Discovery of Benzisothiazolone Derivatives as Bifunctional Inhibitors of HIV-1 Reverse Transcriptase DNA Polymerase and Ribonuclease H Activities.

The ribonuclease H (RNase H) active site of HIV-1 reverse transcriptase (RT) is the only viral enzyme not targeted by approved antiretroviral drugs. Using a fluorescence-based in vitro assay, we screened 65,239 compounds at a final concentration of 10 µM to identify inhibitors of RT RNase H activity. We identified 41 compounds that exhibited 50% inhibitory concentration (i.e., IC50) values < 1.0 µM. Two of these compounds, 2-(4-methyl-3-(piperidin-1-ylsulfonyl)phenyl)benzo[d]isothiazol-3(2H)-one (1) and ethyl 2-(2-(3-oxobenzo[d]isothiazol-2(3H)-yl)thiazol-4-yl)acetate (2), which both share the same benzisothiazolone pharmacophore, demonstrate robust antiviral activity (50% effective concentrations of 1.68 ± 0.94 µM and 2.68 ± 0.54, respectively) in the absence of cellular toxicity. A limited structure-activity relationship analysis identified two additional benzisothiazolone analogs, 2-methylbenzo[d]isothiazol-3(2H)-one (3) and N,N-diethyl-3-(3-oxobenzo[d]isothiazol-2(3H)-yl)benzenesulfonamide (4), which also resulted in the inhibition of RT RNase H activity and virus replication. Compounds 1, 2 and 4, but not 3, inhibited the DNA polymerase activity of RT (IC50 values~1 to 6 µM). In conclusion, benzisothiazolone derivatives represent a new class of multifunctional RT inhibitors that warrants further assessment for the treatment of HIV-1 infection.

HIV-1 Resistance to Islatravir/Tenofovir Combination Therapy in Wild-Type or NRTI-Resistant Strains of Diverse HIV-1 Subtypes.

Tenofovir disoproxil fumarate (TDF) and islatravir (ISL, 4'-ethynyl-2-fluoro-2'-deoxyadensine, or MK-8591) are highly potent nucleoside reverse transcriptase inhibitors. Resistance to TDF and ISL is conferred by K65R and M184V, respectively. Furthermore, K65R and M184V increase sensitivity to ISL and TDF, respectively. Therefore, these two nucleoside analogs have opposing resistance profiles and could present a high genetic barrier to resistance. To explore resistance to TDF and ISL in combination, we performed passaging experiments with HIV-1 WT, K65R, or M184V in the presence of ISL and TDF. We identified K65R, M184V, and S68G/N mutations. The mutant most resistant to ISL was S68N/M184V, yet it remained susceptible to TDF. To further confirm our cellular findings, we implemented an endogenous reverse transcriptase assay to verify in vitro potency. To better understand the impact of these resistance mutations in the context of global infection, we determined potency of ISL and TDF against HIV subtypes A, B, C, D, and circulating recombinant forms (CRF) 01_AE and 02_AG with and without resistance mutations. In all isolates studied, we found K65R imparted hypersensitivity to ISL whereas M184V conferred resistance. We demonstrated that the S68G polymorphism can enhance fitness of drug-resistant mutants in some genetic backgrounds. Collectively, the data suggest that the opposing resistance profiles of ISL and TDF suggest that a combination of the two drugs could be a promising drug regimen for the treatment of patients infected with any HIV-1 subtype, including those who have failed 3TC/FTC-based therapies.

