Tetraenone A: A New ß-Ionone Derivative from Tetraena aegyptia.

In this study, the chemical investigation of Tetraena aegyptia (Zygophyllaceae) led to the identification of a new megastigmene derivative, tetraenone A ((2S, 5R, 6R, 7E)-2-hydroxy-5,6-dihydro-ß-ionone) (1), along with (3S, 5R, 6S, 7E)-3-hydroxy-5,6-epoxy-5,6-dihydro-ß-ionone- (2), 3,4-dihydroxy-cinnamyl alcohol-4-glucoside (3), 3ß,19α-dihydroxy-ursan-28-oic acid (4), quinovic acid (5), p-coumaric acid (6), and ferulic acid (7), for the first time. The chemical structures of 1-7 were confirmed by analysis of their 1D and 2D NMR and HRESIMS spectra and by their comparison with the relevant literature. The absolute configurations of 1 and 2 were assigned based on NOESY interactions and ECD spectra. Conformational analysis showed that 1 existed exclusively in one of the two theoretically possible chair conformers with a predominant s-trans configuration for the 3-oxobut-1-en-1-yl group with the ring, while the half-chair conformer had a pseudo-axial hydroxy group that was predominant over the other half-chair conformation. Boat conformations were not among the most stable conformations, and the s-trans isomerism was in favor of s-cis configuration. In silico investigation revealed that 1 and 2 had more favorable binding interactions with Mpro rather than with TMPRSS2. Accordingly, molecular dynamic simulations were performed on the complexes of compounds 1 and 2 with Mpro to explore the stability of their interaction with the target protein structure. Compounds 1 and 2 might offer a possible starting point for developing covalent inhibitors of Mpro of SARS-CoV-2.

X-ray Structure Characterization of the Selective Recognition of AT Base Pair Sequences.

The rational design of small molecules that target specific DNA sequences is a promising strategy to modulate gene expression. This report focuses on a diamidinobenzimidazole compound, whose selective binding to the minor groove of AT DNA sequences holds broad significance in the molecular recognition of AT-rich human promoter sequences. The objective of this study is to provide a more detailed and systematized understanding, at an atomic level, of the molecular recognition mechanism of different AT-specific sequences by a rationally designed minor groove binder. The specialized method of X-ray crystallography was utilized to investigate how the sequence-dependent recognition properties in general, A-tract, and alternating AT sequences affect the binding of diamidinobenzimidazole in the DNA minor groove. While general and A-tract AT sequences give a narrower minor groove, the alternating AT sequences intrinsically have a wider minor groove which typically constricts upon binding. A strong and direct hydrogen bond between the N-H of the benzimidazole and an H-bond acceptor atom in the minor groove is essential for DNA recognition in all sequences described. In addition, the diamidine compound specifically utilizes an interfacial water molecule for its DNA binding. DNA complexes of AATT and AAAAAA recognition sites show that the diamidine compound can bind in two possible orientations with a preference for water-assisted hydrogen bonding at either cationic end. The complex structures of AAATTT, ATAT, ATATAT, and AAAA are bound in a singular orientation. Analysis of the helical parameters shows a minor groove expansion of about 1 Å across all the nonalternating DNA complexes. The results from this systematic approach will convey a greater understanding of the specific recognition of a diverse array of AT-rich sequences by small molecules and more insight into the design of small molecules with enhanced specificity to AT and mixed DNA sequences.

Small Molecules Incorporating Privileged Amidine Moiety as Potential Hits Combating Antibiotic-Resistant Bacteria.

The continuing need for the discovery of potent antibacterial agents against antibiotic-resistant pathogens is the driving force for many researchers to design and develop such agents. Herein, we report the design, synthesis, and biological evaluation of amidine derivatives as new antibacterial agents. Compound 13d was the most active in this study against a wide range of antibiotic-resistant, and susceptible, Gram-positive, and Gram-negative bacterial strains. Time-kill assay experiments indicated that compound 13d was an effective bactericidal compound against the tested organisms at the log-phase of bacterial growth. Docking simulations were performed to assess in silico its mode of action regarding UPPS, KARI, and DNA as potential bacterial targets. Results unveiled the importance of structural features of compound 13d in its biological activity including central thiophene ring equipped with left and right pyrrolo[2,3-b]pyridine and phenyl moieties and two terminal amidines cyclized into 4,5-dihydro-1H-imidazol-2-yl functionalities. Collectively, compound 13d represents a possible hit for future development of potent antibacterial agents.

