Abstract RNA and DNA biomarkers serve as diagnostic molecules for detecting a specific disease by a variety of methods and technologies. A common approach is based on a fluorescence readout signal generated after hybridization to a target nucleic acid sequence. One such technology is termed Forced-Intercalation Peptide Nucleic Acid (FIT-PNA). In the FIT-PNA design, one of the nucleobases of the PNA sequence is replaced by a fluorescent molecule termed as “surrogate base.” One of the more common fluorophores explored to date is the cyanine dye, Thiazole Orange (TO). In this report, we have designed TO-based FIT-PNAs that are chemically modified with a cyclopentane backbone (cpTO). In addition, we have introduced to the FIT-PNA design either a cyclopentane T (cpT) or a tetrahydrofuran T (THFT) flanking cpTO. In a model system (11-mer FIT-PNA), we observe a dramatic increase in fluorescence (with DNA or RNA complementary sequences) for both cpT-cpTO and THFT-cpTO FIT-PNAs in comparison to the unmodified counterpart (T-TO FIT-PNA). Moreover, sequence specificity for an RNA sequence with a single mismatch is dramatically improved for both cpT-cpTO and THFT-cpTO FIT-PNAs. Molecular simulations of both cpT-cpTO FIT-PNA and TO (unmodified) FIT-PNA further support the superiority of these chemically modified nucleic-acid probes, as corroborated by a greater π–π stacking of cpTO in the PNA-RNA duplex. Lastly, a cpT-cpTO FIT-PNA targeting the oncogenic long noncoding RNA ANRIL (antisense noncoding RNA in the INK4 locus) was shown to detect this RNA biomarker in ovarian cancer cells (OVCAR-8). This probe was superior to the unmodified TO-based FIT-PNA, highlighting the added value of chemically modified TO FIT-PNAs as means for obtaining highly sensitive and sequence-specific nucleic acid sensors.

Cell penetrating thyclotides (CPTs) are synthetic molecules that promote highly efficient cellular uptake and endosomal escape of bioactive peptides. While peptides are valuable as medicinal agents, their translation to therapies is often limited by their inability to cross cell membranes. CPTs have a unique combination of chiral tetrahydrofurans and polar sidechains within a molecular scaffold that can be optimized to efficiently deliver peptide cargo into cells. The cellular uptake and endosomal escape of two peptides with anticancer biological activities but low bioavailabilities were remarkably improved after conjugation to a CPT. Using CPTs to overcome barriers to cellular uptake represents a new direction for the intracellular delivery of bioactive molecules, and will accelerate drug development for new medical therapies.

Covalent modification of target proteins is a well-established mechanism of action for small molecule inhibitors. Cysteine residues in particular have been exploited for their reactivity toward electrophilic molecules. SAMT-247 is a mercaptobenzamide thioester that covalently acetylates cysteines in the zinc-coordinating domains of the HIV nucleocapsid protein. This SAMT-247-promoted reaction leads to loss of zinc binding by the protein, with concomitant loss of protein structure and function. Although it has low cytotoxicity in animal models, recent studies have indicated that it affects other protein targets in uninfected cells, for example leading to increased immune cell functions. In this study, global proteomics approaches have been used to better understand other protein targets of SAMT-247. Minimal effects are observed when unstimulated THP-1 monocyte cells were treated with SAMT-247. In contrast, thermal proteome profiling identified 170 proteins with altered thermal stability when THP-1 cells were stimulated with phorbol 12-myristate 13-acetate/Ionomycin (PMA/Iono) before SAMT-247 treatment. Among the affected proteins, 81 contain a zinc-coordinating domain and/or have been shown to have a reactive cysteine residue. Among these, several play a role in cellular metabolism, and Seahorse assays demonstrated that SAMT-247 significantly increased the anti-metabolic and pro-glycolytic effect of PMA/Iono in THP-1 cells. Two of the most-affected proteins were ZC3H7A, a microRNA-binding protein with four zinc finger domains, and MGMT, a DNA damage repair protein with a reactive cysteine. Both proteins were modified by SAMT-247 when tested alone or in the presence of THP-1 cell lysate, indicating that they are bona fide targets of the inhibitor. The low activity of SAMT-247 in unstimulated THP-1 cells is consistent with its low cytotoxicity. The increased effects of SAMT-247 in stimulated immune cells suggests that this molecule could be developed to target diseases other than HIV.

