Gold(I) and Silver(I) Complexes Containing Hybrid Sulfonamide/Thiourea Ligands as Potential Leishmanicidal Agents.

Leishmaniasis is a group of parasitic diseases with the potential to infect more than 1 billion people; however, its treatment is still old and inadequate. In order to contribute to changing this view, this work consisted of the development of complexes derived from MI metal ions with thioureas, aiming to obtain potential leishmanicidal agents. The thiourea ligands (HLR) were obtained by reactions of p-toluenesulfohydrazide with R-isothiocyanates and were used in complexation reactions with AgI and AuI, leading to the formation of complexes of composition [M(HLR)2]X (M = Ag or Au; X = NO3- or Cl-). All compounds were characterized by FTIR, 1H NMR, UV-vis, emission spectroscopy and elemental analysis. Some representatives were additionally studied by ESI-MS and single-crystal XRD. Their properties were further analyzed by DFT calculations. Their cytotoxicity on Vero cells and the extracellular leishmanicidal activity on Leishmania infantum and Leishmania braziliensis cells were evaluated. Additionally, the interaction of the complexes with the Old Yellow enzyme of the L. braziliensis (LbOYE) was examined. The biological tests showed that some compounds present remarkable leishmanicidal activity, even higher than that of the standard drug Glucantime, with different selectivity for the two species of Leishmania. Finally, the interaction studies with LbOYE revealed that this enzyme could be one of their biological targets.

Interaction between diterpene icetexanes and old yellow enzymes of Leishmania braziliensis and Trypanosoma cruzi.

Old Yellow Enzymes (OYEs) are flavin-dependent redox enzymes that promote the asymmetric reduction of activated alkenes. Due to the high importance of flavoenzymes in the metabolism of organisms, the interaction between OYEs from the parasites Trypanosoma cruzi and Leishmania braziliensis and three diterpene icetexanes (brussonol and two analogs), were evaluated in the present study, and differences in the binding mechanism and inhibition capacity of these molecules were examined. Although the aforementioned compounds showed poor and negligible activities against T. cruzi and L. braziliensis cells, respectively, the experiments with the purified enzymes indicated that the interaction occurs by divergent mechanisms. Overall, the ligands' inhibitory effect depends on their accessibility to the N5 position of the flavin's isoalloxazine ring. The results also indicated that the OYEs found in both parasites share structural similarities and showed affinities for the diterpene icetexanes in the same range. Nevertheless, the interaction between OYEs and ligands is directed by enthalpy and/or entropy in distinct ways. In conclusion, the binding site of both OYEs exhibits remarkable plasticity, and a large range of different molecules, including that can be substrates and inhibitors, can bind this site. This plasticity should be considered in drug design using OYE as a target.

A Short Review of Antimalarial Compounds with Sulfonamide Moiety.

Malaria is a public health problem that causes thousands of deaths, primarily in children in African regions. Artemisinin-based combination therapies (ACTs) have helped to save thousands of lives; however, due to Plasmodium's resistance to available treatments, there is a need to search for new low-cost drugs that act through different mechanisms of action to contain this disease. This review shows that compounds with sulfonamide moiety, possibly, act as inhibitors of P. falciparum carbonic anhydrases, moreover, when linked to a variety of heterocycles potentiate the activities of these compounds and may be used in the design of new antimalarial drugs.

An Overview of Treatment Guidelines and Methods of Synthesis of Drugs Used in Leprosy Chemotherapy.

Leprosy is a Neglected Tropical Disease (NTDs) caused by Mycobacterium leprae (M. leprae). The treatment is considered effective, however, the high dose Multidrug Therapy (MDT) for a long period and its adverse effects result in the abandonment of the treatment by patients. Indeed, antimicrobial resistance is still an obstacle that must be overcome in the treatment of leprosy. In the present article, we reviewed the WHO guidelines for the chemotherapy of leprosy and the methods of synthesis of these drugs.

Corrigendum: Possible Involvement of Hsp90 in the Regulation of Telomere Length and Telomerase Activity During the Leishmania Amazonensis Developmental Cycle and Population Proliferation.

[This corrects the article DOI: 10.3389/fcell.2021.713415.].

Assembly principles of the human R2TP chaperone complex reveal the presence of R2T and R2P complexes.