(E)-5-(4-Meth-oxy-phen-yl)-N'-(2-oxoindolin-3-yl-idene)-1-phenyl-1H-pyrazole-3-carbohydrazide.

The asymmetric unit of the title compound, C25H19N5O3, is composed of two independent mol-ecules with slightly different conformations. The extended structure features N-H⋯O hydrogen bonds as well as π-π inter-actions.

In Silico and In Vitro Evaluation of Some Amidine Derivatives as Hit Compounds towards Development of Inhibitors against Coronavirus Diseases.

Coronaviruses, including SARS-CoV-2, SARS-CoV, MERS-CoV and influenza A virus, require the host proteases to mediate viral entry into cells. Rather than targeting the continuously mutating viral proteins, targeting the conserved host-based entry mechanism could offer advantages. Nafamostat and camostat were discovered as covalent inhibitors of TMPRSS2 protease involved in viral entry. To circumvent their limitations, a reversible inhibitor might be required. Considering nafamostat structure and using pentamidine as a starting point, a small set of structurally diverse rigid analogues were designed and evaluated in silico to guide selection of compounds to be prepared for biological evaluation. Based on the results of in silico study, six compounds were prepared and evaluated in vitro. At the enzyme level, compounds 10-12 triggered potential TMPRSS2 inhibition with low micromolar IC50 concentrations, but they were less effective in cellular assays. Meanwhile, compound 14 did not trigger potential TMPRSS2 inhibition at the enzyme level, but it showed potential cellular activity regarding inhibition of membrane fusion with a low micromolar IC50 value of 10.87 µM, suggesting its action could be mediated by another molecular target. Furthermore, in vitro evaluation showed that compound 14 inhibited pseudovirus entry as well as thrombin and factor Xa. Together, this study presents compound 14 as a hit compound that might serve as a starting point for developing potential viral entry inhibitors with possible application against coronaviruses.

Escaping ESKAPE resistance: in vitro and in silico studies of multifunctional carbamimidoyl-tethered indoles against antibiotic-resistant bacteria.

Combining the hybridization and repurposing strategies, six compounds from our in-house library and having a designed hybrid structure of MBX-1162, pentamidine and MMV688271 were repurposed as potential antibacterial agents. Among, compounds 1a and 1d elicited potential sub-µg ml-1 activity against the high-priority antibiotic-resistant Gram-positive members of ESKAPE bacteria as well as antibiotic-susceptible Gram-positive bacteria. Furthermore, they showed potential low µg ml-1 activity against the explored critical-priority antibiotic-resistant Gram-negative members of ESKAPE bacteria. In time-kill assay, compound 1a has effective 0.5 and 0.25 µg ml-1 antibacterial lethal concentrations against MRSA in exponential growth phase. In silico investigations predicted compounds 1a and 1d as inhibitors of the open conformation of undecaprenyl diphosphate synthase involved in bacterial isoprenoid synthesis. In addition, compounds 1a and 1d were predicted as inhibitors of NADPH-free but not NADPH-bound form of ketol-acid reductoisomerase and may also serve as potential B-DNA minor groove binders with possible differences in the molecular sequence recognition. Overall, compounds 1a and 1d are presented as multifunctional potential antibacterial agents for further development against high- and critical-priority Gram-positive and Gram-negative antibiotic-resistant ESKAPE bacterial pathogens as well as antibiotic-susceptible Gram-positive bacterial pathogens.

A Novel Hydroxyapatite/Vitamin B12 Nanoformula for Treatment of Bone Damage: Preparation, Characterization, and Anti-Arthritic, Anti-Inflammatory, and Antioxidant Activities in Chemically Induced Arthritic Rats.