Nature relies on nucleobase complementation to store and deploy genetic information. Peptide nucleic acids (PNAs) are nucleic acid analogues widely adopted for their high biological stability and robust sequence-specific nucleobase complementation properties. Here, we report a strategy to modify PNAs, affording bioorthogonal analogues that hybridize to one another without binding to complementary nucleic acids under physiological conditions. Chiral cyclopentane and tetrahydrofuran rings are incorporated into the PNA backbone to promote left-handed helical conformations, opposite to the right-handed helix adopted by DNA and RNA. The binding of left-handed PNAs (LH-PNAs) to DNA and RNA is evaluated using melting temperature (Tm) experiments and circular dichroism (CD) experiments. The binding of LH-PNAs in the presence of right-handed PNA (RH-PNA) and DNA is examined using analytical HPLC. Results suggest that only a few left-handed substitutions at the center of PNA sequences attain bioorthogonal properties. These findings may facilitate the use of LH-PNAs for a range of applications in bioorthogonal chemical space. Peptide nucleic acids (PNAs) are valued for their stability and strong binding to complementary sequences, yet their interactions with natural nucleic acids can limit applications. Here, the authors introduce cyclic chiral modifications to PNAs, creating left-handed analogues that selectively recognize other left-handed complementary PNA strands while avoiding natural DNA and RNA. These left-handed PNAs broaden the toolkit for bioorthogonal applications in chemical biology.

We assess the combined effect of sustained mucosal delivery of the anti-HIV small molecule S-acyl-2-mercaptobenzamide thioester 247 (SAMT-247) via an intravaginal ring (IVR) and ΔV1DNA/ALVAC-SIV/ΔV1gp120/alum (ΔV1-SIV) immunization in female macaques. The combined approach reduces the risk of vaginal SIVmac251 infection by 82.8%, with 58% of animals remaining uninfected following 14 weekly low-dose challenges; about half of the initially protected animals also remain uninfected during a second challenge phase. Compared with a historical vaccine-only cohort, continued IVR use shows a trend toward improved protection. Protection correlates with the expansion of tolerogenic CD73+ dendritic cells, IL-10+ macrophages, IL-17-producing NKp44+ innate lymphoid cells (ILCs), and antibody-mediated natural killer (NK) cytotoxic activity targeting the V2 helical conformation. In vitro studies support ex vivo observations that SAMT-247 enhances gp120-reactive anti-inflammatory mucosal immunity and NK activity. These findings support sustained IVR-based delivery of SAMT-247 combined with V1-deleted immunogens as a promising strategy to prevent vaginal HIV transmission.

Peptide nucleic acids (PNAs) have attracted considerable attention in biomedical research due to their strong binding properties toward complementary oligonucleotides and complete resistance to enzymatic degradation. However, the applications of PNAs may be limited by poor cellular uptake and low water solubility. To this end, we introduced rigid tetrahydrofurans (thfs) into the PNA backbone to develop tetrahydrofuran peptide nucleic acids (thfPNAs) with significant improvements in binding properties, water solubility, and cellular uptake. Herein we describe the protocols for preparing thfPNA monomers, thfPNA oligomers, and methods to study their cellular uptake using FACS experiments.

