With the growing number and diversity of known genome sequences, there is an increasing opportunity to regulate gene expression through synthetic, cell-permeable small molecules. Enhancing the DNA sequence recognition abilities of minor groove compounds has the potential to broaden their therapeutic applications with significant implications for areas such as modulating transcription factor activity. While various classes of minor groove binding agents can selectively identify pure AT and mixed AT and GC base pair(s) containing sequences, there remains a lack of compounds capable of distinguishing between different AT sequences. In this work, we report on the design compounds that exhibit selective binding to -TTAA- or -TATA- containing DNA minor groove sequences compared with other AT ones. Several studies have shown that the -AATT- and -TTAA- sequences have distinct physical and interaction properties, especially in terms of their different requirements for recognition in the minor groove. Achieving strong, selective minor groove binding at -TTAA- sequences has been challenging, but DB1003, a benzimidazole-furan-furan diamidine, has demonstrated cooperative dimeric binding activity at -TTAA-. It has significantly less binding preference for AATT. To better understand and modify the selectivity, we synthesized a set of rationally designed analogs of DB1003 by altering the position of the five-membered heterocyclic structure. Binding affinities and stoichiometries obtained from biosensor-surface plasmon resonance experiments show that DB1992, a benzimidazolefuran-thiophene diamidine, binds strongly to -TTAA- as a positive cooperative dimer with high cooperativity. The high-resolution crystal structure of the TTAA-DNA-DB1992 complex reveals that DB1992 binds as an antiparallel π-stacked dimer with numerous diverse contacts to the DNA minor groove. This distinctive binding arrangement and the properties of diamidines at the -TTAA- minor groove demonstrate that benzimidazole-furan-thiophene is a unique DNA binding pharmacophore. Competition mass spectroscopy and circular dichroism studies confirmed the binding stoichiometry and selectivity preference of the compounds for the -TTAA- sequence.

Transcription factor (TF) DNA-binding dynamics govern cell fate and identity. However, our ability to pharmacologically control TF localization is limited. Here we leverage chemically driven binding site restriction leading to robust and DNA-sequence-specific redistribution of PU.1, a pioneer TF pertinent to many hematopoietic malignancies. Through an innovative technique, 'CLICK-on-CUT&Tag', we characterize the hierarchy of de novo PU.1 motifs, predicting occupancy in the PU.1 cistrome under binding site restriction. Temporal and single-molecule studies of binding site restriction uncover the pioneering dynamics of native PU.1 and identify the paradoxical activation of an alternate target gene set driven by PU.1 localization to second-tier binding sites. These transcriptional changes were corroborated by genetic blockade and site-specific reporter assays. Binding site restriction and subsequent PU.1 network rewiring causes primary human leukemia cells to differentiate. In summary, pharmacologically induced TF redistribution can be harnessed to govern TF localization, actuate alternate gene networks and direct cell fate.

The recognition of specific genomic arrangements by rationally designed small molecules is fundamental for the expansion of targeted gene expression. Here, we report the first X-ray crystal structures that demonstrate single G (guanine) recognition by a highly selective diamidine (DB2447) in a mixed DNA sequence. The study presents detailed structural information on the mechanism of single G recognition by D2447 and its various interactions in the DNA minor groove. Molecular dynamics and binding studies were used to evaluate the details of our reported structures. The study provides structural insight and resources necessary for understanding single G selection in genomic sequences.

The mainstay of acute myeloid leukemia (AML) treatment still relies on traditional chemotherapy, with a survival rate of approximately 30% for patients under 65 years of age and as low as 5% for those beyond. This unfavorable prognosis primarily stems from frequent relapses, resistance to chemotherapy, and limited approved targeted therapies for specific AML subtypes. Around 70% of all AML cases show overexpression of the transcription factor HOXA9, which is associated with a poor prognosis, increased chemoresistance, and higher relapse rates. However, direct targeting of HOXA9 in a clinical setting has not been achieved yet. The dysregulation caused by the leukemic HOXA9 transcription factor primarily results from its binding activity to DNA, leading to differentiation blockade. Our previous investigations have identified two HOXA9/DNA binding competitors, namely DB1055 and DB818. We assessed their antileukemic effects in comparison to HOXA9 knockdown or cytarabine treatment. Using human AML cell models, DB1055 and DB818 induced in vitro cell growth reduction, death, differentiation, and common transcriptomic deregulation but did not impact human CD34+ bone marrow cells. Furthermore, DB1055 and DB818 exhibited potent antileukemic activities in a human THP-1 AML in vivo model, leading to the differentiation of monocytes into macrophages. In vitro assays also demonstrated the efficacy of DB1055 and DB818 against AML blasts from patients, with DB1055 successfully reducing leukemia burden in patient-derived xenografts in NSG immunodeficient mice. Our findings indicate that inhibiting HOXA9/DNA interaction using DNA ligands may offer a novel differentiation therapy for the future treatment of AML patients dependent on HOXA9.