R2TP is a highly conserved chaperone complex formed by two AAA+ ATPases, RUVBL1 and RUVBL2, that associate with PIH1D1 and RPAP3 proteins. R2TP acts in promoting macromolecular complex formation. Here, we establish the principles of R2TP assembly. Three distinct RUVBL1/2-based complexes are identified: R2TP, RUVBL1/2-RPAP3 (R2T), and RUVBL1/2-PIH1D1 (R2P). Interestingly, we find that PIH1D1 does not bind to RUVBL1/RUVBL2 in R2TP and does not function as a nucleotide exchange factor; instead, RPAP3 is found to be the central subunit coordinating R2TP architecture and linking PIH1D1 and RUVBL1/2. We also report that RPAP3 contains an intrinsically disordered N-terminal domain mediating interactions with substrates whose sequences are primarily enriched for Armadillo repeat domains and other helical-type domains. Our work provides a clear and consistent model of R2TP complex structure and gives important insights into how a chaperone machine concerned with assembly of folded proteins into multisubunit complexes might work.

Possible Involvement of Hsp90 in the Regulation of Telomere Length and Telomerase Activity During the Leishmania amazonensis Developmental Cycle and Population Proliferation.

The Leishmania developmental cycle comprises three main life forms in two hosts, indicating that the parasite is continually challenged due to drastic environmental changes. The disruption of this cycle is critical for discovering new therapies to eradicate leishmaniasis, a neglected disease that affects millions worldwide. Telomeres, the physical ends of chromosomes, maintain genome stability and cell proliferation and are potential antiparasitic drug targets. Therefore, understanding how telomere length is regulated during parasite development is vital. Here, we show that telomeres form clusters spread in the nucleoplasm of the three parasite life forms. We also observed that amastigotes telomeres are shorter than metacyclic and procyclic promastigotes and that in parasites with continuous in vitro passages, telomere length increases over time. These observed differences in telomere length among parasite's life stages were not due to lack/inhibition of telomerase since enzyme activity was detected in all parasite life stages, although the catalysis was temperature-dependent. These data led us to test if, similar to other eukaryotes, parasite telomere length maintenance could be regulated by Hsp83, the ortholog of Hsp90 in trypanosomatids, and Leishmania (LHsp90). Parasites were then treated with the Hsp90 inhibitor 17AAG. The results showed that 17AAG disturbed parasite growth, induced accumulation into G2/M phases, and telomere shortening in a time-dependent manner. It has also inhibited procyclic promastigote's telomerase activity. Besides, LHsp90 interacts with the telomerase TERT component as shown by immunoprecipitation, strongly suggesting a new role for LHsp90 as a parasite telomerase component involved in controlling telomere length maintenance and parasite life span.

Human heat shock cognate protein (HSC70/HSPA8) interacts with negatively charged phospholipids by a different mechanism than other HSP70s and brings HSP90 into membranes.

Heat shock proteins (HSP) are critical elements for the preservation of cellular homeostasis by participating in an array of biological processes. In addition, HSP play an important role in cellular protection from various environmental stresses. HSP are part of a large family of different molecular mass polypeptides, displaying various expression patterns, subcellular localizations, and diversity functions. An unexpected observation was the detection of HSP on the cell surface. Subsequent studies have demonstrated that HSP have the ability to interact and penetrate lipid bilayers by a process initiated by the recognition of phospholipid heads, followed by conformational changes, membrane insertion, and oligomerization. In the present study, we described the interaction of HSPA8 (HSC70), the constitutive cytosolic member of the HSP70 family, with lipid membranes. HSPA8 showed high selectivity for negatively charged phospholipids, such as phosphatidylserine and cardiolipin, and low affinity for phosphatidylcholine. Membrane insertion was mediated by a spontaneous process driven by increases in entropy and diminished by the presence of ADP or ATP. Finally, HSPA8 was capable of driving into the lipid bilayer HSP90 that does not display any lipid biding capacity by itself. This observation suggests that HSPA8 may act as a membrane chaperone.

Purification and characterization of a novel and conserved TPR-domain protein that binds both Hsp90 and Hsp70 and is expressed in all developmental stages of Leishmania major.