BackgroundTopical administration of SAMT-247, a mercaptobenzamide thioester zinc finger inhibitor targeting the HIV nucleocapsid Gag zinc finger protein, generates multiple metabolites in mucosal tissues, including Met-A, Met-B, Met-C, and Met-D. Prior studies established that SAMT-247, delivered either as a vaginal gel or via an intravaginal ring (IVR), synergizes with the ΔV1DNA/ALVAC-SIV/ΔV1gp120/alum vaccine regimen to markedly reduce vaginal SIVmac251 acquisition risk. This enhanced protection is associated with augmented protective mucosal immunity and reduced inflammatory responses that promote viral acquisition.MethodsWe performed in vivo characterization of SAMT-247 metabolite distribution in the vaginal compartment of macaques and evaluate the biological activity of individual metabolites using ex vivo rectal mucosal biopsies from vaccinated animals.ResultsHigh concentrations of Met-B, Met-C, and Met-D were detected in vaginal secretions, whereas vaginal tissues contained predominantly Met-D, low levels of Met-A, and no detectable Met-B or Met-C. Functionally, Met-D most closely recapitulated and reinforced the protective immune profile associated with SAMT-247, preserving or expanding IL-17+NKp44+ innate lymphoid cells (ILCs) and CD107a+NKG2A+ natural killer (NK) cells, increasing CD73+ ILCs, NK and dendritic cell as well as IL-10+ dendritic cell and monocyte populations, and reducing inflammatory TNF-α-producing myeloid subsets. Met-B and Met-C partially reproduced this profile, enhancing CD73+ NK/ILC populations and promoting anti-inflammatory myeloid responses, but with more limited effects on antiviral NK/ILC activity and attenuated NKp44+ ILC responses relative to Met-D. In contrast, Met-A, which lacks virucidal activity, failed to induce regulatory CD73+ and IL-10+ responses and diminished both protective IL-17+NKp44+ ILCs and cytolytic CD107a+NKG2A+ NK cells, consistent with inflammatory skewing. In vaccinated macaques receiving SAMT-247-releasing IVRs, plasma Met-A showed limited correlation with rectal mucosal immune responses, partially supporting the ex vivo findings.ConclusionWe observed distinct immunomodulatory effects associated with different SAMT-247 metabolites. These findings may guide future delivery strategies that favor tissue-available Met-D while limiting Met-A accumulation. More broadly, this study underscores the importance of metabolite-specific analyses for defining the biological activity and mechanisms of action of therapeutic agents.

FIT-PNAs (forced intercalation-Peptide Nucleic Acids) are promising RNA sensors due to the enhanced fluorescence gained by such molecules upon RNA hybridization. In this report we describe a chemical approach that leads to unprecedented brightness for a FIT-PNA where the neighbouring Guanine base (G) to the fluorophore (a.k.a. surrogate base) is chemically modified with a cyclopentane (cp) backbone and is N-methylated, leading to a positively charged (G+) base. A series of G modified bases (G+, cpG, and cpG+) were introduced as the neighbouring base to BisQ (surrogate base) in 15-mer FIT-PNAs designed to sense the oncogenic long-noncoding RNA, colon cancer associated transcript 1 (lncRNA CCTA-1). Using synthetic RNA, the combination denoted as cpG+ led to a two-fold increase in brightness (BR = 16.9) compared to the unmodified G base (BR = 8.4). Introducing a G mismatch in RNA sequence that is opposite to the G base (G, G+, cpG, or cpG+) in the FIT-PNA, led to an increase in fluorescence that was not observed for synthetic DNA. Molecular simulations confirmed these observations and further correlated fluorescence data for FIT-PNAs with synthetic DNA and RNA with/out mismatches. Importantly, in ovarian cancer cells overexpressing CCAT1, only the cpG+ modified FIT-PNA produced a bright fluorescent signal, confirmed by FACS and confocal microscopy. Our results demonstrate that strategic chemical modifications of the neighboring G base in FIT-PNA significantly enhance their brightness and specificity for RNA detection in biological systems.

Molecules that inhibit HIV by mechanisms other than inhibition of viral enzymes may be helpful in the treatment of infections where viral resistance has developed. Simple sulfanylbenzamides inactivate HIV by disrupting the coordination of zinc in the viral nucleocapsid protein, resulting in immature and noninfectious virus. HIV nucleocapsid protein (NCp7) is highly conserved across all viral strains, and there is a very high barrier for the virus to develop resistance to sulfanylbenzamide inactivators. These types of molecules act as topical microbicides to prevent HIV infections in animal models, but their systemic use has not been possible due to rapid metabolism in the blood. In this article, we describe a strategy to chemically modify the side chains of sulfanylbenzamides with unique groups to protect the molecules from metabolism while preserving antiviral activity and low toxicity. In vivo pharmacokinetics demonstrates the success of this approach, leading to a new route of systemic administration for this class of molecules.