Development of Human Immunodeficiency Virus Type 1 Resistance to 4'-Ethynyl-2-Fluoro-2'-Deoxyadenosine Starting with Wild-Type or Nucleoside Reverse Transcriptase Inhibitor-Resistant Strains.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA, MK-8591, islatravir) is a nucleoside reverse transcriptase translocation inhibitor (NRTTI) with exceptional potency against wild-type (WT) and drug-resistant HIV-1 in phase III clinical trials. EFdA resistance is not well characterized. To study EFdA resistance patterns that may emerge in naive or tenofovir (TFV)-, emtricitabine/lamivudine (FTC/3TC)-, or zidovudine (AZT)-treated patients, we performed viral passaging experiments starting with WT, K65R, M184V, or D67N/K70R/T215F/K219Q HIV-1. Regardless of the starting viral sequence, all selected EFdA-resistant variants included the M184V reverse transcriptase (RT) mutation. Using recombinant viruses, we validated the role for M184V as the primary determinant of EFdA resistance; none of the observed connection subdomain (R358K and E399K) or RNase H domain (A502V) mutations significantly contributed to EFdA resistance. A novel EFdA resistance mutational pattern that included A114S was identified in the background of M184V. A114S/M184V exhibited higher EFdA resistance (∼24-fold) than either M184V (∼8-fold) or A114S alone (∼2-fold). Remarkably, A114S/M184V and A114S/M184V/A502V resistance mutations were up to 50-fold more sensitive to tenofovir than was WT HIV-1. These mutants also had significantly lower specific infectivities than did WT. Biochemical experiments confirmed decreases in the enzymatic efficiency (kcat/Km) of WT versus A114S (2.1-fold) and A114S/M184V/A502V (6.5-fold) RTs, with no effect of A502V on enzymatic efficiency or specific infectivity. The rather modest EFdA resistance of M184V or A114S/M184V (8- and 24-fold), their hypersusceptibility to tenofovir, and strong published in vitro and in vivo data suggest that EFdA is an excellent therapeutic candidate for naive, AZT-, FTC/3TC-, and especially tenofovir-treated patients.

Conformational Changes in HIV-1 Reverse Transcriptase that Facilitate Its Maturation.

HIV-1 reverse transcriptase (RT) is translated as part of the Gag-Pol polyprotein that is proteolytically processed by HIV-1 protease (PR) to finally become a mature heterodimer, composed of a p66 and a p66-derived 51-kDa subunit, p51. Our previous work suggested that tRNALys3 binding to p66/p66 introduces conformational changes in the ribonuclease (RNH) domain of RT that facilitate efficient cleavage of p66 to p51 by PR. In this study, we characterized the conformational changes in the RNH domain of p66/p66 imparted by tRNALys3 using NMR. Moreover, the importance of tRNALys3 in RT maturation was confirmed in cellulo by modulating the levels of Lys-tRNA synthetase, which affects recruitment of tRNALys3 to the virus. We also employed nonnucleoside RT inhibitors, to modulate the p66 dimer-monomer equilibrium and monitor the resulting structural changes. Taken together, our data provide unique insights into the conformational changes in p66/p66 that drive PR cleavage.

Nanocrystal Formulation Improves Vaginal Delivery of CSIC for HIV Prevention.

5-Chloro-3-phenylsulfonylindole-2-carboxamide (CSIC) is a highly potent non-nucleoside reverse transcriptase inhibitor (NNRTI) with potential for use in topical prophylaxis against HIV transmission. However, the hydrophobic nature of CSIC limits its administration through vaginal route. In this study, we developed nanocrystals of CSIC to potentially improve the aqueous solubility and intracellular uptake of CSIC in vitro and in vivo. CSIC nanocrystals were manufactured and stabilized with Pluronic F98 and hydroxypropyl methylcellulose E5. Transmission electron microscopy showed CSIC nanocrystals to be needle-like. Dynamic light scattering measurements showed a hydrodynamic size of 243 nm (polydispersity index < 0.3) and near neutral surface charge (- 7.8 mV). Particle size was maintained for at least 7 days in the liquid state and for at least 5 months after lyophilization. Drug content in the CSIC nanocrystal formulation (nanosuspension) was 0.8 mg/mL, which is 1000 times higher than the aqueous solubility of CSIC. In vitro release study showed that over 90% of CSIC was released from the nanocrystal formulation in a linear fashion over a period of 4 days. Importantly, CSIC nanocrystals showed equivalent cell-based anti-HIV activity (EC50 ~ 1 nM) as that of non-formulated drug. In vitro studies demonstrated rapid macrophage uptake of CSIC nanocrystals via both energy-dependent (endocytosis) and independent processes. In vivo studies in Swiss Webster female mice showed that the nanocrystal formulation significantly improved CSIC delivery to mouse cervicovaginal tissues following intravaginal instillation. In summary, nanocrystals are a promising formulation approach for topical delivery of CSIC for protection against HIV sexual transmission.

Pharmacophore-based design of novel 3-hydroxypyrimidine-2,4-dione subtypes as inhibitors of HIV reverse transcriptase-associated RNase H: Tolerance of a nonflexible linker.