The usage of nanomaterials for rheumatoid arthritis (RA) treatment can improve bioavailability and enable selective targeting. The current study prepares and evaluates the in vivo biological effects of a novel hydroxyapatite/vitamin B12 nanoformula in Complete Freund's adjuvant-induced arthritis in rats. The synthesized nanoformula was characterized using XRD, FTIR, BET analysis, HERTEM, SEM, particle size, and zeta potential. We synthesized pure HAP NPs with 71.01% loading weight percentages of Vit B12 and 49 mg/g loading capacity. Loading of vitamin B12 on hydroxyapatite was modeled by Monte Carlo simulation. Anti-arthritic, anti-inflammatory, and antioxidant effects of the prepared nanoformula were assessed. Treated arthritic rats showed lower levels of RF and CRP, IL-1ß, TNF-α, IL-17, and ADAMTS-5, but higher IL-4 and TIMP-3 levels. In addition, the prepared nanoformula increased GSH content and GST antioxidant activity while decreasing LPO levels. Furthermore, it reduced the expression of TGF-ß mRNA. Histopathological examinations revealed an improvement in joint injuries through the reduction of inflammatory cell infiltration, cartilage deterioration, and bone damage caused by Complete Freund's adjuvant. These findings indicate that the anti-arthritic, antioxidant, and anti-inflammatory properties of the prepared nanoformula could be useful for the development of new anti-arthritic treatments.

Investigation of the effect of structure modification of furamidine on the DNA minor groove binding and antiprotozoal activity.

New analogs of the antiprotozoal agent Furamidine were prepared utilizing Stille coupling reactions and amidation of the bisnitrile intermediate using lithium bis-trimethylsilylamide. Both the phenyl groups and the furan moiety of furamidine were replaced by heterocycles including thiophene, selenophene, indole or benzimidazole. Based upon the ΔTm and the CD results, the new compounds showed strong binding to the DNA minor groove. The new analogues are also more active both in vitro and in vivo than furamidine. Compounds 7a, 7b, and 7f showed the highest activity in vivo by curing 75% of animals, and this merits further evaluation.

Selective Sensing of Darolutamide and Thalidomide in Pharmaceutical Preparations and in Spiked Biofluids.

Selective spectrofluorometric sensing is introduced for the analysis of non-steroidal anti-androgens, darolutamide, and thalidomide in pharmaceutical preparations and biofluids. An organic fluorophore, 2,4,8,10-tetramethylpyrido[2',3':3,4]pyrazolo[1,5-a]pyrimidine 2 was synthesized in our laboratories by new simple methods to act as a fluorescent reagent for the analysis of the studied drugs. Elemental and spectral analyses were performed to approve the fluorophore structure. The fluorophore possesses a fluorescence at λem 422 nm when excited at 328 nm. The interaction between the studied drugs and the fluorophore was found to be quenching. The quenching mechanisms were studied and interpreted through the Stern-Volmer relationship. Moreover, the Stern-Volmer constants were calculated for the quenching interactions of both drugs. The introduced method was validated for the estimation of darolutamide and thalidomide in dosage forms, plasma, and urine, offering good percentage recoveries.

Thermodynamic Factors That Drive Sequence-Specific DNA Binding of Designed, Synthetic Minor Groove Binding Agents.

Ken Breslauer began studies on the thermodynamics of small cationic molecules binding in the DNA minor groove over 30 years ago, and the studies reported here are an extension of those ground-breaking reports. The goals of this report are to develop a detailed understanding of the binding thermodynamics of pyridine-based sequence-specific minor groove binders that have different terminal cationic groups. We apply biosensor-surface plasmon resonance and ITC methods to extend the understanding of minor groove binders in two directions: (i) by using designed, heterocyclic dicationic minor groove binders that can incorporate a G•C base pair (bp), with flanking AT base pairs, into their DNA recognition site, and bind to DNA sequences specifically; and (ii) by using a range of flanking AT sequences to better define molecular recognition of the minor groove. A G•C bp in the DNA recognition site causes a generally more negative binding enthalpy than with most previously used pure AT binding sites. The binding is enthalpy-driven at 25 °C and above. The flanking AT sequences also have a large effect on the binding energetics with the -AAAGTTT- site having the strongest affinity. As a result of these studies, we now have a much better understanding of the effects of the DNA sequence and compound structure on the molecular recognition and thermodynamics of minor groove complexes.

Engineered modular heterocyclic-diamidines for sequence-specific recognition of mixed AT/GC base pairs at the DNA minor groove.

