Iron-based nanoscale coordination polymers synergistically induce immunogenic ferroptosis by blocking dihydrofolate reductase for cancer immunotherapy.

Iron is indispensable for cancer cell survival and cancer cells are more vulnerable to ferroptosis than normal cells. Ferroptosis holds promise for overcoming chemoresistance and inducing tumor immunogenic cell death, which offers new possibilities for cancer immunotherapy. However, the prevalence of immunogenic ferroptosis in cancer cells is diminished because of the high levels of reducing substances within tumor microenvironments. Ferroptosis-needed iron is overdose for livings, which is also an obstacle for effective immune responses. In this study, we construct self-assembled carrier-free nanoscale coordination polymers based on iron and methotrexate (MFe-NCPs). The low-dose-iron-induced immunogenic ferroptosis is obviously enhanced by methotrexate via inhibiting dihydrofolate reductase and abating substance reduction, respectively. Of note, MFe-NCPs sequentially promoted antigen presentation, immune activation, T cell infiltration and boosted the therapeutic effect of immune checkpoint blockade therapy.

Chemotherapeutics for Toxoplasma gondii: Molecular Biotargets, Binding Modes, and Structure-Activity Relationship Investigations.

Toxoplasmosis, an infectious zoonotic disease caused by the apicomplexan parasite Toxoplasma gondii (T. gondii), is a major worldwide health problem. However, there are currently no effective options (chemotherapeutic drugs or prophylactic vaccines) for treating chronic latent toxoplasmosis infection. Accordingly, seeking more effective and safer chemotherapeutics for combating this disease remains a long-term and challenging objective. In this paper, we summarize possible molecular biotargets, with an emphasis on those that are druggable and promising, including, without limitation, calcium-dependent protein kinase 1, bifunctional thymidylate synthase-dihydrofolate reductase, and farnesyl diphosphate synthase. Meanwhile, as important components of medicinal chemistry, the binding modes and structure-activity relationship profiles of the corresponding inhibitors were also illuminated. We anticipate that this information will be helpful for further identification of more effective chemotherapeutic interventions to prevent and treat zoonotic infections caused by T. gondii.

Methotrexate and its mechanisms of action in inflammatory arthritis.

Despite the introduction of numerous biologic agents for the treatment of rheumatoid arthritis (RA) and other forms of inflammatory arthritis, low-dose methotrexate therapy remains the gold standard in RA therapy. Methotrexate is generally the first-line drug for the treatment of RA, psoriatic arthritis and other forms of inflammatory arthritis, and it enhances the effect of most biologic agents in RA. Understanding the mechanism of action of methotrexate could be instructive in the appropriate use of the drug and in the design of new regimens for the treatment of RA. Although methotrexate is one of the first examples of intelligent drug design, multiple mechanisms potentially contribute to the anti-inflammatory actions of methotrexate, including the inhibition of purine and pyrimidine synthesis, transmethylation reactions, translocation of nuclear factor-κB (NF-κB) to the nucleus, signalling via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway and nitric oxide production, as well as the promotion of adenosine release and expression of certain long non-coding RNAs.

In vitro apoptotic effect on human lymphatic filarial parasite by piperidine derivatives and thymidine reversal study.

A novel library of synthetic piperidine derivatives was used to screen against human lymphatic filarial parasite Brugia malayi. Piperidine has earlier been reported to have effect against parasites including rodent filarial nematodes. Compounds with hydroxyl substitutions (4Q and 4H) showed marked antifilarial effect. Molecular docking of 4H derivative showed more favorable thermodynamic parameters against thymidylate synthase of B. malayi than human counterpart. A wide difference between IC50 and LD50 ensured the therapeutic safety of the candidates against the filarial parasites. Addition of thymidine to the treatment regimen led to a significant reversal of antifilarial effect of 4H that confirmed inhibition of thymidylate synthase as pharmacological rationale. Apoptosis induced in the parasite as a consequence of probable inhibition of thymidylate synthase was studied by acridine orange/ethidium bromide fluorescent staining and poly (ADP-ribose) polymerase activity inhibition. Involvement of mitochondria was confirmed by decreased 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) conversion and increased cytosolic cytochrome c level in 4H treated microfilariae, compared with the untreated microfilariae. Moreover, Michael adduct of chalcone targeting dihydrofolate reductase and piperidine targeting thymidylate synthase demonstrated synergistic effect on the parasite, indicating the importance of inhibition of DNA synthesis by combined effect. In conclusion, piperidine derivatives with hydroxyl substitution have a great therapeutic potential with an apoptotic rationale involving mitochondrial pathway, due to possible inhibition of parasitic thymidylate synthase.

