Competitive Binding of Viral Nuclear Localization Signal Peptide and Inhibitor Ligands to Importin-α Nuclear Transport Protein.

Venezuelan equine encephalitis virus (VEEV) is a highly virulent pathogen whose nuclear localization signal (NLS) sequence from capsid protein binds to the host importin-α transport protein and blocks nuclear import. We studied the molecular mechanisms by which two small ligands, termed I1 and I2, interfere with the binding of VEEV's NLS peptide to importin-α protein. To this end, we performed all-atom replica exchange molecular dynamics simulations probing the competitive binding of the VEEV coreNLS peptide and I1 or I2 ligand to the importin-α major NLS binding site. As a reference, we used our previous simulations, which examined noncompetitive binding of the coreNLS peptide or the inhibitors to importin-α. We found that both inhibitors completely abrogate the native binding of the coreNLS peptide, forcing it to adopt a manifold of nonnative loosely bound poses within the importin-α major NLS binding site. Both inhibitors primarily destabilize the native coreNLS binding by masking its amino acids rather than competing with it for binding to importin-α. Because I2, in contrast to I1, binds off-site localizing on the edge of the major NLS binding site, it inhibits fewer coreNLS native binding interactions than I1. Structural analysis is supported by computations of the free energies of the coreNLS peptide binding to importin-α with or without competition from the inhibitors. Specifically, both inhibitors reduce the free energy gain from coreNLS binding, with I1 causing significantly larger loss than I2. To test our simulations, we performed AlphaScreen experiments measuring IC50 values for both inhibitors. Consistent with in silico results, the IC50 value for I1 was found to be lower than that for I2. We hypothesize that the inhibitory action of I1 and I2 ligands might be specific to the NLS from VEEV's capsid protein.

Binding of viral nuclear localization signal peptides to importin-α nuclear transport protein.

Using all-atom replica-exchange molecular dynamics simulations, we mapped the mechanisms of binding of the nuclear localization signal (NLS) sequence from Venezuelan equine encephalitis virus (VEEV) capsid protein to importin-α (impα) transport protein. Our objective was to identify the VEEV NLS sequence fragment that confers native, experimentally resolved binding to impα as well as to study associated binding energetics and conformational ensembles. The two selected VEEV NLS peptide fragments, KKPK and KKPKKE, show strikingly different binding mechanisms. The minNLS peptide KKPK binds non-natively and nonspecifically by adopting five diverse conformational clusters with low similarity to the x-ray structure 3VE6 of NLS-impα complex. Despite the prevalence of non-native interactions, the minNLS peptide still largely binds to the impα major NLS binding site. In contrast, the coreNLS peptide KKPKKE binds specifically and natively, adopting a largely homogeneous binding ensemble with a dominant, highly native-like conformational cluster. The coreNLS peptide retains most of native binding interactions, including π-cation contacts and a tryptophan cage. While KKPK binding is governed by a complex multistate free energy landscape featuring transitions between multiple binding poses, the coreNLS peptide free energy map is simple, exhibiting a single dominant native-like bound basin. We argue that the origin of the coreNLS peptide binding specificity is several electrostatic interactions formed by the two C-terminal amino acids, Lys10 and Glu11, with impα. The coreNLS sequence is then sufficient for native binding, but none of the amino acids flanking minNLS, including Lys10 and Glu11, are strictly necessary for the native pose. Our analyses indicate that the VEEV coreNLS sequence is virtually unique among human and viral proteins interacting with impα making it a potential target for VEEV-specific inhibitors.

Can Free Energy Perturbation Simulations Coupled with Replica-Exchange Molecular Dynamics Study Ligands with Distributed Binding Sites?