Nucleic acid detection is commonly used to diagnose pathogenic and genetic diseases. Diagnostics based on polymerase chain reaction (PCR) are routinely used to amplify and signal the presence of a target nucleic acid, but PCR-based methods are difficult to deploy at the patient's point-of-care and in resource-limited environments. This study reports a novel nucleic acid detection assay that utilizes chemically modified PNAs to directly detect a target RNA without enzymatic amplification. By incorporating trans-3,4-diaminotetrahydrofuran units into a PNA backbone, THF-PNAs (also called thyclotides) were designed with both enhanced binding affinity to target nucleic acids and bio-orthogonal properties that promote thyclotide-to-thyclotide binding over nucleic acid binding. Specifically, incorporation of R,R-THF monomers within the thyclotide backbone promotes a right-handed helix favoring binding to target nucleic acids. Bio-orthogonal thyclotide containing S,S-THF monomers promotes a left-handed helix, preventing their binding to natural nucleic acids. Complementary bio-orthogonal thyclotides can bind to each other in the presence of competing RNA sequences, and these unique properties were used in combination with gold nanoparticles to develop a detection signal. By employing both THF stereochemistries in different thyclotide sequences, a prototype microfluidic assay was developed to detect synthetic HIV-1 RNA and signal its presence using silver-based enhancement of surface-bound gold nanoparticles. The limit of detection for this assay was 0.5 pM of synthetic HIV-1 RNA, which is a significant (~100-fold) improvement over earlier PNA-based detection system. Concentration-dependent variation in detection signal intensity allows for semi-quantitative determination of different RNA concentrations. A scrambled RNA control sequence does not interfere with detection. All results were obtained without using enzymatic amplification. The thyclotides in this study may be used in more advanced diagnostic technologies.

The evolution of drug resistance to many antimalarial drugs in the lethal strain of malaria (Plasmodium falciparum) has been a great concern over the past 50 years. Among these drugs, artemisinin has become less effective for treating malaria. Indeed, several P. falciparum variants have become resistant to this drug, as elucidated by specific mutations in the pfK13 gene. This study presents the development of a diagnostic kit for the detection of a common point mutation in the pfK13 gene of P. falciparum, namely, the C580Y point mutation. FIT-PNAs (forced-intercalation peptide nucleic acid) are DNA mimics that serve as RNA sensors that fluoresce upon hybridization to their complementary RNA. Herein, FIT-PNAs were designed to sense the C580Y single nucleotide polymorphism (SNP) and were conjugated to biotin in order to bind these molecules to streptavidin-coated plates. Initial studies with synthetic RNA were conducted to optimize the sensing system. In addition, cyclopentane-modified PNA monomers (cpPNAs) were introduced to improve FIT-PNA sensing. Lastly, total RNA was isolated from red blood cells infected with P. falciparum (WT strain - NF54-WT or mutant strain - NF54-C580Y). Streptavidin plates loaded with either FIT-PNA or cpFIT-PNA were incubated with the total RNA. A significant difference in fluorescence for mutant vs WT total RNA was found only for the cpFIT-PNA probe. In summary, this study paves the way for a simple diagnostic kit for monitoring artemisinin drug resistance that may be easily adapted to malaria endemic regions.

TP53 is commonly mutated in cancer, giving rise to loss of wild-type tumor suppressor function and increases in gain-of-function oncogenic roles. Thus, inhibition of mutant p53 and reactivation of wild-type function represents a potential means to target diverse tumor types. (E)-1-(4-Methylpiperazin-1-yl)-3-(5-nitrofuran-2-yl)prop-2-en-1-one (NSC59984), first identified from a high-throughput screen, induces wild-type p53 signaling and antiproliferative effects while inhibiting mutant p53 gain-of-function activities. Here, we investigate the specific mechanism of action of NSC59984 against p53. We found that NSC59984 reacts with thiols via an unusual Michael addition at the alpha-carbon. Covalent modification of p53 Cys124 and Cys229 was observed both following in vitro reaction and upon treatment of cells. Finally, we used a biotinylated form of NSC59984 and, separately, thermal proteome profiling to examine off-target effects, identifying several metabolic proteins involved in cellular metabolism as potential targets. These results demonstrate that covalent modification of p53 by NSC59984 leads to increased wild-type activity and suggest that potential reaction with metabolic enzymes may contribute to antiproliferative function.