Design and synthesis of novel pyrimidine derivatives as potent antitubercular agents.

The emergence of various drug-resistant Mycobacterium tuberculosis (Mtb) strains has necessitated the exploration of new drugs that lack cross-resistance with existing therapeutics. By screening the MedChemExpress bioactive compound library, ceritinib was identified as a compound with activity against Mtb H37Ra. Ceritinib had a MIC value of 9.0 µM in vitro and demonstrated in vivo efficacy in a BALB/c mouse model infected with autoluminescent H37Ra. Then, 32 novel ceritinib derivatives were synthesized, and their antimycobacterial activities were evaluated in vitro. The antimycobacterial activities of the synthesized compounds were drastically affected by substitutions at position 4 of the pyrimidine nucleus and were enhanced by the presence of 2-isopropoxy-5-methyl-4-(piperidin-4-yl)aniline at position 2 of the pyrimidine nucleus. The in vivo antitubercular activities of the three most potent compounds were evaluated. 5-Chloro-N2-(2-isopropoxy-5-methyl-4-(piperidin-4-yl) phenyl)-N4-(naph thalen-1-yl) pyrimidine-2,4-diamine (16j) remarkably reduced the Mtb burden of mice. This result suggested the potential of 16j as a novel drug with superior antitubercular activities. The results of experiments on the combination of sulfamethoxazole with 16j and in silico modeling suggest that dihydrofolate reductase is the potential molecular target of 16j.

Quantification of Cellular Folate Species by LC-MS after Stabilization by Derivatization.

Folate cofactors play a key role in one-carbon metabolism. Analysis of individual folate species is hampered by the low chemical stability and high interconvertibility of folates, which can lead to severe experimental bias. Here, we present a complete workflow that employs simultaneous extraction and stabilization of folates by derivatization. We perform reductive methylation employing stable isotope labeled reagents to retain information on the position and redox state of one-carbon units as well as the redox state of the pteridine ring. The derivatives are analyzed by a targeted LC(HILIC)-MS/MS method without the need for deconjugation, thereby also preserving the glutamation state of folates. The presented method does not only improve analyte coverage and sensitivity as compared to other published methods, it also greatly simplifies sample handling and storage. Finally, we report differences in the response of bacterial and mammalian systems to pharmacological inhibition of dihydrofolate reductase.

Homology modelling, molecular docking, and molecular dynamics simulations reveal the inhibition of Leishmania donovani dihydrofolate reductase-thymidylate synthase enzyme by Withaferin-A.

OBJECTIVE: Present in silico study was carried out to explore the mode of inhibition of Leishmania donovani dihydrofolate reductase-thymidylate synthase (Ld DHFR-TS) enzyme by Withaferin-A, a withanolide isolated from Withania somnifera. Withaferin-A (WA) is known for its profound multifaceted properties, but its antileishmanial activity is not well understood. The parasite's DHFR-TS enzyme is diverse from its mammalian host and could be a potential drug target in parasites. RESULTS: A 3D model of Ld DHFR-TS enzyme was built and verified using Ramachandran plot and SAVES tools. The protein was docked with WA-the ligand, methotrexate (MTX)-competitive inhibitor of DHFR, and dihydrofolic acid (DHFA)-substrate for DHFR-TS. Molecular docking studies reveal that WA competes for active sites of both Hu DHFR and TS enzymes whereas it binds to a site other than active site in Ld DHFR-TS. Moreover, Lys 173 residue of DHFR-TS forms a H-bond with WA and has higher binding affinity to Ld DHFR-TS than Hu DHFR and Hu TS. The MD simulations confirmed the H-bonding interactions were stable. The binding energies of WA with Ld DHFR-TS were calculated using MM-PBSA. Homology modelling, molecular docking and MD simulations of Ld DHFR-TS revealed that WA could be a potential anti-leishmanial drug.

Characterizing the Antimicrobial Activity of N2,N4-Disubstituted Quinazoline-2,4-Diamines toward Multidrug-Resistant Acinetobacter baumannii.

