytostatic agents have been developed over the years, aiming at an improved antineoplastic activity coupled to decreased acquired resistance and deleterious side effects, including Pt- and Pd-based compounds introduced upon the discovery of cisplatin. Among these, Pt(II) and Pd(II) polynuclear chelates with polyamines (Fig. 1A) constitute a specific class of DNA-damaging anticancer agents 1 . However, low bioavailability, toxicity and acquired resistance of metallodrugs are still severe handicaps in oncology practice. Intracellular inactivation of the drug via coordination to biomolecules apart from the target (DNA) is one of the mechanisms underlying acquired resistance, namely involving metallothioneins (MTs). These ubiquitous cysteine-rich proteins are linked to cellular protection against metal toxicity and have a high affinity for soft transitionmetal ions such as Pt 2+ and Pd 2+ , thus being involved in cisplatin´s resistance through the formation of Pt-thiolate clusters. Human SerumAlbumin (HSA) is themost abundant protein in human serum, playing a key role in the transport andmetabolismof exogenous compounds. Pt-based agents, in particular cisplatin, are known to reversibly bind to HSA upon intravenous administration, yielding stable adducts that circulate in the bloodstream. This will help to elucidate the drug´s pharmacokinetic profile, specifically the competition between S- or N-donor ligands from proteins and DNA´s purine bases. Fourier Transform Infrared (FTIR) spectroscopy coupled to synchrotron radiation constitutes an unmatched tool for studying biological systems 2 . In addition, terahertz (THz) absorption spectroscopy, covering the 3 – 100 cm -1 frequency range, is particularly sensitive to very low frequency vibrational modes. THz measurements on proteins are highly sensitive to intra- and intermolecular interactions, providing reliable information on protein´s conformational rearrangements, average flexibility and functionality. The use of a high-flux terahertz Coherent Synchrotron Radiation (CSR) emission at the B22 beamline ensures a greatly enhanced intensity of the exciting beamwhich, coupled to highly sensitivebroadbanddetectors, provides amuchbetter quality of the acquireddata. In particular, the low-alpha mode available at Diamond allows to probe the very low frequency spectral range (10 – 65 cm -1 ). Inelastic Neutron Scattering (INS) spectroscopy performed in the TOSCA spectrometer at the ISIS Neutron and Muon Source is a non-optical technique complementary to THz spectroscopy that has proved to be extremely useful for monitoring drugs’ impact on biological samples 3 . INS enables the observation of all the fundamental vibrational modes, overtones and combination bands of a system, and is especially sensitive to hydrogenous materials particularly in the low energy range (0 – 400 cm -1 ). This complementarity between THz absorption and neutron scattering allows to probe the same energy modes of a sample in two completely different ways – respectively via an optical method (subject to selection rules) and via inelastic scattering (according to the nuclear cross section). The present study aimed at studying the interaction between the anticancer agents Pt 2 Spm, Pd 2 Spm and cisplatin, and the proteins HSA and MT. Two crucial processes for drug activity were investigated: (i) drug transport through the blood stream, for these types of intravenously administered chemotherapeutic agents – using a drug–HSAmodel; (ii) acquired resistance, due to drug binding to sulphur- containing intracellular proteins such as MT – using a drug–MTmodel. The measurements performed covered the 10 – 660 cm -1 (0.3 – 20 THz) spectral range, with special emphasis to the low energy excitations (below 65 cm -1 ). This region comprises global collective vibrational modes of the biopolymer, which are strongly dependent on the protein´s overall conformation and are prone to be affected by the drug-protein interplay see Fig 1B. Therefore, any changes in these conformational preferences will be clearly reflected in the corresponding THz profile. While for the HSA systems the absorbance increased with frequency up to ca. 40 cm -1 then becoming nearly constant, mainly for the Pt-containing systems (Fig. 2A), for the MT conjugates there was an almost linear increase from 20 to 65 cm -1 (Fig. 2B). This slope increment, more pronounced for the drug- protein conjugates as compared to the free proteins (particularly for the Pd 2 Spm adducts), reveals a conformational rearrangement of the protein consistent with an enhanced flexibility, giving rise to a greater density of low energy vibrational modes. This plasticity enhancement within a protein due to binding to an