The pharmacophore of active site inhibitors of human immunodeficiency virus (HIV) reverse transcriptase (RT)-associated RNase H typically entails a flexible linker connecting the chelating core and the hydrophobic aromatics. We report herein that novel 3-hydroxypyrimidine-2,4-dione (HPD) subtypes with a nonflexible C-6 carbonyl linkage exhibited potent and selective biochemical inhibitory profiles with strong RNase H inhibition at low nM, weak to moderate integrase strand transfer (INST) inhibition at low µM, and no to marginal RT polymerase (pol) inhibition up to 10 µM. A few analogues also demonstrated significant antiviral activity without cytotoxicity. The overall inhibitory profile is comparable to or better than that of previous HPD subtypes with a flexible C-6 linker, suggesting that the nonflexible carbonyl linker can be tolerated in the design of novel HIV RNase H active site inhibitors.

6-Arylthio-3-hydroxypyrimidine-2,4-diones potently inhibited HIV reverse transcriptase-associated RNase H with antiviral activity.

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function not targeted by current drugs. Although a few chemotypes have been reported to inhibit HIV RNase H in biochemical assays, their general lack of significant antiviral activity in cell culture necessitates continued efforts in identifying highly potent RNase H inhibitors to confer antiviral activity. We report herein the design, synthesis, biochemical and antiviral evaluations of a new 6-arylthio subtype of the 3-hydroxypyrimidine-2,4-dione (HPD) chemotype. In biochemical assays these new analogues inhibited RT RNase H in single-digit nanomolar range without inhibiting RT polymerase (pol) at concentrations up to 10 µM, amounting to exceptional biochemical inhibitory selectivity. Many analogues also inhibited integrase strand transfer (INST) activity in low to sub micromolar range. More importantly, most analogues inhibited HIV in low micromolar range without cytotoxicity. In the end, compound 13j (RNase H IC50 = 0.005 µM; RT pol IC50 = 10 µM; INST IC50 = 4.0 µM; antiviral EC50 = 7.7 µM; CC50 > 100 µM) represents the best analogues within this series. These results characterize the new 6-arylthio-HPD subtype as a promising scaffold for HIV RNase H inhibitor discovery.

6-Biphenylmethyl-3-hydroxypyrimidine-2,4-diones potently and selectively inhibited HIV reverse transcriptase-associated RNase H.

Human immunodeficiency virus (HIV) reverse transcriptase (RT)-associated ribonuclease H (RNase H) remains an unvalidated drug target. Reported HIV RNase H inhibitors generally lack significant antiviral activity. We report herein the design, synthesis, biochemical and antiviral evaluations of a new 6-biphenylmethyl subtype of the 3-hydroxypyrimidine-2,4-dione (HPD) chemotype. In biochemical assays, analogues of this new subtype potently inhibited RT RNase H in low nanomolar range without inhibiting RT polymerase (pol) or integrase strand transfer (INST) at the highest concentrations tested. In cell-based assays, a few analogues inhibited HIV in low micromolar range without cytotoxicity at concentrations up to 100 µM.

Effect of tRNA on the Maturation of HIV-1 Reverse Transcriptase.

The mature HIV-1 reverse transcriptase is a heterodimer that comprises 66 kDa (p66) and 51 kDa (p51) subunits. The latter is formed by HIV-1 protease-catalyzed removal of a C-terminal ribonuclease H domain from a p66 subunit. This proteolytic processing is a critical step in virus maturation and essential for viral infectivity. Here, we report that tRNA significantly enhances in vitro processing even at a substoichiometric tRNA:p66/p66 ratio. Other double-stranded RNAs have considerably less pronounced effect. Our data support a model where interaction of p66/p66 with tRNA introduces conformational asymmetry in the two subunits, permitting specific proteolytic processing of one p66 to provide the mature RT p66/p51 heterodimer.

Preformulation and Vaginal Film Formulation Development of Microbicide Drug Candidate CSIC for HIV prevention.