Free energy perturbation coupled with replica exchange with solute tempering (FEP/REST) offers a rigorous approach to compute relative free energy changes for ligands. To determine the applicability of FEP/REST for the ligands with distributed binding poses, we considered two alchemical transformations involving three putative inhibitors I0, I1, and I2 of the Venezuelan equine encephalitis virus nuclear localization signal sequence binding to the importin-α (impα) transporter protein. I0 → I1 and I0 → I2 transformations, respectively, increase or decrease the polarity of the parent molecule. Our objective was three-fold─(i) to verify FEP/REST technical performance and convergence, (ii) to estimate changes in binding free energy ΔΔG, and (iii) to determine the utility of FEP/REST simulations for conformational binding analysis. Our results are as follows. First, our FEP/REST implementation properly follows FEP/REST formalism and produces converged ΔΔG estimates. Due to ligand inherent unbinding, the better FEP/REST strategy lies in performing multiple independent trajectories rather than extending their length. Second, I0 → I1 and I0 → I2 transformations result in overall minor changes in inhibitor binding free energy, slightly strengthening the affinity of I1 and weakening that of I2. Electrostatic interactions dominate binding interactions, determining the enthalpic changes. The two transformations cause opposite entropic changes, which ultimately govern binding affinities. Importantly, we confirm the validity of FEP/REST free energy estimates by comparing them with our previous REST simulations, directly probing binding of three ligands to impα. Third, we established that FEP/REST simulations can sample binding ensembles of ligands. Thus, FEP/REST can be applied (i) to study the energetics of the ligand binding without defined poses and showing minor differences in affinities |ΔΔG| ≲ 0.5 kcal/mol and (ii) to collect ligand binding conformational ensembles.

Binding of Venezuelan Equine Encephalitis Virus Inhibitors to Importin-α Receptors Explored with All-Atom Replica Exchange Molecular Dynamics.

Although Venezuelan equine encephalitis virus (VEEV) is a life-threatening pathogen with a capacity for epidemic outbreaks, there are no FDA-approved VEEV antivirals for humans. VEEV cytotoxicity is partially attributed to the formation of a tetrameric complex between the VEEV capsid protein, the nuclear import proteins importin-α and importin-ß, and the nuclear export protein CRM1, which together block trafficking through the nuclear pore complex. Experimental studies have identified small molecules from the CL6662 scaffold as potential inhibitors of the viral nuclear localization signal (NLS) sequence binding to importin-α. However, little is known about the molecular mechanism of CL6662 inhibition. To address this issue, we employed all-atom replica exchange molecular dynamics simulations to probe, in atomistic detail, the binding mechanism of CL6662 ligands to importin-α. Three ligands, including G281-1485 and two congeners with varying hydrophobicities, were considered. We investigated the distribution of ligand binding poses, their locations, and ligand specificities measured by the strength of binding interactions. We found that G281-1485 binds nonspecifically without forming well-defined binding poses throughout the NLS binding site. Binding of the less hydrophobic congener becomes strongly on-target with respect to the NLS binding site but remains nonspecific. However, a more hydrophobic congener is a strongly specific binder and the only ligand out of three to form a well-defined binding pose, while partially overlapping with the NLS binding site. On the basis of free energy estimates, we argue that all three ligands weakly compete with the viral NLS sequence for binding to importin-α in an apparent compromise to preserve host NLS binding. We further show that all-atom replica exchange binding simulations are a viable tool for studying ligands binding nonspecifically without forming well-defined binding poses.

Testing Precision and Accuracy of an Upper Extremity Proprioceptive Targeting Task Assessment.