Ovarian cancer (OC) is one of the most lethal gynecologic cancers that is typically diagnosed at the very late stage of disease progression. Thus, there is an unmet need to develop diagnostic probes for early detection of OC. One approach may rely on RNA as a molecular biomarker. In this regard, FLJ22447 lncRNA is an RNA biomarker that is over-expressed in ovarian cancer (OC) and in cancer-associated fibroblasts (CAFs). CAFs appear early on in OC as they provide a metastatic niche for OC progression. FIT-PNAs (forced intercalation-peptide nucleic acids) are DNA analogs that are designed to fluoresce upon hybridization to their complementary RNA target sequence. In recent studies, we have shown that the introduction of cyclopentane PNAs into FIT-PNAs (cpFIT-PNA) results in superior RNA sensors. Herein, we report the design and synthesis of cpFIT-PNAs for the detection of this RNA biomarker in living OC cells (OVCAR8) and in CAFs. cpFIT-PNA was compared to FIT-PNA and the cell-penetrating peptide (CPP) of choice was either a simple one (four L-lysines) or a CPP with enhanced cellular uptake (CLIP6). The combination of CLIP6 with cpFIT-PNA resulted in a superior sensing of FLJ22447 lncRNA in OVCAR8 cells as well as in CAFs. Moreover, incubation of CLIP6-cpFIT-PNA in OVCAR8 cells leads to a significant decrease (ca. 60%) in FLJ22447 lncRNA levels and in cell viability, highlighting the potential theranostic use of such molecules.

Protein phosphatase 1D (PPM1D, Wip1) is induced by the tumor suppressor p53 during DNA damage response signaling and acts as an oncoprotein in several human cancers. Although PPM1D is a potential therapeutic target, insights into its atomic structure were challenging due to flexible regions unique to this family member. Here, we report the first crystal structure of the PPM1D catalytic domain to 1.8 Å resolution. The structure reveals the active site with two Mg2+ ions bound, similar to other structures. The flap subdomain and B-loop, which are crucial for substrate recognition and catalysis, were also resolved, with the flap forming two short helices and three short β-strands that are followed by an irregular loop. Unexpectedly, a nitrogen-oxygen-sulfur bridge was identified in the catalytic domain. Molecular dynamics simulations and kinetic studies provided further mechanistic insights into the regulation of PPM1D catalytic activity. In particular, the kinetic experiments demonstrated a magnesium concentration-dependent lag in PPM1D attaining steady-state velocity, a feature of hysteretic enzymes that show slow transitions compared with catalytic turnover. All combined, these results advance the understanding of PPM1D function and will support the development of PPM1D-targeted therapeutics.

Selective incorporation of conformational constraints into thyclotides can be used to modulate their binding to complementary oligonucleotides, increase polarity, and optimize uptake into HCT116 cells without assistance from moieties known to promote cell uptake. The X-ray structure and biophysical studies of a thyclotide-DNA duplex reveal that incorporation of tetrahydrofurans into an aegPNA backbone promotes a helical conformation that enhances binding to complementary DNA and RNA. Selective incorporation of tetrahydrofurans into the aegPNA backbone allows polarity to be increased incrementally so that uptake into HCT116 cells can be optimized. The enhanced binding, polarity, and cellular uptake properties of thyclotides were used to demonstrate effective inhibition of microRNA-21 in HCT116 cells.

Correction for 'Cyclopentane FIT-PNAs: bright RNA sensors' by Odelia Tepper et al., Chem. Commun., 2021, 57, 540-543, https://doi.org/10.1039/D0CC07400D.