PURPOSE: 5-chloro-3-[phenylsulfonyl] indole-2-carboxamide (CSIC) is a highly potent non-nucleoside reverse transcriptase inhibitor (NNRTI) of HIV-1 which has been shown to have a more desirable resistance profile than other NNRTIs in development as HIV prevention strategies. This work involves generation of preformulation data for CSIC and systematic development of a cosolvent system to effectively solubilize this hydrophobic drug candidate. This system was then applied to produce a polymeric thin film solid dosage form for vaginal administration of CSIC for use in prevention of sexual acquisition of HIV. METHODS: Extensive preformulation, formulation development, and film characterization studies were conducted. An HPLC method was developed for CSIC quantification. Preformulation tests included solubility, crystal properties, stability, and drug-excipient compatibility. Cytotoxicity was evaluated using both human epithelial and mouse macrophage cell lines. Ternary phase diagram methodology was used to identify a cosolvent system for CSIC solubility enhancement. Following preformulation evaluation, a CSIC film formulation was developed and manufactured using solvent casting technique. The developed film product was assessed for physicochemical properties, anti-HIV bioactivity, and Lactobacillus biocompatibility during 12-month stability testing period. RESULTS: Preformulation studies showed CSIC to be very stable. Due to its hydrophobicity, a cosolvent system consisting of polyethylene glycol 400, propylene glycol, and glycerin (5:2:1, w/w/w) was developed, which provided a uniform dispersion of CSIC in the film formulation. The final film product met target specifications established for vaginal microbicide application. CONCLUSIONS: The hydrophobic drug candidate CSIC was successfully formulated with high loading capacity in a vaginal film by means of a cosolvent system. The developed cosolvent strategy is applicable for incorporation of other hydrophobic drug candidates in the film platform.

A 2-Hydroxyisoquinoline-1,3-Dione Active-Site RNase H Inhibitor Binds in Multiple Modes to HIV-1 Reverse Transcriptase.

The RNase H (RNH) function of HIV-1 reverse transcriptase (RT) plays an essential part in the viral life cycle. We report the characterization of YLC2-155, a 2-hydroxyisoquinoline-1,3-dione (HID)-based active-site RNH inhibitor. YLC2-155 inhibits both polymerase (50% inhibitory concentration [IC50] = 2.6 µM) and RNH functions (IC50 = 0.65 µM) of RT but is more effective against RNH. X-ray crystallography, nuclear magnetic resonance (NMR) analysis, and molecular modeling were used to show that YLC2-155 binds at the RNH-active site in multiple conformations.

Double-Winged 3-Hydroxypyrimidine-2,4-diones: Potent and Selective Inhibition against HIV-1 RNase H with Significant Antiviral Activity.

Human immunodeficiency virus (HIV) reverse transcriptase (RT)-associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function yet to be exploited as an antiviral target. One of the possible challenges may be that targeting HIV RNase H is confronted with a steep substrate barrier. We have previously reported a 3-hydroxypyrimidine-2,4-dione (HPD) subtype that potently and selectively inhibited RNase H without inhibiting HIV in cell culture. We report herein a critical redesign of the HPD chemotype featuring an additional wing at the C5 position that led to drastically improved RNase H inhibition and significant antiviral activity. Structure-activity relationship (SAR) concerning primarily the length and flexibility of the two wings revealed important structural features that dictate the potency and selectivity of RNase H inhibition as well as the observed antiviral activity. Our current medicinal chemistry data also revealed that the RNase H biochemical inhibition largely correlated the antiviral activity.

Synthesis, biological evaluation and molecular modeling of 2-Hydroxyisoquinoline-1,3-dione analogues as inhibitors of HIV reverse transcriptase associated ribonuclease H and polymerase.

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function not clinically validated as an antiviral target. 2-Hydroxyisoquinoline-1,3-dione (HID) is known to confer active site directed inhibition of divalent metal-dependent enzymatic functions, such as HIV RNase H, integrase (IN) and hepatitis C virus (HCV) NS5B polymerase. We report herein the synthesis and biochemical evaluation of a few C-5, C-6 or C-7 substituted HID subtypes as HIV RNase H inhibitors. Our data indicate that while some of these subtypes inhibited both the RNase H and polymerase (pol) functions of RT, potent and selective RNase H inhibition was achieved with subtypes 8-9 as exemplified with compounds 8c and 9c.

3-Hydroxypyrimidine-2,4-Diones as Novel Hepatitis B Virus Antivirals Targeting the Viral Ribonuclease H.