Objective: To develop and test an assessment measuring extended physiological proprioception (EPP). EPP is a learned skill that allows one to extend proprioception to an external tool, which is important for controlling prosthetic devices. The current study examines the ability of this assessment to measure EPP in a nonamputee population for translation into the affected population. Design: Measuring precision and accuracy of an upper extremity (UE) proprioceptive targeting task assessment. Participants completed 2 sessions of a targeting task while seated at a table. The targeting was completed with the dominant and nondominant hand and with eyes open and eyes closed during the task. Participants completed 2 sessions of the clinical test with a 1-week washout period to simulate reasonable time between clinical visits. Setting: Research laboratory. Participants: Twenty right-handed participants (N=20) with no neurologic or orthopedic deficits that would interfere with proprioception, median age of 25 years (range, 19-33 years), completed the assessment (10 men, 10 women). Interventions: Not applicable. Main Outcome Measures: Precision (consistency in targeting) and accuracy (distance between the intended target and participant result) in UE targeting task using EPP; test-retest repeatability between sessions. Results: Both precision and accuracy were significantly decreased in the eyes-closed condition compared with the eyes-open condition regardless of targeting with dominant or nondominant hand (all P<.001). In the eyes-open condition, there was a dominance effect relating to the accuracy; however, in the eyes-closed condition, accuracy between dominant and nondominant hands was statistically equivalent. Based on minimum detectable change with 95% confidence, there was no change in either metric between the first and second sessions. Conclusions: The results of this study support the feasibility of using this assessment to measure EPP-based on the definition of EPP as a learned skill that indicates control over an external, simple tool-because they demonstrate reliance on proprioception in the eyes-closed condition, symmetry in proprioceptive accuracy between hands for within-participant control, and test-retest reliability for longitudinal measurements. The results also establish normative values for this assessment in young, healthy adults. Further research is required in a clinical population to evaluate the UE proprioceptive targeting task assessment further and collect objective data on EPP.

Substrate-dependent modulation of the leukotriene A4 hydrolase aminopeptidase activity and effect in a murine model of acute lung inflammation.

The aminopeptidase activity (AP) of the leukotriene A4 hydrolase (LTA4H) enzyme has emerged as a therapeutic target to modulate host immunity. Initial reports focused on the benefits of augmenting the LTA4H AP activity and clearing its putative pro-inflammatory substrate Pro-Gly-Pro (PGP). However, recent reports have introduced substantial complexity disconnecting the LTA4H modulator 4-methoxydiphenylmethane (4MDM) from PGP as follows: (1) 4MDM inhibits PGP hydrolysis and subsequently inhibition of LTA4H AP activity, and (2) 4MDM activates the same enzyme target in the presence of alternative substrates. Differential modulation of LTA4H by 4MDM was probed in a murine model of acute lung inflammation, which showed that 4MDM modulates the host neutrophilic response independent of clearing PGP. X-ray crystallography showed that 4MDM and PGP bind at the zinc binding pocket and no allosteric binding was observed. We then determined that 4MDM modulation is not dependent on the allosteric binding of the ligand, but on the N-terminal side chain of the peptide. In conclusion, our study revealed that a peptidase therapeutic target can interact with its substrate and ligand in complex biochemical mechanisms. This raises an important consideration when ligands are designed to explain some of the unpredictable outcomes observed in therapeutic discovery targeting LTA4H.

Developing a Breast Reconstruction Program in a Resource-Constrained Ghanaian Teaching Hospital: Needs Assessment and Implementation.

ABSTRACT: As the detection of breast cancer in Ghana improves, the incidence of mastectomy has increased and the outcomes have improved. As a secondary result, the need for breast reconstruction is increasing. The cultural hesitation to undergo a mastectomy and live without a breast can be decreased by making breast reconstruction available, cost-effective, and acceptable. Cultural, economic, and technical factors were considered in choosing the best method of breast reconstruction. Discussions, lectures, and cadaver dissections investigated the various reconstructive options. Operative cases were performed using a latissimus musculocutaneous flap, a lower abdominal transverse rectus abdominis myocutaneous (TRAM) flap, and a midabdominal TRAM flap. The midabdominal TRAM was found to be the best choice at Komfo Anokye Teaching Hospital. It is a reliable, robust, well-perfused, single-stage flap that produced excellent patient satisfaction.

Changes in Step Characteristics Over a Known Outdoor Surface Transition: The Effect of Parkinson Disease.