We previously reported a series of N2,N4-disubstituted quinazoline-2,4-diamines as dihydrofolate reductase inhibitors with potent in vitro and in vivo antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) strains. In this work, we extended our previous study to the Gram-negative pathogen Acinetobacter baumannii We determined that optimized N2,N4-disubstituted quinazoline-2,4-diamines are strongly antibacterial against multidrug-resistant A. baumannii strains when the 6-position is replaced with a halide or an alkyl substituent. Such agents display potent antibacterial activity, with MICs as low as 0.5 µM, while proving to be strongly bactericidal. Interestingly, these compounds also possess the potential for antibiofilm activity, eradicating 90% of cells within a biofilm at or near MICs. Using serial passage assays, we observed a limited capacity for the development of resistance toward these molecules (4-fold increase in MIC) compared to existing folic acid synthesis inhibitors, such as trimethoprim (64-fold increase) and sulfamethoxazole (128-fold increase). We also identified limited toxicity toward human cells, with 50% lethal doses (LD50s) of ≤23 µM for lead agents 4 and 5. Finally, we demonstrated that our lead agents have excellent in vivo efficacy, with lead agent 5 proving more efficacious than tigecycline in a murine model of A. baumannii infection (90% survival versus 66%), despite being used at a lower dose (2 versus 30 mg kg-1). Together, our results demonstrate that N2,N4-disubstituted quinazoline-2,4-diamines have strong antimicrobial and antibiofilm activities against both Gram-positive organisms and Gram-negative pathogens, suggesting strong potential for their development as antibacterial agents.

Design, Synthesis, and Biological Evaluation of Coupled Bioactive Scaffolds as Potential Anticancer Agents for Dual Targeting of Dihydrofolate Reductase and Thioredoxin Reductase.

The dihydrofolate reductase (DHFR) and thioredoxin reductase (TrxR) enzymes are involved in the process of tumor cell growth and survival. The 4,6-diamino-1,2-dihydro-1,3,5-triazine scaffold is well-established as a useful scaffold for DHFR inhibition, while chalcones have been reported to be inhibitors of TrxR. In this study, 15 novel compounds designed by the structural combination of the 4,6-diamino-1,2-dihydro-1,3,5-triazine and chalcone scaffolds via a diether linker were successfully synthesized and characterized. All of the compounds demonstrated dual inhibition against DHFR and TrxR when they were assessed by in vitro enzyme assays. The compounds also exhibited antiproliferative activity against the MCF-7 and HCT116 cells. The more potent analogs 14 and 15 were found to inhibit cellular DHFR and TrxR activities in HCT116 cells. Therefore, this study provided compelling evidence that 14 and 15 could exert their anticancer property via multitarget inhibition at the cellular level.

Identifying antimalarial compounds targeting dihydrofolate reductase-thymidylate synthase (DHFR-TS) by chemogenomic profiling.

The mode of action of many antimalarial drugs is unknown. Chemogenomic profiling is a powerful method to address this issue. This experimental approach entails disruption of gene function and phenotypic screening for changes in sensitivity to bioactive compounds. Here, we describe the application of reverse genetics for chemogenomic profiling in Plasmodium. Plasmodium falciparum parasites harbouring a transgenic insertion of the glmS ribozyme downstream of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene were used for chemogenomic profiling of antimalarial compounds to identify those which target DHFR-TS. DHFR-TS expression can be attenuated by exposing parasites to glucosamine. Parasites with attenuated DHFR-TS expression were significantly more sensitive to antifolate drugs known to target DHFR-TS. In contrast, no change in sensitivity to other antimalarial drugs with different modes of action was observed. Chemogenomic profiling was performed using the Medicines for Malaria Venture (Switzerland) Malaria Box compound library, and two compounds were identified as novel DHFR-TS inhibitors. We also tested the glmS ribozyme in Plasmodium berghei, a rodent malaria parasite. The expression of reporter genes with downstream glmS ribozyme could be attenuated in transgenic parasites comparable with that obtained in P. falciparum. The chemogenomic profiling method was applied in a P. berghei line expressing a pyrimethamine-resistant Toxoplasma gondii DHFR-TS reporter gene under glmS ribozyme control. Parasites with attenuated expression of this gene were significantly sensitised to antifolates targeting DHFR-TS, but not other drugs with different modes of action. In conclusion, these data show that the glmS ribozyme reverse genetic tool can be applied for identifying primary targets of antimalarial compounds in human and rodent malaria parasites.

Targeting folate metabolism for therapeutic option: A bioinformatics approach.