Arthropod-borne members of the genus Orthoflavivirus cause significant human disease. Four serotypes of dengue virus are endemic globally, and approximately 50 percent of the world's population lives in a dengue-affected area. Complications from immunoenhancement occurring after a secondary infection with a different dengue serotype make vaccine development challenging. Antiviral therapies that target features conserved in all four serotypes would, therefore, be beneficial. Computational studies identified multiple potential G-quadruplex sites that are conserved in the RNA genome sequences of members of the genus Orthoflavivirus. Biophysical studies confirmed that the NS5-B quadruplex sequences obtained from viruses of each dengue serotype can form quadruplexes in vitro, and binding data showed that known quadruplex binders stabilized NS5-B quadruplexes for all four dengue serotypes.

Aberrant transcriptional networks are hallmarks of aging and cancer, yet our ability to target these aberrations is poor. PU.1 is one such transcription factor (TF) who's transcriptional networks are corrupted in disease, including in >50% of Acute Myeloid Leukemia (AML) cases. In this study we investigate an unappreciated pharmacological approach to target the aberrant PU.1 network, by employing novel small molecules which competitively inhibit PU.1:DNA interactions. We performed an extensive multiomics-driven, molecular characterization of AML cells following exposure to PU.1-DNA binding inhibitors (e.g. DB2115). Unexpectedly, we found that such compounds not only led to a reduction of many canonical PU.1 transcripts, but also (seemingly paradoxically) caused increases in alternative PU.1-driven transcripts. We discovered that this two-sided response was a result of robust cistromic repositioning of PU.1 chromatin binding rather than global inhibition. From kinetic CUT&Tag and ATAC sequencing studies, we identified that PU.1 redistribution involved rapid PU.1 loss (~1hr) followed by delayed PU.1 gain (~4-12hrs), causing subsequent opening of the chromatin, thus demonstrating endogenous PU.1's powerful pioneering ability. Through development of CLICK-on-CUT&Tag, we identified exclusive drug binding at displaced PU.1 binding sites, and identified unique, sequence-specific ETS motifs dictating PU.1 gain or loss. By employing these motifs in eGFP reporter assays and CRISPRd systems, we identified direct PU.1-mediated transcriptional control of known and novel functionally relevant genes, including MYC, POMP and STRAP. Finally, we establish that primary AML samples are sensitive to pharmacological PU.1 redistribution upon exposure to DB2115, and the rewiring of the PU.1 network in these cells drives differentiation down the myeloid lineage (Figure 1). Overall, we uncover a novel biological phenomenon we describe as: Pharmacological transcription factor (TF) redistribution. “TF Redistributors”, such as DB2115 and next generation derivatives, are an unprecedented investigative and pharmacological tool which allow the study of complex and fast transcription factor dynamics without disrupting the structure or levels of the TF itself. Furthermore, we discover that pharmacological binding-site restriction and consecutive repositioning of the PU.1 network, cascades into an extensive rewiring of transcriptional circuits which ultimately drives myeloid differentiation of AML cells, potentially providing a novel therapeutic intervention for PU.1-corrupted AML.

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.

Chagas disease (CD) affects over 6 million people worldwide and can be transmitted iatrogenically. Crystal violet (CV) was previously used for pathogen reduction but has harmful side-effects. In the present study, three arylimidamides (AIAs) and CV were used to sterilize mice blood samples experimentally contaminated with bloodstream trypomastigotes (BT) of Trypanosoma cruzi, at non hemolytic doses. All AIAs were not toxic to mouse blood cells until the highest tested concentration (96 µM). The previous treatment of BT with the AIAs impaired the infection establishment of cardiac cell cultures. In vivo assays showed that pre-incubation of mouse blood samples with the AIAs and CV (96 µM) significantly suppressed the parasitemia peak, but only the AIA DB1831 gave ≥90% animal survival, while vehicle treated samples reached 0%. Our findings support further studies regarding the potential use of AIAs for blood bank purposes.