This report describes a breakthrough in a project to design minor groove binders to recognize any sequence of DNA. A key goal is to invent synthetic chemistry for compound preparation to recognize an adjacent GG sequence that has been difficult to target. After trying several unsuccessful compound designs, an N-alkyl-benzodiimidazole structure was selected to provide two H-bond acceptors for the adjacent GG-NH groups. Flanking thiophenes provide a preorganized structure with strong affinity, DB2831, and the structure is terminated by phenyl-amidines. The binding experimental results for DB2831 with a target AAAGGTTT sequence were successful and include a high ΔT m, biosensor SPR with a K D of 4 nM, a similar K D from fluorescence titrations and supporting competition mass spectrometry. MD analysis of DB2831 bound to an AAAGGTTT site reveals that the two unprotonated N of the benzodiimidazole group form strong H-bonds (based on distance) with the two central G-NH while the central -CH of the benzodiimidazole is close to the -C[double bond, length as m-dash]O of a C base. These three interactions account for the strong preference of DB2831 for a -GG- sequence. Surprisingly, a complex with one dynamic, interfacial water is favored with 75% occupancy.

Dissecting Dynamic and Hydration Contributions to Sequence-Dependent DNA Minor Groove Recognition.

Sequence selectivity is a critical attribute of DNA-binding ligands and underlines the need for detailed molecular descriptions of binding in representative sequence contexts. We investigated the binding and volumetric properties of DB1976, a model bis(benzimidazole)-selenophene diamidine compound with emerging therapeutic potential in acute myeloid leukemia, debilitating fibroses, and obesity-related liver dysfunction. To sample the scope of cognate DB1976 target sites, we evaluated three dodecameric duplexes spanning >103-fold in binding affinity. The attendant changes in partial molar volumes varied substantially, but not in step with binding affinity, suggesting distinct modes of interactions in these complexes. Specifically, whereas optimal binding was associated with loss of hydration water, low-affinity binding released more hydration water. Explicit-atom molecular dynamics simulations showed that minor groove binding perturbed the conformational dynamics and hydration at the termini and interior of the DNA in a sequence-dependent manner. The impact of these distinct local dynamics on hydration was experimentally validated by domain-specific interrogation of hydration with salt, which probed the charged axial surfaces of oligomeric DNA preferentially over the uncharged termini. Minor groove recognition by DB1976, therefore, generates dynamically distinct domains that can make favorable contributions to hydration release in both high- and low-affinity binding. Because ligand binding at internal sites of DNA oligomers modulates dynamics at the termini, the results suggest both short- and long-range dynamic effects along the DNA target that can influence their effectiveness as low-MW competitors of protein binding.

Extending the σ-Hole Motif for Sequence-Specific Recognition of the DNA Minor Groove.

The majority of current drugs against diseases, such as cancer, can bind to one or more sites in a protein and inhibit its activity. There are, however, well-known limits on the number of druggable proteins, and complementary current drugs with compounds that could selectively target DNA or RNA would greatly enhance the availability of cellular probes and therapeutic progress. We are focusing on the design of sequence-specific DNA minor groove binders that, for example, target the promoter sites of transcription factors involved in a disease. We have started with AT-specific minor groove binders that are known to enter human cells and have entered clinical trials. To broaden the sequence-specific recognition of these compounds, several modules that have H-bond acceptors that strongly and specifically recognize G·C base pairs were identified. A lead module is a thiophene-N-alkyl-benzimidazole σ-hole-based system with terminal phenyl-amidines that have excellent affinity and selectivity for a G·C base pair in the minor groove. Efforts are now focused on optimizing this module. In this work, we are evaluating modifications to the compound aromatic system with the goal of improving GC selectivity and affinity. The lead compounds retain the thiophene-N-alkyl-BI module but have halogen substituents adjacent to an amidine group on the terminal phenyl-amidine. The optimum compounds must have strong affinity and specificity with a residence time of at least 100 s.

Small Sequence-Sensitive Compounds for Specific Recognition of the G⋠C Base Pair in DNA Minor Groove.