Lymphatic filariasis, commonly called elephantiasis, poses a burden of estimated level of 5.09 million disability adjusted life year. Limitations of its sole drug, diethylcarbamazine (DEC) drive exploration of effective filarial target. A few plant extracts having polyphenolic ingredients and some synthetic compounds possess potential dihydrofolate reductase (DHFR) inhibitory effect. Here, we postulated a plausible link between folates and polyphenolics based on their common precursor in shikimate metabolism. Considering its implication in structural resemblance based antagonism, we have attempted to validate parasitic DHFR protein as a target. The bioinformatics approach, in the absence of crystal structure of the proposed target, used to authenticate and for virtual docking with suitable tested compounds, showed remarkably lower thermodynamic parameters as opposed to the positive control. A comparative docking analysis between human and Brugia malayi DHFR also showed effective binding parameters with lower inhibition constants of these ligands with parasitic target, but not with human counterpart highlighting safety and efficacy. This study suggests that DHFR could be a valid drug target for lymphatic filariasis, and further reveal that bioinformatics may be an effective tool in reverse pharmacological approach for drug design.

Protein design algorithms predict viable resistance to an experimental antifolate.

Methods to accurately predict potential drug target mutations in response to early-stage leads could drive the design of more resilient first generation drug candidates. In this study, a structure-based protein design algorithm (K* in the OSPREY suite) was used to prospectively identify single-nucleotide polymorphisms that confer resistance to an experimental inhibitor effective against dihydrofolate reductase (DHFR) from Staphylococcus aureus. Four of the top-ranked mutations in DHFR were found to be catalytically competent and resistant to the inhibitor. Selection of resistant bacteria in vitro reveals that two of the predicted mutations arise in the background of a compensatory mutation. Using enzyme kinetics, microbiology, and crystal structures of the complexes, we determined the fitness of the mutant enzymes and strains, the structural basis of resistance, and the compensatory relationship of the mutations. To our knowledge, this work illustrates the first application of protein design algorithms to prospectively predict viable resistance mutations that arise in bacteria under antibiotic pressure.

Origins of the specificity of inhibitor P218 toward wild-type and mutant PfDHFR: a molecular dynamics analysis.

Molecular dynamics simulations were performed to evaluate the origin of the antimalarial effect of the lead compound P218. The simulations of the ligand in the cavities of wild-type, mutant Plasmodium falciparum Dihydrofolate Reductase (PfDHFR) and the human DHFR revealed the differences in the atomic-level interactions and also provided explanation for the specificity of this ligand toward PfDHFR. The binding free energy estimation using Molecular Mechanics Poisson-Boltzmann Surface Area method revealed that P218 has higher binding affinity (~ -30 to -35 kcal/mol) toward PfDHFR (both in wild-type and mutant forms) than human DHFR (~ -22 kcal/mol), corroborating the experimental observations. Intermolecular hydrogen bonding analysis of the trajectories showed that P218 formed two stable hydrogen bonds with human DHFR (Ile7 and Glu30), wild-type and double-mutant PfDHFR's (Asp54 and Arg122), while it formed three stable hydrogen bonds with quadruple-mutant PfDHFR (Asp54, Arg59, and Arg122). Additionally, P218 binding in PfDHFR is stabilized by hydrogen bonds with residues Ile14 and Ile164. It was found that mutant residues do not reduce the binding affinity of P218 to PfDHFR, in contrast, Cys59Arg mutation strongly favors inhibitor binding to quadruple-mutant PfDHFR. The atomistic-level details explored in this work will be highly useful for the design of non-resistant novel PfDHFR inhibitors as antimalarial agents.

Identification of novel potential antibiotics against Staphylococcus using structure-based drug screening targeting dihydrofolate reductase.