Heat shock proteins (Hsps) are involved in several important aspects of the cell proteostasis. Hsp90 interacts with at least a tenth of the cell proteome helping a large number of proteins to fold correctly. Hsp90 function is modulated by several co-chaperones having TPR (tetratricopeptide repeat) domains that allow for interaction with the C-terminal MEEVD motif of the chaperone. Another important chaperone, Hsp70, has a C-terminal EEVD motif that binds to TPR. Leishmania is a protozoan that causes leishmaniasis, a neglected disease in humans and other animals. There is still no effective treatment for leishmaniasis, however the study of structure and function of the proteins of the parasite may generate potential targets for future therapeutic intervention studies. In this work, the genome of Leishmania major was searched for a novel TPR-domain gene, which is conserved in Leishmania. The recombinant protein, LmTPR, was produced in pure and folded state and was characterized by biophysical tools as a monomer with an elongated conformation. Studies in Leishmania major were also preformed to complement these in vitro experiments. Splice Leader RNA-seq analysis and Western blot indicated that the protein was expressed in all developmental stages of the parasite. Binding assays confirmed that both Hsp90 and Hsp70 bind specifically to LmTPR. Finally, sequence and structural predictions indicated a C-terminal region as a RPAP3 domain. Altogether, this study identified a novel TPR-domain co-chaperone of Hsp90 that is conserved and expressed in all developmental stages of Leishmania major.

Human HSPA9 (mtHsp70, mortalin) interacts with lipid bilayers containing cardiolipin, a major component of the inner mitochondrial membrane.

Mitochondrial Hsp70 (HSPA9, mtHsp70, mortalin) in conjunction with a complex set of other proteins is involved in the transport of polypeptides across the mitochondrial matrix. This observation allows us to hypothesize that HSPA9 might interact with membranes directly, similarly to other Hsp70s. Thus, we investigated whether human HSPA9 could also get inserted into lipid membranes. Human HSPA9 was incubated with liposomes made of lipids found within the mitochondrial membrane, such as 1', 3'-bis [1, 2-dimyristoyl-sn-glycero-3-phospho]-glycerol (CL), palmitoyl-oleoyl phosphocholine (POPC), palmitoyl-oleoyl phosphoserine (POPS), and palmitoyl-oleoyl phosphoethanolamine (POPE). HSPA9 displayed a predilection for CL and POPS, and low affinity for POPC and POPE, suggesting that the proteins have high specificity for negatively charged phospholipids. Then, liposomes were made with a composition resembling either the outer or inner mitochondrial membrane (OMM or IMM, respectively). We observed that HSPA9 has a higher affinity for IMM than OMM, which is consistent with the higher content of CL in the IMM. A comparison for the incorporation into POPS or CL liposomes by HSPA9 or HSPA1 indicated that both proteins behaved very similarly when exposed to CL liposomes, but differently with POPS liposomes, which was further corroborated by their susceptibility to proteinase K digestion after incorporation into liposomes. The measurement of thermodynamic parameters also showed that the interaction of both proteins with CL and POPS liposomes was different. Overall, our data showed that HSPA9 is prone to interact with membranes resembling the IMM that may be important for its role in the translocation of proteins into the mitochondria.

Interaction of HSPA5 (Grp78, BIP) with negatively charged phospholipid membranes via oligomerization involving the N-terminal end domain.

Heat shock proteins (HSPs) are ubiquitous polypeptides expressed in all living organisms that participate in several basic cellular processes, including protein folding, from which their denomination as molecular chaperones originated. There are several HSPs, including HSPA5, also known as 78-kDa glucose-regulated protein (GRP78) or binding immunoglobulin protein (BIP) that is an ER resident involved in the folding of polypeptides during their translocation into this compartment prior to the transition to the Golgi network. HSPA5 is detected on the surface of cells or secreted into the extracellular environment. Surface HSPA5 has been proposed to have various roles, such as receptor-mediated signal transduction, a co-receptor for soluble ligands, as well as a participant in tumor survival, proliferation, and resistance. Recently, surface HSPA5 has been reported to be a potential receptor of some viruses, including the novel SARS-CoV-2. In spite of these observations, the association of HSPA5 within the plasma membrane is still unclear. To gain information about this process, we studied the interaction of HSPA5 with liposomes made of different phospholipids. We found that HSPA5 has a high affinity for negatively charged phospholipids, such as palmitoyl-oleoyl phosphoserine (POPS) and cardiolipin (CL). The N-terminal and C-terminal domains of HSPA5 were independently capable of interacting with negatively charged phospholipids, but to a lesser extent than the full-length protein, suggesting that both domains are required for the maximum insertion into membranes. Interestingly, we found that the interaction of HSPA5 with negatively charged liposomes promotes an oligomerization process via intermolecular disulfide bonds in which the N-terminus end of the protein plays a critical role.