A series of small diamidines with thiophene and modified N-alkylbenzimidazole σ-hole module represent specific binding to single G⋅C base pair (bp) DNA sequence. The variation of N-alkyl or aromatic rings were sensitive to microstructures of the DNA minor groove. Thirteen new compounds were synthesized to test their binding affinity and selectivity. The dicyanobenzimidazoles needed to synthesize the target diamidines were made via condensation/cyclization reactions of different aldehydes with different 3-amino-4-(alkyl- or phenyl-amino) benzonitriles. The final diamidines were synthesized using lithium bis-trimethylsilylamide (LiN[Si(CH3 )3 ]2 ) or Pinner methods. The newly synthesized compounds showed strong binding and selectivity to AAAGTTT compared to similar sequences AAATTT and AAAGCTTT investigated by several biophysical methods including biosensor-SPR, fluorescence spectroscopy, DNA thermal melting, ESI-MS spectrometry, circular dichroism, and molecular dynamics. The binding affinity results determined by fluorescence spectroscopy are in accordance with those obtained by biosensor-SPR. These small size single G⋅C bp highly specific binders extend the compound database for future biological applications.

Heterocyclic Diamidine DNA Ligands as HOXA9 Transcription Factor Inhibitors: Design, Molecular Evaluation, and Cellular Consequences in a HOXA9-Dependant Leukemia Cell Model.

Most transcription factors were for a long time considered as undruggable targets because of the absence of binding pockets for direct targeting. HOXA9, implicated in acute myeloid leukemia, is one of them. To date, only indirect targeting of HOXA9 expression or multitarget HOX/PBX protein/protein interaction inhibitors has been developed. As an attractive alternative by inhibiting the DNA binding, we selected a series of heterocyclic diamidines as efficient competitors for the HOXA9/DNA interaction through binding as minor groove DNA ligands on the HOXA9 cognate sequence. Selected DB818 and DB1055 compounds altered HOXA9-mediated transcription in luciferase assays, cell survival, and cell cycle, but increased cell death and granulocyte/monocyte differentiation, two main HOXA9 functions also highlighted using transcriptomic analysis of DB818-treated murine Hoxa9-transformed hematopoietic cells. Altogether, these data demonstrate for the first time the propensity of sequence-selective DNA ligands to inhibit HOXA9/DNA binding both in vitro and in a murine Hoxa9-dependent leukemic cell model.

DNA recognition by linear indole-biphenyl DNA minor groove ligands.

Linear heterocyclic cations are interesting DNA minor groove ligands due to their lack of isohelical curvature classically associated with groove-binding compounds. We determined the DNA binding properties of four related dications harboring a linear indole-biphenyl core: the diamidine DB1883, a ditetrahydropyrimidine derivative (DB1804), and their monocationic counterparts (DB1944 and DB2627). These compounds exhibit heterogeneity in binding in accordance with their structures. Whereas the monocations exhibit salt-sensitive 1:1 binding to the duplex 5'-CGCGAATTCGCG-3' (A2T2), the dications show a marked preference for a salt-insensitive 2:1 complex. The two binding modes are differentially modulated by salt and specific non-ionic co-solutes. For both dications, 2-methyl-2,4-pentanediol enforces 1:1 binding as observed crystallographically. Fluorescence quenching studies show self-association without DNA in a relative order that is correlated with preference for the 2:1 complex. The data support a structure-binding relationship in which favorable cation-π interactions drive dimer formation via antiparallel stacking of the linear indole-biphenyl cation motif.

Compound Shape Effects in Minor Groove Binding Affinity and Specificity for Mixed Sequence DNA.

AT specific heterocyclic cations that bind in the DNA duplex minor groove have had major successes as cell and nuclear stains and as therapeutic agents which can effectively enter human cells. Expanding the DNA sequence recognition capability of the minor groove compounds could also expand their therapeutic targets and have an impact in many areas, such as modulation of transcription factor biological activity. Success in the design of mixed sequence binding compounds has been achieved with N-methylbenzimidazole ( N-MeBI) thiophenes which are preorganized to fit the shape of the DNA minor groove and H-bond to the -NH of G·C base pairs that project into the minor groove. Initial compounds bind strongly to a single G·C base pair in an AT context with a specificity ratio of 50 ( KD AT-GC/ KD AT) or less and this is somewhat low for biological use. We felt that modifications of compound shape could be used to probe local DNA microstructure in target mixed base pair sequences of DNA and potentially improve the compound binding selectivity. Modifications were made by increasing the size of the benzimidazole N-substituent, for example, by using N-isobutyl instead of N-Me, and by changing the molecular twist by introducing substitutions at specific positions on the aromatic core of the compounds. In both cases, we have been able to achieve a dramatic increase in binding specificity, including no detectible binding to pure AT sequences, without a significant loss in affinity to mixed base pair target sequences.