The emergence of multidrug-resistant Staphylococcus aureus (S. aureus) makes the treatment of infectious diseases in hospitals more difficult and increases the mortality of the patients. In this study, we attempted to identify novel potent antibiotic candidate compounds against S. aureus dihydrofolate reductase (saDHFR). We performed three-step in silico structure-based drug screening (SBDS) based on the crystal structure of saDHFR using a 154,118 chemical compound library. We subsequently evaluated whether candidate chemical compounds exhibited inhibitory effects on the growth of the model bacterium: Staphylococcus epidermidis (S. epidermidis). The compound KB1 showed a strong inhibitory effect on the growth of S. epidermidis. Moreover, we rescreened chemical structures similar to KB1 from a 461,397 chemical compound library. Three of the four KB1 analogs (KBS1, KBS3, and KBS4) showed inhibitory effects on the growth of S. epidermidis and enzyme inhibitory effects on saDHFR. We performed structure-activity relationship (SAR) analysis of active chemical compounds and observed a correlative relationship among the IC50 values, interaction residues, and structure scaffolds. In addition, the active chemical compounds (KB1, KBS3, and KBS4) had no inhibitory effects on the growth of model enterobacteria (E. coli BL21 and JM109 strains) and no toxic effects on cultured mammalian cells (MDCK cells). Results obtained from Protein Ligand Interaction Fingerprint (PLIF) and Ligand Interaction (LI) analyses suggested that all of the active compounds exhibited potential inhibitory effects on mutated saDHFR of the drug-resistant strains. The structural and experimental information concerning these novel chemical compounds will likely contribute to the development of new antibiotics for both wild-type and drug-resistant S. aureus.

Propargyl-linked antifolates are dual inhibitors of Candida albicans and Candida glabrata.

Species of Candida, primarily C. albicans and with increasing prevalence, C. glabrata, are responsible for the majority of fungal bloodstream infections that cause morbidity, especially among immune compromised patients. While the development of new antifungal agents that target the essential enzyme, dihydrofolate reductase (DHFR), in both Candida species would be ideal, previous attempts have resulted in antifolates that exhibit inconsistencies between enzyme inhibition and antifungal properties. In this article, we describe the evaluation of pairs of propargyl-linked antifolates that possess similar physicochemical properties but different shapes. All of these compounds are effective at inhibiting the fungal enzymes and the growth of C. glabrata; however, the inhibition of the growth of C. albicans is shape-dependent with extended para-linked compounds proving more effective than compact, meta-linked compounds. Using crystal structures of DHFR from C. albicans and C. glabrata bound to lead compounds, 13 new para-linked compounds designed to inhibit both species were synthesized. Eight of these compounds potently inhibit the growth of both fungal species with three compounds displaying dual MIC values less than 1 µg/mL. Analysis of the active compounds shows that shape and distribution of polar functionality is critical in achieving dual antifungal activity.

Hybrid-drug design targeting Pseudomonas aeruginosa dihydropteroate synthase and dihydrofolate reductase

In this study, we successfully present the dual-target design hypothesis to inhibit both dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) enzymes using a novel scheme that integrates our previous antibiotic-phytochemical interaction data, fragment combination and knowledge-based methods. Both the enzymes are well established antibacterial targets from folate biosynthesis pathway and their synergistic modulation by a single hybrid entity may have profound therapeutic benefits. Evaluation of the designed hybrid compounds based on their physico-chemical properties has indicated them as promising drug candidates with drug-like pharmacotherapeutic profiles. In addition, the stereo-electronic properties such as HOMO, LUMO and MEP maps calculated by quantum chemical methods gave a good correlation with the common pharmacophoric features required for dual-site interactions. Furthermore, docking and dynamics simulation studies reveal that the designed hybrid compounds have favorable binding affinity and stability in both pterin-binding site of DHPS and folate-binding site of DHFR by forming strong hydrogen bonds and hydrophobic interactions with key active-site residues. Looking forward this study could serve as a prospective lead in the process of new natural-product based hybrid-drugs development.

Sulfadiazine resistance in Toxoplasma gondii: no involvement of overexpression or polymorphisms in genes of therapeutic targets and ABC transporters.

Several treatment failures have been reported for the treatment of toxoplasmic encephalitis, chorioretinitis, and congenital toxoplasmosis. Recently we found three Toxoplasma gondii strains naturally resistant to sulfadiazine and we developed in vitro two sulfadiazine resistant strains, RH-R(SDZ) and ME-49-R(SDZ), by gradual pressure. In Plasmodium, common mechanisms of drug resistance involve, among others, mutations and/or amplification within genes encoding the therapeutic targets dhps and dhfr and/or the ABC transporter genes family. To identify genotypic and/or phenotypic markers of resistance in T. gondii, we sequenced and analyzed the expression levels of therapeutic targets dhps and dhfr, three ABC genes, two Pgp, TgABC.B1 and TgABC.B2, and one MRP, TgABC.C1, on sensitive strains compared to sulfadiazine resistant strains. Neither polymorphism nor overexpression was identified. Contrary to Plasmodium, in which mutations and/or overexpression within gene targets and ABC transporters are involved in antimalarial resistance, T. gondii sulfadiazine resistance is not related to these toxoplasmic genes studied.