The factors that contribute to the difficulties persons with Parkinson Disease (PwPD) have when negotiating transitions in walking surfaces are not completely known. The authors investigated if PwPD adjusted their step characteristics when negotiating a familiar outdoor surface transition between synthetic concrete and synthetic turf. Force plate and motion capture data were collected for 10 participants with mild to moderate Parkinson disease and 5 healthy older control participants ambulating bidirectionally across the transition between synthetic concrete and synthetic turf. Between groups, PwPD had a significantly higher minimum toe clearance (P = .007) for both directions of travel compared with the healthy control group. Within groups, PwPD significantly increased their hip (P < .001) and ankle (P = .016) range of motion walking from concrete to turf, while the healthy control participants significantly increased their minimum toe clearance (P = .013), margin of stability (P = .019), hip (P < .001) and ankle (P = .038) range of motion, and step length (P < .001). Walking from turf to concrete, both the Parkinson disease group (P = .014) and the healthy control group (P < .001) increased their knee range of motion. Both groups adjusted their step characteristics when negotiating known surface transitions, indicating that surface transitions result in step changes regardless of health status. However, PwPD exhibited overcompensations, particularly in their minimum toe clearance.

Novel, Self-Assembling Dimeric Inhibitors of Human ß Tryptase.

ß-Tryptase, a homotetrameric serine protease, has four identical active sites facing a central pore, presenting an optimized setting for the rational design of bivalent inhibitors that bridge two adjacent sites. Using diol, hydroxymethyl phenols or benzoyl methyl hydroxamates, and boronic acid chemistries to reversibly join two [3-(1-acylpiperidin-4-yl)phenyl]methanamine core ligands, we have successfully produced a series of self-assembling heterodimeric inhibitors. These heterodimeric tryptase inhibitors demonstrate superior activity compared to monomeric modes of inhibition. X-ray crystallography validated the dimeric mechanism of inhibition, and compounds demonstrated high selectivity against related proteases, good target engagement, and tryptase inhibition in HMC1 xenograft models. Screening 3872 possible combinations from 44 boronic acid and 88 diol derivatives revealed several combinations that produced nanomolar inhibition, and seven unique pairs produced greater than 100-fold improvement in potency over monomeric inhibition. These heterodimeric tryptase inhibitors demonstrate the power of target-driven combinatorial chemistry to deliver bivalent drugs in a small molecule form.

Ankle strength, muscle size, and adipose content following unilateral tibiotalar arthrodesis.

Tibiotalar arthrodesis is commonly used to treat end-stage ankle osteoarthritis. Post-operative impairments are often attributed to limited ankle motion. However, whether muscular deficits also exist, thereby potentially contributing to impairments, is unknown. This study aimed to identify post-operative deficits in ankle musculature by examining range of motion, strength (maximum isometric ankle joint torque), and leg composition (cross-sectional area of individual tissue types: bone, subcutaneous adipose, intramuscular adipose, muscle). Ten individuals with unilateral tibiotalar arthrodesis participated. Paired t-tests (p < 0.05) identified differences between the fused and contralateral, control limb. The results indicate that individuals with tibiotalar arthrodesis have profound losses of range of motion and strength. Across participants, range of motion in the fused limb was 53.5 ± 11.7%, 66.8 ± 6.3%, 38.2 ± 18.7%, 37.8 ± 13.6% less than the control for dorsiflexion, plantarflexion, inversion, and eversion, respectively. The largest strength deficit was in dorsiflexion, with the fused limb producing 47.2 ± 9.4% less torque than the control. The quantity and quality of muscle tissue was also negatively affected in individuals following tibiotalar arthrodesis. The total cross-sectional area of the fused limb was 11.4 ± 5.4% smaller than the control limb. This change was primarily due to the 16.1 ± 6.7% decrease in muscle cross-sectional area. However, intramuscular adipose was significantly increased. Although the posterior compartment demonstrated a significant decrease in cross-sectional area, when accounting for differences in total cross-sectional area, the relative sizes of the four muscle compartments were consistent between limbs. This cross-sectional study motivates longitudinal research examining muscular deficits and whether these deficits are reversible with exercise and rehabilitation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Target-Directed Self-Assembly of Homodimeric Drugs Against ß-Tryptase.