external ligand has been previously reported, namely regarding the interaction of the anticancer drugmethotrexate with its target enzyme dihydrofolate reductase 4 . Each of the currently tested metallodrugs induced distinct conformational changes in either HSAorMT.The effect of the dinuclear agents Pt 2 SpmandPd 2 Spm followed a similar trend for each of these biomolecules, with either a lower or higher intensity relative to free HSA andMT, respectively. In turn, themononuclear agent cisplatinwas found tohave opposite impacts onHSAas compared toMT, and always inverse to the effects of the Pt- and Pd-spermine compounds. Also, Pd 2 Spm showed amore significant influence onMT relative to HSA, and an overall stronger impact on both proteins as compared to its Pt-counterpart. Regarding the INS profile, clear changes of the drug-HSA conjugates relative to both the free protein and the isolated Pd-agent were detected (Fig. 2C), revealing the perturbationonprotein´s conformationdue todrug-bindingwhich takes place via metal coordination to the N and S sites from HSA´s histidine and methionine residues. A drug impact on HSA´s skeletal torsions (Amide VII mode) and NH ... O interactions within the peptide backbone, responsible for signals centered at ca. 160 cm -1 , was evidenced in the cisplatin-adducts for which these features virtually disappeared – this may be due to drug-elicited protein aggregation. Additionally, the band from tryptophan (ca. 750 cm -1 ) slightly lowered its intensity upon drug interaction, this variation beingmore significant for the Pd 2 Spm-HSA system. Similarly to former observations on the effect of this type of metallodrugs on DNA, aclear impact of thePt- andPd-agentsonprotein´s structure, conformational behaviour and overall flexibility, both for HSA and MT was revealed. Furthermore, drug effects on proteins often lead to aggregation via cross-linking interactions, as previously reported for lysozyme and HSA 5 . This type of protein aggregation has been identified as one of the pre-DNA binding mechanisms responsible for acquired drug resistance. Coupled to previous data on the impact of Pt- and Pd-spermine compounds on human cancer cells and DNA, the present results are expected to contribute to a better understanding of the drug´s mode of action, particularly of the inactivation processes associated to drug-protein linkage prior to DNA binding, which underlie resistance and toxicity. This will assist in the design of improved platinum- and palladium-derived anticancer agents, with higher bioavailability, lower acquired resistance and decreased adverse side effects, thus enhancing chemotherapy effectiveness. References: 1. Farrell, N. P. Multi-platinumanti-cancer agents. Substitution-inert compounds for tumor selectivity and new targets. Chemical Society Reviews, 44, 8773–8785 (2015). DOI : 10.1039/C5CS00201J 2. Batista de Carvalho, A. L. M. et al. Anticancer drug impact on DNA – a study by neutron spectroscopy coupledwith synchrotron-based FTIR and EXAFS. Physical Chemistry Chemical Physics, 21, 4162–4175 (2019). DOI: 10.1039/ C8CP05881D 3. Marques, M. P. M. et al. A new look into themode of action of metal- based anticancer drugs. Molecules, 25, 246 (2020). DOI: 10.3390/ molecules25020246 4. Balog, E. et al. Direct determination of vibrational density of states change on ligand binding to a protein. Physical ReviewLetters, 9, 028103 (2004). DOI: 10.1103/PhysRevLett.93.028103 5. Pinato, O. et al. Platinum-based drugs and proteins: Reactivity and relevance to DNA adduct formation. Journal of Inorganic Biochemistry, 12, 27–37 (2013). DOI: 10.1016/j.jinorgbio.2013.01.007 Funding acknowledgement: The author thanks financial support fromPOCentro, COMPETE 2020, Portugal 2020 and European Community through the FEDER and the Portuguese Foundation for Science andTechnology (UIDB/00070/2020 and PhD Grant SFRH/ BD/137001/2018). Diamond Light Source (UK) and the STFC Rutherford Appleton Laboratory are thanked for access to the B22/MIRIAMbeamline (SM21620) and to neutron beam facilities (TOSCA/RB2010013, DOI 10.5286/ISIS.E.RB2010013). Corresponding author: Dr Ana Batista de Carvalho, University of Coimbra,
[email protected] Soft CondensedMatter Group Beamline B22 Figure 1: (A) Structural representation of cisplatin and Pt/Pd 2 Spm; (B) Vibrational spectra – THz-ATR, Raman and INS (below 600 cm -1 ) – of the anticancer agent cisplatin. Figure 2: (A) CSR-THz-ATR spectra (acquired in low-alpha mode) of human serum albumin (HSA) and its drug adducts; (B) CSR-THz-ATR spectra (acquired in low-alpha mode) of metallothionein (MT) and its drug adducts; (C) INS spectra (at 10 K) of human serum albumin (HSA), cisplatin-HSA and Pd 2 Spm-HSA (0 – 2000 cm -1 ).