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.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are common forms of adult onset muscular dystrophy. Pathogenesis in both diseases is largely driven by production of toxic-expanded repeat RNAs that sequester MBNL RNA-binding proteins, causing mis-splicing. Given this shared pathogenesis, we hypothesized that diamidines, small molecules that rescue mis-splicing in DM1 models, could also rescue mis-splicing in DM2 models. While several DM1 cell models exist, few are available for DM2 limiting research and therapeutic development. Here, we characterize DM1 and DM2 patient-derived fibroblasts for use in small molecule screens and therapeutic studies. We identify mis-splicing events unique to DM2 fibroblasts and common events shared with DM1 fibroblasts. We show that diamidines can partially rescue molecular phenotypes in both DM1 and DM2 fibroblasts. This study demonstrates the potential of fibroblasts as models for DM1 and DM2, which will help meet an important need for well-characterized DM2 cell models.

The rapid and persistent emergence of drug-resistant bacteria poses a looming public health crisis. The possible task of developing new sets of antibiotics to replenish the existing ones is daunting to say the least. Searching for adjuvants that restore or even enhance the potency of existing antibiotics against drug-resistant strains of bacteria represents a practical and cost-effective approach. Herein, we describe the discovery of potent adjuvants that extend the antimicrobial spectrum of existing antibiotics and restore their effectiveness toward drug-resistant strains including mcr-1-expressing strains. From a library of cationic compounds, MD-100, which has a diamidine core structure, was identified as a potent antibiotic adjuvant against Gram-negative bacteria. Further optimization efforts including the synthesis of ∼20 compounds through medicinal chemistry work led to the discovery of a much more potent compound MD-124. MD-124 was shown to sensitize various Gram-negative bacterial species and strains, including multidrug resistant pathogens, toward existing antibiotics with diverse mechanisms of action. We further demonstrated the efficacy of MD-124 in an ex vivo skin infection model and in an in vivo murine systemic infection model using both wild-type and drug-resistant Escherichia coli strains. MD-124 functions through selective permeabilization of the outer membrane of Gram-negative bacteria. Importantly, bacteria exhibited low-resistance frequency toward MD-124. In-depth computational investigations of MD-124 binding to the bacterial outer membrane using equilibrium and steered molecular dynamics simulations revealed key structural features for favorable interactions. The very potent nature of such adjuvants distinguishes them as very useful leads for future drug development in combating bacterial drug resistance.

The understanding of sequence-specific DNA minor groove interactions has recently made major steps forward and as a result, the goal of development of compounds that target the minor groove is an active research area. In an effort to develop biologically active minor groove agents, we are preparing and exploring the DNA interactions of diverse diamidine derivatives with a 5 '-GAATTC-3 ' binding site using a powerful array of methods including, biosensor-SPR methods, and X-ray crystallography. The benzimidazole-thiophene module provides an excellent minor groove recognition component. A central thiophene in a benzimidazole-thiophene-phenyl aromatic system provides essentially optimum curvature for matching the shape of the minor groove. Comparison of that structure to one with the benzimidazole replaced with an indole shows that the two structures are very similar, but have some interesting and important differences in electrostatic potential maps, the DNA minor groove binding structure based on x-ray crystallographic analysis, and inhibition of the major groove binding PU.1 transcription factor complex. The binding KD for both compounds is under 10 nM and both form amidine Hbonds to DNA bases. They both have bifurcated H-bonds from the benzimidazole or indole groups to bases at the center of the -AATT- binding site. Analysis of the comparative results provides an excellent understanding of how thiophene compounds recognize the minor groove and can act as transcription factor inhibitors.

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.

Aberrant transcriptional networks are a hallmark of cancer, yet our knowledge of the intricacies of transcription factor behavior is poor. PU.1 is an ETS family transcription factor that is essential for hematopoiesis, however more than 50% of AML patients display a disruption of the PU.1 transcriptional network. In this study we implement novel molecular probes to competitively displace PU.1 from canonical DNA binding sites, allowing us to understand PU.1-chromatin binding dynamics, as well as identify the consequences of PU.1 binding site blockade upon PU.1-driven gene transcription and chromatin accessibility. We treated human PU.1low AML cells with a tool PU.1-DNA binding inhibitor, DB2115, and performed PU.1 CUT&Tag, ATAC sequencing and transcriptional profiling. We found, unexpectedly, that DB2115 not only led to inhibition of some canonical PU.1 targets but also mediated concurrent increases of other PU.1 targets. This two-sided response correlated strongly with a robust redistribution of PU.1 chromatin binding rather than a global inhibition - and included losses at regulatory regions of MYC, POMP and gains at CSF1R and TREM2. In fact, most of these redistributed sites (78% of PU.1 gained sites) display subsequent increases in chromatin accessibility and elevated target gene expression, highlighting the pioneering and transcriptional control exerted by repositioned PU.1. Kinetic analyses of PU.1 redistribution reveal that PU.1 losses occur rapidly after DB2115 exposure (1-4hrs), whereas PU.1 gains occur more slowly (4-12hrs) indicative of a loci searching phase prior to novel site binding. Development of an experimental approach combining CLICK-chemistry compound mapping with PU.1 CUT&Tag identified selective drug binding at displaced PU.1 binding sites compared to unchanged/gained sites, with both lost and gained regions being locally enriched for specific and distinct surrounding nucleotide sequences including A/T enrichment. Furthermore, CRISPR-dCas9 blockade and binding site-driven reporter investigations into specific PU.1 cistromic elements revealed important PU.1-mediated direct and rapid control of the POMP, CSF1R and STRAP genes. Overall, PU.1-DNA binding inhibition causes a robust perturbation of PU.1 transcriptional circuits via a novel phenomenon we describe as "Pharmacological transcription factor repositioning". Further investigations with binding site inhibitors such as these represent an unprecedented investigative approach to study complex and fast transcription factor dynamics without disrupting the structure or levels of the factor itself. Furthermore, exploitation of the pharmacological TF repositioning phenomenon may provide novel avenues for therapeutic intervention in PU.1-driven hematologic disorders and other transcriptionally-aberrant diseases.