Tryptase, a serine protease released from mast cells, is implicated in many allergic and inflammatory disorders. Human tryptase is a donut-shaped tetramer with the active sites facing inward forming a central pore. Bivalent ligands spanning two active sites potently inhibit this configuration, but these large compounds have poor drug-like properties. To overcome some of these challenges, we developed self-assembling molecules, called coferons, which deliver a larger compound in two parts. Using a pharmacophoric core and reversibly binding linkers to span two active sites, we have successfully produced three novel homodimeric tryptase inhibitors. Upon binding to tryptase, compounds reassembled into flexible homodimers, with significant improvements in IC50 (0.19 ± 0.08 µM) over controls (5.50 ± 0.09 µM), and demonstrate good activity in mast cell lines. These studies provide validation for this innovative technology that is especially well-suited for the delivery of dimeric drugs to modulate intracellular macromolecular targets.

Effects of mercury addition on microbial community composition and nitrate removal inside permeable reactive barriers.

Permeable reactive barriers (PRBs) remove nitrogen from groundwater by enhancing microbial denitrification. The PRBs consist of woodchips that provide carbon for denitrifiers, but these woodchips also support other anaerobic microbes, including sulfate-reducing bacteria. Some of these anaerobes have the ability to methylate inorganic mercury present in groundwater. Methylmercury is hazardous to human health, so it is essential to understand whether PRBs promote mercury methylation. We examined microbial communities and geochemistry in fresh water and sulfate-enriched PRB flow-through columns by spiking replicates of both treatments with mercuric chloride. We hypothesized that mercury addition could alter bacterial community composition to favor higher abundances of genera containing known methylating taxa and that the sulfate-rich columns would produce more methylmercury after mercury addition, due mainly to an increase in abundance of sulfate reducing bacteria (SRB). However, methylmercury output at the end of the experiment was not different from output at the beginning, due in part to coupled Hg methylation and demethylation. There was a transient reduction in nitrate removal after mercury addition in the sulfate enriched columns, but nitrate removal returned to initial rates after two weeks, demonstrating resilience of the denitrifying community. Since methylmercury output did not increase and nitrate removal was not permanently affected, PRBs could be a low cost approach to combat eutrophication.

Quadriceps weakness preferentially predicts detrimental gait compensations among common impairments after total knee arthroplasty.

Patients with total knee arthroplasty (TKA) have large deficits in physical performance in comparison to their healthy age-matched peers. Limb asymmetry stemming from less relative load borne by the surgical limb during daily mobility is associated with diminished performance and worsens with greater mobility demands. How common targets of postoperative care, such as muscle weakness, lower limb extension power, residual knee pain, and poor balance confidence can influence asymmetrical limb loading remains unclear. Forty-six patients with unilateral TKA underwent testing of impairments and motion analysis during 10° decline walking at 3 and 6 months postoperatively. At 3 months, only quadriceps femoris strength asymmetry was found to be significantly related to both total support moment (MT ) (ß = 0.431; p < 0.001) and knee extensor moment (MK ) (ß = 0.493; p < 0.001) asymmetry. Again at 6 months, only quadriceps strength asymmetry was related to MT (ß = 0.432; p < 0.001) and MK (ß = 0.534; p < 0.001) asymmetry. Quadriceps strength significantly improved over time in both limbs, however, deficits between limbs remained. Persistent quadriceps weakness is a key factor associated with walking compensation patterns that are limiting the capacity for greater physical performance of patients with TKA. The pronounced asymmetry in limb and knee loading at 3 months remains unchanged until at least 6 months after surgery, and its association with quadriceps strength asymmetry does not substantially change over time. While other factors may also prompt gait compensations, emphasis on improved quadriceps strength should be a focus of efforts to resolve gait compensations and enhance physical performance outcomes. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2355-2363, 2018.