Exploration of 2, 4-diaminopyrimidine and 2, 4-diamino-s-triazine derivatives as potential antifilarial agents.

In view of the mandate from the World Health Organization (WHO) for developing novel drug candidates against human lymphatic filariasis, dihydrofolate reductase (DHFR) inhibitors are explored as potential antifilarial agents. The in vitro biological evaluation of an in-house library of 12 diverse antifolate compounds with 2,4-diaminopyrimidine and 2,4-diamino-s-triazine structural features against Brugia malayi is reported. To confirm the DHFR inhibitory potential of these compounds, reversal studies using folic acid and folinic acid were undertaken. Inhibition of DHFR can induce apoptosis; in this light, preliminary evidence of apoptosis by test compounds was detected using ethidium bromide-acridine orange staining and the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibition assay. Among the evaluated compounds, 3 showed significant activity against both microfilariae and adult worms. The effects of 2 of these compounds were mostly reversed by folic acid, validating DHFR inhibitory activity. Partial reversal of the effect of 2 compounds by folinic acid and non-reversal of the effect of the third compound both by folic and folinic acids are discussed. This study opens new avenues for the discovery of lead molecules by exploiting the folate pathway against one of the major neglected tropical diseases, filariasis.

Kinetic and structural analysis for potent antifolate inhibition of Pneumocystis jirovecii, Pneumocystis carinii, and human dihydrofolate reductases and their active-site variants.

A major concern of immunocompromised patients, in particular those with AIDS, is susceptibility to infection caused by opportunistic pathogens such as Pneumocystis jirovecii, which is a leading cause of pneumonia in immunocompromised patients. We report the first kinetic and structural data for 2,4-diamino-6-[(2',5'-dichloro anilino)methyl]pyrido[2,3-d]pyrimidine (OAAG324), a potent inhibitor of dihydrofolate reductase (DHFR) from P. jirovecii (pjDHFR), and also for trimethoprim (TMP) and methotrexate (MTX) with pjDHFR, Pneumocystis carinii DHFR (pcDHFR), and human DHFR (hDHFR). OAAG324 shows a 9.0-fold selectivity for pjDHFR (Ki, 2.7 nM) compared to its selectivity for hDHFR (Ki, 24.4 nM), whereas there is only a 2.3-fold selectivity for pcDHFR (Ki, 6.3 nM). In order to understand the determinants of inhibitory potency, active-site mutations of pj-, pc-, and hDHFR were explored to make these enzymes more like each other. The most unexpected observations were that the variant pcDHFR forms with K37Q and K37Q/F69N mutations, which made the enzyme more like the human form, also made these enzymes more sensitive to the inhibitory activity of OAAG324, with Ki values of 0.26 and 0.71 nM, respectively. A similar gain in sensitivity was also observed for the hDHFR N64F variant, which showed a lower Ki value (0.58 nM) than native hDHFR, pcDHFR, or pjDHFR. Structural data are reported for complexes of OAAG324 with hDHFR and its Q35K and Q35S/N64F variants and for the complex of the K37S/F69N variant of pcDHFR with TMP. These results provide useful insight into the role of these residues in the optimization of highly selective inhibitors of DHFR against the opportunistic pathogen P. jirovecii.

Protein interface remodeling in a chemically induced protein dimer.

Although the development of chemically induced, self-assembled protein-based materials is rapidly expanding, methods for directing their assembly in solution are sparse, and problems of population heterogeneity remain. By exerting control over the assembly of advanced protein structures, new classes of ordered protein nanomaterials become feasible, affecting numerous applications ranging from therapeutics to nanostructural engineering. Focusing on a protein-based method for modulating the stability of a chemically induced dihydrofolate reductase (DHFR) dimer, we demonstrate the sensitivity of a methotrexate competition assay in determining the change in DHFR-DHFR binding cooperativity via interfacial mutations over a 1.3 kcal/mol range. This represents a change of more than 40% of the dimer complex binding energy conferred from protein-protein cooperativity (~3.1 kcal/mol). With the development of this investigative system and refinement of protein-based techniques for complex stability modulation, the directed assembly of protein nanomaterials into heterocomplexes and a concomitant decrease in population heterogeneity becomes a realizable goal.