Indole and Benzimidazole Bichalcophenes: Synthesis, DNA Binding and Antiparasitic Activity.

A novel series of indole and benzimidazole bichalcophene diamidine derivatives were prepared to study their antimicrobial activity against the tropical parasites causing African sleeping sickness and malaria. The dicyanoindoles needed to synthesize the target diamidines were obtained through Stille coupling reactions while the bis-cyanobenzimidazoles intermediates were made via condensation/cyclization reactions of different aldehydes with 4-cyano-1,2-diaminobenzene. Different amidine synthesis methodologies namely, lithium bis-trimethylsilylamide (LiN[Si(CH3)3]2) and Pinner methods were used to prepare the diamidines. Both types (indole and benzimidazole) derivatives of the new diamidines bind strongly with the DNA minor groove and generally show excellent in vitro antitrypanosomal activity. The diamidino-indole derivatives also showed excellent in vitro antimalarial activity while their benzimidazole counterparts were generally less active. Compound 7c was highly active in vivo and cured all mice infected with Trypanosoma brucei rhodesiense, a model that mimics the acute stage of African sleeping sickness, at a low dose of 4 × 5 mg/kg i.p. and hence 7c is more potent in vivo than pentamidine.

Antileishmanial Efficacy and Pharmacokinetics of DB766-Azole Combinations.

Given the limitations of current antileishmanial drugs and the utility of oral combination therapy for other infections, developing an oral combination against visceral leishmaniasis should be a high priority. In vitro combination studies with DB766 and antifungal azoles against intracellular Leishmania donovani showed that posaconazole and ketoconazole, but not fluconazole, enhanced DB766 potency. Pharmacokinetic analysis of DB766-azole combinations in uninfected Swiss Webster mice revealed that DB766 exposure was increased by higher posaconazole and ketoconazole doses, while DB766 decreased ketoconazole exposure. In L. donovani-infected BALB/c mice, DB766-posaconazole combinations given orally for 5 days were more effective than DB766 or posaconazole alone. For example, 81% ± 1% (means ± standard errors) inhibition of liver parasite burden was observed for 37.5 mg/kg of body weight DB766 plus 15 mg/kg posaconazole, while 37.5 mg/kg DB766 and 15 mg/kg posaconazole administered as monotherapy gave 40% ± 5% and 21% ± 3% inhibition, respectively. Combination index (CI) analysis indicated that synergy or moderate synergy was observed in six of nine combined dose groups, while the other three were nearly additive. Liver concentrations of DB766 and posaconazole increased in almost all combination groups compared to monotherapy groups, although many increases were not statistically significant. For DB766-ketoconazole combinations evaluated in this model, two were antagonistic, one displayed synergy, and one was nearly additive. These data indicate that the efficacy of DB766-posaconazole and DB766-ketoconazole combinations in vivo is influenced in part by the pharmacokinetics of the combination, and that the former combination deserves further consideration in developing new treatment strategies against visceral leishmaniasis.

G-quadruplex-binding small molecules ameliorate C9orf72 FTD/ALS pathology in vitro and in vivo.

Intronic GGGGCC repeat expansions in C9orf72 are the most common known cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), which are characterised by degeneration of cortical and motor neurons, respectively. Repeat expansions have been proposed to cause disease by both the repeat RNA forming foci that sequester RNA-binding proteins and through toxic dipeptide repeat proteins generated by repeat-associated non-ATG translation. GGGGCC repeat RNA folds into a G-quadruplex secondary structure, and we investigated whether targeting this structure is a potential therapeutic strategy. We performed a screen that identified three structurally related small molecules that specifically stabilise GGGGCC repeat G-quadruplex RNA We investigated their effect in C9orf72 patient iPSC-derived motor and cortical neurons and show that they significantly reduce RNA foci burden and the levels of dipeptide repeat proteins. Furthermore, they also reduce dipeptide repeat proteins and improve survival in vivo, in GGGGCC repeat-expressing Drosophila Therefore, small molecules that target GGGGCC repeat G-quadruplexes can ameliorate the two key pathologies associated with C9orf72 FTD/ALS These data provide proof of principle that targeting GGGGCC repeat G-quadruplexes has therapeutic potential.