A general model for predicting the binding affinity of reversibly and irreversibly dimerized ligands.

Empirical data has shown that bivalent inhibitors can bind a given target protein significantly better than their monomeric counterparts. However, predicting the corresponding theoretical fold improvements has been challenging. The current work builds off the reacted-site probability approach to provide a straightforward baseline reference model for predicting fold-improvements in effective affinity of dimerized ligands over their monomeric counterparts. For the more familiar irreversibly linked bivalents, the model predicts a weak dependence on tether length and a scaling of the effective affinity with the 3/2 power of the monomer's affinity. For the previously untreated case of the emerging technology of reversibly linking dimers, the effective affinity is also significantly improved over the affinity of the non-dimerizing monomers. The model is related back to experimental quantities, such as EC50s, and the approaches to fully characterize the system given the assumptions of the model. Because of the predicted significant potency gains, both irreversibly and reversibly linked bivalent ligands offer the potential to be a disruptive technology in pharmaceutical research.

Morphometry of the lower lumbar intervertebral discs and endplates: comparative analyses of new MRI data with previous findings.

PURPOSE: Variability of the human lower lumbar geometry is related to complications of disc arthroplasty surgery. Accurate morphometric descriptions are essential for the design of artificial intervertebral discs to ensure good prothesis-vertebra contact and better load distribution, and can improve spinal biomechanics. Unfortunately, current knowledge of the lower lumbar geometry is limited either in the representativeness of sample populations or the accuracy and comprehensiveness of measurements. The objective of this study was to establish an accurate and reliable measurement protocol, provide a comprehensive database of lower lumbar geometry, and compare and summarize geometric data as reported in the literature. METHODS: T2-weighted magnetic resonance imaging (MRI) scans of lower lumbar spine (L3-S1), taken from 109 adult subjects, were anonymized from the digital archive of a local hospital. A total of 318 intervertebral discs and 590 endplates met the inclusion criteria and were studied. Linear and planar measurements were performed using OsiriX software, and analyzed using split plot factorial (SPF) analysis of variance (ANOVA), independent student t tests, paired sample t tests, and Tukey's honest significant difference (HSD) post hoc tests. RESULTS: Excellent intra- and inter-observer reliabilities were achieved using the proposed measurement protocol. The results of this study indicated that male subjects had significantly larger geometric dimensions. L5/S1 discs had the smallest geometric dimensions compared to the discs at other two levels. Significant craniocaudal differences were found in endplate morpohometry. The error associated with using ellipsoid methods was quantified at each lower lumbar level. A large comprehensive database compiling lower lumbar geometry from many studies was established. This study provides geometric data for the female subjects at the L5/S1 level, previously lacking in the literature. CONCLUSION: This study demonstrates the potential of using MRI data to establish a standard measurement protocol for morphometric quantification of the lower lumbar intervertebral discs and vertebral endplates. These results are invaluable in characterizing comprehensive lower lumbar morphometry, which may provide crucial information for planning spinal surgeries, designing artificial intervertebral discs, and for biomechanical modeling of the low lack.

Permeable Reactive Barriers Designed To Mitigate Eutrophication Alter Bacterial Community Composition and Aquifer Redox Conditions.