Solution structure of Plasmodium falciparum Hsp90 indicates a high flexible dimer.

Hsp90 is a ubiquitous, homodimer and modular molecular chaperone. Each Hsp90 protomer has three different domains, named the N-terminal domain (NTD), middle domain (MD) and C-terminal domain (CTD). The Hsp90 molecular cycle involves ATP binding and hydrolysis, which drive conformational changes. Hsp90 is critical for the viability of eukaryotic organisms, including the protozoan that causes the severe form of malaria, Plasmodium falciparum, the growth and differentiation of which are compromised when Hsp90 is inhibited. Here, we characterize the structure of a recombinant P. falciparum Hsp90 (PfHsp90) protein, as well as its MD (PfHsp90MD) and NTD plus MD (PfHsp90NMD) constructs. All the proteins were obtained with high purity and in the folded state. PfHsp90 and PfHsp90NMD interacted with adenosine nucleotides via the NTD, and Mg2+ was critical for strong binding. PfHsp90 behaved mostly as elongated and flexible dimers in solution, which dissociate with a sub-micromolar dissociation constant. The PfHsp90MD and PfHsp90NMD constructs behaved as globular and elongated monomers, respectively, confirming the importance of the CTD for dimerization. Small angle X-ray scattering data were obtained for all the constructs, and ab initio models were constructed, revealing PfHsp90 in an open conformation and as a greatly elongated and flexible protein.

Discovery of small molecule inhibitors of Leishmania braziliensis Hsp90 chaperone.

Leishmaniasis is a neglected disease caused by the protozoa Leishmania ssp. Environmental differences found by the parasites in the vector and the host are translated into cellular stress, leading to the production of heat shock proteins (Hsp). These are molecular chaperones involved in the folding of nascent proteins as well as in the regulation of gene expression, signalling events and proteostasis. Since Leishmania spp. use Hsp90 to trigger important transitions between their different stages of the life cycle, this protein family becomes a profitable target in anti-parasite drug discovery. In this work, we implemented a multidisciplinary strategy coupling molecular modelling with in vitro assays to identify small molecules able to inhibit Hsp90 from L. braziliensis (LbHsp90). Overall, we identified some compounds able to kill the promastigote form of the L. braziliensis, and to inhibit LbHsp90 ATPase activity.

Thermal aggregates of human mortalin and Hsp70-1A behave as supramolecular assemblies.

The Hsp70 family of heat shock proteins plays a critical function in maintaining cellular homeostasis within various subcellular compartments. The human mitochondrial Hsp70 (HSPA9) has been associated with cellular death, senescence, cancer and neurodegenerative diseases, which is the rational for the name mortalin. It is well documented that mortalin, such as other Hsp70s, is prone to self-aggregation, which is related to mitochondria biogenesis failure. Here, we investigated the assembly, structure and function of thermic aggregates/oligomers of recombinant human mortalin and Hsp70-1A (HSPA1A). Summarily, both Hsp70 thermic aggregates have characteristics of supramolecular assemblies. They display characteristic organized structures and partial ATPase activity, despite their nanometric size. Indeed, we observed that the interaction of these aggregates/oligomers with liposomes is similar to monomeric Hsp70s and, finally, they were non-toxic over neuroblastoma cells. These findings revealed that high molecular mass oligomers of mortalin and Hsp70-1A preserved some of the fundamental functions of these proteins.

Structural studies of the Hsp70/Hsp90 organizing protein of Plasmodium falciparum and its modulation of Hsp70 and Hsp90 ATPase activities.