The human immunodeficiency virus epidemic continues in sub-Saharan Africa, and particularly affects adolescent girls and women who have limited access to antiretroviral therapy. Here we report that the risk of vaginal simian immunodeficiency virus (SIV) mac251 acquisition is reduced by more than 90% using a combination of a vaccine comprising V1-deleted (V2 enhanced) SIV envelope immunogens with topical treatment of the zinc-finger inhibitor SAMT-247. Following 14 weekly intravaginal exposures to the highly pathogenic SIV mac251 , 80% of a cohort of 20 macaques vaccinated and treated with SAMT-247 remained uninfected. In an arm of 18 vaccinated-only animals without microbicide, 40% of macaques remained uninfected. The combined SAMT-247/vaccine regimen was significantly more effective than vaccination alone. By analysing immune correlates of protection, we show that, by increasing zinc availability, SAMT-247 increases natural killer cytotoxicity and monocyte efferocytosis, and decreases T-cell activation to augment vaccine-induced protection.

The tumor suppressor p53 is mutated (mt-p53) in over 50% of human cancers causing gain-of-function oncogenic effects, including metabolic changes that reduce tumor responsiveness to radio/chemotherapy. Common hot-spot mutations within the DNA-binding domain can be categorized as conformational (R175H) or DNA binding (R248W). NSC59984 has been characterized as a small molecule that targets mt-p53 for degradation and restores wt-p53 signaling. Using esophageal adenocarcinoma cells and CRISPR generated isogenic cell lines bearing matching hot-spot p53 mutations, we aim to understand how the molecular features of mt-p53 affect drug efficiency and enable the development of targeted therapies to limit cancer cell growth. We found that NSC59984 covalently modifies p53 by Michael addition at cysteine residues 124 and 229, which promote interactions that would stabilize the protein/DNA complex leading to increased p53 transcriptional activity. In cells, the effects of NSC59984 were substantially greater in cells harboring the R248W mutation compared with the R175H mutation. Treatment with NSC59984 reduced proliferation and increased apoptosis via the intrinsic mitochondrial pathway. It also induced changes in OXPHOS, ATP level, mitochondrial membrane potential, glycolysis, and lactate production. Furthermore, treatment of cells with NSC59984 increased reactive oxygen species production and decreased glutathione levels; effects were enhanced by the addition of buthionine sulfoximine and inhibited by N-acetyl cysteine. NSC59984 treatment increased G6PD activity, total NADPH levels, and expression of TIGAR. Knockout of TIGAR partially removed the antiproliferative effects of the drug and reduced G6PD levels in the p53-R248W cells. Incorporation of [13C6] into cellular metabolites suggests that p53-regulated transcription of TIGAR increased utilization of the pentose phosphate pathway and inhibited glycolysis at the fructose-6-P fructose-1,6-bisphosphate junction, supported by an increase in Hexokinase 2 and a decrease of phosphofructokinase-1. Thermal proteome profiling identified TIGAR as an additional reaction target of NSC59984, suggesting increased involvement in modulating these metabolic effects. Combining currently available therapeutic metabolic inhibitors with NSC59984 enhanced the antiproliferative effects in cells harboring p53-R248W creating a therapeutic window when compared to the wt-p53 expressing cells. This suggests these combinations could be used in a clinically relevant setting. Overall, this work has identified a distinctive mode of action for p53 reactivation resulting in not only transcriptional activity, but also a unique effect on cellular energetics. This study shows evidence of variation in responsiveness of different mt-p53 forms and allows the development of specific therapeutics directed to individuals for patient-centered precision medicine. Importantly, we have shown that targeting p53 signaling has significant effects on the metabolic profiles of cancer cells rendering them more vulnerable to neoadjuvant therapy. Citation Format: Kate Brown, Lisa Jenkins, Dan Crooks, Deborah Surman, Sharlyn Mazur, Yuan Xu, Bhargav Arimilli, Ye Yang, Andrew Lane, Stewart Durell, Teresa Fan, David Schrump, Marston Marston, Taylor Ripley, Ettore Appella, Gaelyn Lyons, Andrew Perciaccante, Jerry Dinan, Marco Robello, Herman Nikolayevskiy, Robert O’Connor, Daniel Appella. Targeting mutant p53-R248W reactivates WT p53 function and alters the onco-metabolic profile [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A103.