Permeable reactive barriers (PRBs) consist of a labile carbon source that is positioned to intercept nitrate-laden groundwater to prevent eutrophication. Decomposition of carbon in the PRB drives groundwater anoxic, fostering microbial denitrification. Such PRBs are an ideal habitat to examine microbial community structure under high-nitrate, carbon-replete conditions in coastal aquifers. We examined a PRB installed at the Waquoit Bay National Estuarine Research Reserve in Falmouth, MA. Groundwater within and below the PRB was depleted in oxygen compared to groundwater at sites upgradient and at adjacent reference sites. Nitrate concentrations declined from a high of 25 µM upgradient and adjacent to the barrier to <0.1 µM within the PRB. We analyzed the total and active bacterial communities filtered from groundwater flowing through the PRB using amplicons of 16S rRNA and of the 16S rRNA genes. Analysis of the 16S rRNA genes collected from the PRB showed that the total bacterial community had high relative abundances of bacteria thought to have alternative metabolisms, such as fermentation, including candidate phyla OD1, OP3, TM7, and GN02. In contrast, the active bacteria had lower abundances of many of these bacteria, suggesting that the bacterial taxa that differentiate the PRB groundwater community were not actively growing. Among the environmental variables analyzed, dissolved oxygen concentration explained the largest proportion of total community structure. There was, however, no significant correlation between measured environmental parameters and the active microbial community, suggesting that controls on the active portion may differ from the community as a whole.

Does Dopamine Replacement Medication Affect Postural Sequence Learning in Parkinson's Disease?

Deficits in sequence-specific learning (SSL) may be a product of Parkinson's disease (PD) but this deficit could also be related to dopamine replacement. The purpose of this study was to determine whether dopamine replacement affected acquisition and retention of a standing Continuous Tracking Task in individuals with PD. SSL (difference between random/repeated Root Mean Square Error across trials) was calculated over 2 days of practice and 1 day of retention for 4 groups; 10 healthy young (HY), 10 healthy elders, 10 individuals with PD on, 9 individuals with PD off their usual dosage of dopamine replacement. Improvements in acquisition were observed for all groups; however, only the HY demonstrated retention. Therefore, age appeared to have the largest effect on SSL with no significant effect of medication. Additional research is needed to understand the influence of factors such as practice amount, task difficulty, and dopamine replacement status on SSL deficits during postural tasks.

Reversible linkage of two distinct small molecule inhibitors of Myc generates a dimeric inhibitor with improved potency that is active in myc over-expressing cancer cell lines.

We describe the successful application of a novel approach for generating dimeric Myc inhibitors by modifying and reversibly linking two previously described small molecules. We synthesized two directed libraries of monomers, each comprised of a ligand, a connector, and a bioorthogonal linker element, to identify the optimal dimer configuration required to inhibit Myc. We identified combinations of monomers, termed self-assembling dimeric inhibitors, which displayed synergistic inhibition of Myc-dependent cell growth. We confirmed that these dimeric inhibitors directly bind to Myc blocking its interaction with Max and affect transcription of MYC dependent genes. Control combinations that are unable to form a dimer do not show any synergistic effects in these assays. Collectively, these data validate our new approach to generate more potent and selective inhibitors of Myc by self-assembly from smaller, lower affinity components. This approach provides an opportunity for developing novel therapeutics against Myc and other challenging protein:protein interaction (PPI) target classes.

C-terminal domain of SMYD3 serves as a unique HSP90-regulated motif in oncogenesis.

The SMYD3 histone methyl transferase (HMTase) and the nuclear chaperone, HSP90, have been independently implicated as proto-oncogenes in several human malignancies. We show that a degenerate tetratricopeptide repeat (TPR)-like domain encoded in the SMYD3 C-terminal domain (CTD) mediates physical interaction with HSP90. We further demonstrate that the CTD of SMYD3 is essential for its basal HMTase activity and that the TPR-like structure is required for HSP90-enhanced enzyme activity. Loss of SMYD3-HSP90 interaction leads to SMYD3 mislocalization within the nucleus, thereby losing its chromatin association. This results in reduction of SMYD3-mediated cell proliferation and, potentially, impairment of SMYD3's oncogenic activity. These results suggest a novel approach for blocking HSP90-driven malignancy in SMYD3-overexpressing cells with a reduced toxicity profile over current HSP90 inhibitors.