HOP is a cochaperone belonging to the foldosome, a system formed by the cytoplasmic Hsp70 and Hsp90 chaperones. HOP acts as an adapter protein capable of transferring client proteins from the first to the second molecular chaperone. HOP is a modular protein that regulates the ATPase activity of Hsp70 and Hsp90 to perform its function. To obtain more detailed information on the structure and function of this protein, we produced the recombinant HOP of Plasmodium falciparum (PfHOP). The protein was obtained in a folded form, with a high content of α-helix secondary structure. Unfolding experiments showed that PfHOP unfolds through two transitions, suggesting the presence of at least two domains with different stabilities. In addition, PfHOP primarily behaved as an elongated dimer in equilibrium with the monomer. Small-angle X-ray scattering data corroborated this interpretation and led to the reconstruction of a PfHOP ab initio model as a dimer. Finally, the PfHOP protein was able to inhibit and to stimulate the ATPase activity of the recombinant Hsp90 and Hsp70-1, respectively, of P. falciparum. Our results deepened the knowledge of the structure and function of PfHOP and further clarified its participation in the P. falciparum foldosome.

Heterobimetallic nickel(II) and palladium(II) complexes derived from S-benzyl-N- (ferrocenyl)methylenedithiocarbazate: Trypanocidal activity and interaction with Trypanosoma cruzi Old Yellow Enzyme (TcOYE).

Reactions of Ni(II) and Pd(II) precursors with S-benzyl-N-(ferrocenyl)methylenedithiocarbazate (HFedtc) led to the formation of heterobimetallic complexes of the type [MII(Fedtc)2] (M = Ni and Pd). The characterization of the compounds involved the determination of melting point, FTIR, UV-Vis, 1H NMR, elemental analysis and electrochemical experiments. Furthermore, the crystalline structures of HFedtc and [NiII(Fedtc)2] were determined by single crystal X-ray diffraction. The compounds were evaluated against the intracellular form of Trypanosoma cruzi (Tulahuen Lac-Z strain) and the cytotoxicity assays were assessed using LLC-MK2 cells. The results showed that the coordination of HFedtc to Ni(II) or Pd(II) decreases the in vitro trypanocidal activity while the cytotoxicity against LLC-MK2 cells does not change significantly. [PdII(Fedtc)2] showed the greater potential between the two complexes studied, showing an SI value of 8.9. However, this value is not better than that of the free ligand with an SI of 40, a similar value to that of the standard drug benznidazole (SI = 48). Additionally, molecular docking simulations were performed with Trypanosoma cruzi Old Yellow Enzyme (TcOYE), which predicted that HFedtc binds to the protein, almost parallel to the flavin mononucleotide (FMN) prosthetic group, while the [NiII(Fedtc)2] complex was docked into the enzyme binding site in a significantly different manner. In order to confirm the hypothetical interaction, in vitro experiments of fluorescence quenching and enzymatic activity were performed which indicated that, although HFedtc was not processed by the enzyme, it was able to act as a competitive inhibitor, blocking the hydride transfer from the FMN prosthetic group of the enzyme to the menadione substrate.

Studies on the effect of the J-domain on the substrate binding domain (SBD) of Hsp70 using a chimeric human J-SBD polypeptide.

DnaJ/Hsp40 chaperones deliver unfolded proteins and stimulate the ATPase activity of DnaK/Hsp70 via their J-domain. However, the interaction is transient, creating a challenge for detailed analysis. We investigated whether it would be possible to gain further understanding of this interaction by engineering a chimeric polypeptide where the J-domain of Hsp40 was covalently attached to the substrate binding domain (SBD) of Hsp70 by a flexible linker. The rationale is to increase the proximity between the interacting partners to promote their natural interaction and facilitate the characterization of the interaction. The resulting chimera, termed J-SBD, was properly folded and had properties not present in the full-length Hsp70 or in the SBD alone, for instance a higher protective effect against aggregation and being a monomer. Substrate binding also appear to exceed that of SBD alone as revealed by a decreased binding to bis-ANS, a probe for hydrophobic patches. This hypothesis is supported by the structural model created by small angle X-ray scattering, suggesting that the lid subdomain (SBDα) is partially opened in the J-SBD. Collectively, our results suggest a model in which J-domain binding may shift the Hsp70 equilibrium towards the monomer state, exposing hydrophobic sites prone to substrate accommodation.

Structural and functional studies of the Leishmania braziliensis SGT co-chaperone indicate that it shares structural features with HIP and can interact with both Hsp90 and Hsp70 with similar affinities.