Pharmacological targeting of aberrant transcriptional networks, such as PU.1 in Acute Myeloid Leukemia, is a highly desirable yet currently unrealized prospect. First-in-class PU.1 binding site inhibitors have been reported with promising therapeutic potential, however the exact molecular and transcriptomic consequences of PU.1 binding site inhibition have not been characterized. We use one of these novel inhibitors, DB2115, as a molecular probe to study PU.1-chromatin binding dynamics, and identify the consequences of PU.1 binding site blockade upon PU.1-driven gene transcription and chromatin accessibility.We found, unexpectedly, that DB2115 not only led to inhibition of expression of some canonical PU.1 targets but also mediated concurrent increases of other PU.1 targets. This two-sided response correlated strongly with a robust redistribution of PU.1 chromatin binding detected by CUT&Tag. Kinetic analyses of PU.1 redistribution reveal that PU.1 losses occur rapidly after DB2115 exposure, whereas PU.1 gains occur more slowly suggestive of a loci searching phase prior to novel site binding. Development of a novel approach combining CLICK-chemistry with PU.1 CUT&Tag identified selective drug binding at displaced PU.1 binding sites compared to unchanged sites, with both lost and gained sites being locally enriched for distinct surrounding nucleotide sequences including A/T enrichment. Furthermore, we have functionally validated the importance of specific gained and lost PU.1 binding sites for driving expression of target genes via CRISPRd and eGFP-reporter assays.Pharmacological transcription factor redistribution via genomic binding site inhibition represents an unprecedented therapeutic and investigative approach, and allows for the study of complex and fast transcription factor dynamics without disrupting the structure or levels of the factor itself.

Dicationic diamidines have been well established as potent antiparasitic agents with proven activity against tropical diseases like trypanosomiasis and malaria. This work presents the synthesis of new mono and diflexible triaryl amidines (6a-c, 13a,b and 17), their aza analogues (23 and 27) and respective methoxyamidine prodrugs (5, 7, 12a,b, 22 and 26). All diamidines were assessed in vitro against Trypanosoma brucei rhodesiense (T. b. r.) and Plasmodium falciparum (P. f.) where they displayed potent to moderate activities at the nanomolar level with IC(50)s = 11-378 nM for T. b. r. and 4-323 nM against P. f.. In vivo efficacy testing against T. b. r. STIB900 has shown the monoflexible diamidine 6c as the most potent derivative in this study eliciting 4/4 cures of infected mice for a treatment period of >60 days upon a 4 x 5 mg/kg dose i. p. treatment. Moreover, thermal melting analysis measurement Delta T-m for this series of diamidines/poly (dA-dT) complexes fell between 0.5 and 19 degrees C with 6c showing the highest binding to the DNA minor groove. Finally, a 50 ns molecular dynamics study of an AT-rich DNA dodecamer with compound 6c revealed a strong binding complex supported by vdW and electrostatic interactions. (C) 2021 Elsevier Masson SAS. All rights reserved.

This report describes a breakthrough in a project to design minor groove binders to recognize any sequence of DNA.

The synthesis of bisnitrile derivatives of benzobisimidazole and bibenzimidazole in a good yield is described in detail for the first time. Nucleophilic substitution of 1,5-difluoro-2,4-dinitrobenzene using different amines produced the intermediate diamines that were reduced using sodium borohydride/Pd(C) to produce the tetramines. These tetramines were allowed to couple with different aldehydes to produce the final benzimidazoles. In a different investigation, these bisnitrile derivatives will be used to make benzimidazole diamidines that could be used as potential mixed sequence minor groove binders.