Molecular chaperones and co-chaperones play an essential role in the life cycles of protozoa belonging to the genus Leishmania. The small glutamine-rich TPR-containing protein (SGT) is a co-chaperone that can be divided into three domains: N-terminal, tetratricopeptide (TPR) and C-terminal. The TPR domain is responsible for interactions with both Hsp70 and Hsp90; however, the mechanism of interaction and the functionality of SGT are unclear. In this context, we present the structural and functional characterization of Leishmania braziliensis SGT (LbSGT), aiming to elucidate how this co-chaperone interacts with the Hsp90/Hsp70 chaperone machinery. Structurally, the recombinant LbSGT behaves as an α-helical, multidomain and elongated dimer in solution. Despite their low amino acid sequence identity and similarity, LbSGT shares structural properties and domain organization with the Hsp70-interacting protein (HIP) co-chaperone. Functionally, LbSGT is a cognate protein in L. braziliensis promastigote cells and interacts indiscriminately, with similar affinities, with both Hsp90 and Hsp70 chaperones, capable of working as an adaptor protein. Sequence analysis indicates that LbSGT interacts via a dicarboxylate clamp, the same mechanism used by the Hsp90-Hsp70-organizing protein (HOP) co-chaperone. These results suggest that SGT can develop the same function as HOP but using the HIP structural scaffold.

Comparative studies of the low-resolution structure of two p23 co-chaperones for Hsp90 identified in Plasmodium falciparum genome.

The p23 proteins are small acidic proteins that aid the functional cycle of the Hsp90 molecular chaperone. Such co-chaperone acts by temporarily inhibiting the ATPase activity of Hsp90 and exhibits intrinsic chaperone activity, suggesting independent roles. A search for p23 in the Plasmodium falciparum genome led to the identification of two putative proteins showing 13% identity to each other and approximately 20% identity to human p23. To understand the presence of two p23 proteins in this organism, we generated recombinant p23 proteins (Pfp23A and Pfp23B) and investigated their structure and function. The proteins presented some similarities and dissimilarities in structural contents and showed different chemical and thermal stabilities, with Pfp23A being more stable than Pfp23B, suggesting that these proteins may present different functions in this organism. Both Pfp23 proteins behaved as elongated monomers in solution and were capable of preventing the thermal-induced aggregation of model client proteins with different efficiencies. Finally, the Pfp23 proteins inhibited the ATPase activity of recombinant P. falciparum Hsp90 (PfHsp90). These results validate the studied proteins as p23 proteins and co-chaperones of PfHsp90.

PtII, PdII and AuIII complexes with a thiosemicarbazone derived from diacethylmonooxime: Structural analysis, trypanocidal activity, cytotoxicity and first insight into the antiparasitic mechanism of action.

New complexes of composition [MX(HL1)] (M = PtII, PdII, X = Cl- or I-) and [MX(L1)] (M = AuIII, X = Cl-; M = PtII, PdII, X = PPh3) have been synthesized using a potentially tridentate thiosemicarbazone (H2L1) containing an additional oxime binding site. Among other analytical methods, all the seven complexes have been structurally characterized by single crystal X-ray diffractometry. Interesting structural features such as the influence of the halide ligands on hydrogen bonds and the formation of supramolecular structures for the phosphine derivatives are discussed. The in vitro trypanocidal activity of the free ligand H2L1 and its derivatives against both extracellular trypomastigote and intracellular amastigote (IC50try/ama) forms of Trypanosoma cruzi (Tulahuen Lac-Z strain) and the cytotoxicity was assessed on LLC-MK2 cell line. The results showed that complexation of the thiosemicarbazone ligand H2L1 to PtII, PdII and AuIII metal centers enhances the in vitro trypanocidal activity and that the cytotoxicity is dependent on both the metal center and coligands. Within the studied series, the AuIII complex showed the greatest potential, being not the most active but the most selective compound with a similar selectivity index to that of the standard drug benznidazole. In order to get a preliminary insight into the mechanism of action of these compounds, in vitro experiments of fluorescence quenching and enzymatic activity were performed using the AuIII complex and Trypanosoma cruzi Old Yellow Enzyme (TcOYE) which indicated that the gold derivative was capable of abstracting the hydride from the prosthetic FMN group of the enzyme. Additionally, molecular docking studies followed by semiempirical simulations showed that the [AuCl(L1)] binds to the binary complex TcOYE/FMN, almost parallel to the FMN prosthetic group, in a close distance that an electron/proton transfer might occur among them.