Hepatitis B virus (HBV) RNase H (RNH) is an appealing therapeutic target due to its essential role in viral replication. RNH inhibitors (RNHIs) could help to more effectively control HBV infections. Here, we report 3-hydroxypyrimidine-2,4-diones as novel HBV RNHIs with antiviral activity. We synthesized and tested 52 analogs and found 4 that inhibit HBV RNH activity in infected cells. Importantly, 2 of these compounds inhibited HBV replication in the low micromolar range.

Structural basis of HIV inhibition by translocation-defective RT inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA).

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is the most potent nucleoside analog inhibitor of HIV reverse transcriptase (RT). It retains a 3'-OH yet acts as a chain-terminating agent by diminishing translocation from the pretranslocation nucleotide-binding site (N site) to the posttranslocation primer-binding site (P site). Also, facile misincorporation of EFdA-monophosphate (MP) results in difficult-to-extend mismatched primers. To understand the high potency and unusual inhibition mechanism of EFdA, we solved RT crystal structures (resolutions from 2.4 to 2.9 Å) that include inhibition intermediates (i) before inhibitor incorporation (catalytic complex, RT/DNA/EFdA-triphosphate), (ii) after incorporation of EFdA-MP followed by dT-MP (RT/DNAEFdA-MP(P)• dT-MP(N) ), or (iii) after incorporation of two EFdA-MPs (RT/DNAEFdA-MP(P)• EFdA-MP(N) ); (iv) the latter was also solved with EFdA-MP mismatched at the N site (RT/DNAEFdA-MP(P)• EFdA-MP(*N) ). We report that the inhibition mechanism and potency of EFdA stem from interactions of its 4'-ethynyl at a previously unexploited conserved hydrophobic pocket in the polymerase active site. The high resolution of the catalytic complex structure revealed a network of ordered water molecules at the polymerase active site that stabilize enzyme interactions with nucleotide and DNA substrates. Finally, decreased translocation results from favorable interactions of primer-terminating EFdA-MP at the pretranslocation site and unfavorable posttranslocation interactions that lead to observed localized primer distortions.

Efficient Inhibition of HIV Replication in the Gastrointestinal and Female Reproductive Tracts of Humanized BLT Mice by EFdA.

BACKGROUND: The nucleoside reverse transcriptase inhibitor (NRTI) 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) in preclinical development exhibits improved safety and antiviral activity profiles with minimal drug resistance compared to approved NRTIs. However, the systemic antiviral efficacy of EFdA has not been fully evaluated. In this study, we utilized bone marrow/liver/thymus (BLT) humanized mice to investigate the systemic effect of EFdA treatment on HIV replication and CD4+ T cell depletion in the peripheral blood (PB) and tissues. In particular, we performed a comprehensive analysis of the female reproductive tract (FRT) and gastrointestinal (GI) tract, major sites of transmission, viral replication, and CD4+ T cell depletion and where some current antiretroviral drugs have a sub-optimal effect. RESULTS: EFdA treatment resulted in reduction of HIV-RNA in PB to undetectable levels in the majority of treated mice by 3 weeks post-treatment. HIV-RNA levels in cervicovaginal lavage of EFdA-treated BLT mice also declined to undetectable levels demonstrating strong penetration of EFdA into the FRT. Our results also demonstrate a strong systemic suppression of HIV replication in all tissues analyzed. In particular, we observed more than a 2-log difference in HIV-RNA levels in the GI tract and FRT of EFdA-treated BLT mice compared to untreated HIV-infected control mice. In addition, HIV-RNA was also significantly lower in the lymph nodes, liver, lung, spleen of EFdA-treated BLT mice compared to untreated HIV-infected control mice. Furthermore, EFdA treatment prevented the depletion of CD4+ T cells in the PB, mucosal tissues and lymphoid tissues. CONCLUSION: Our findings indicate that EFdA is highly effective in controlling viral replication and preserving CD4+ T cells in particular with high efficiency in the GI and FRT tract. Thus, EFdA represents a strong potential candidate for further development as a part of antiretroviral therapy regimens.

Design, Synthesis, and Biological Evaluations of Hydroxypyridonecarboxylic Acids as Inhibitors of HIV Reverse Transcriptase Associated RNase H.

Targeting the clinically unvalidated reverse transcriptase (RT) associated ribonuclease H (RNase H) for human immunodeficiency virus (HIV) drug discovery generally entails chemotypes capable of chelating two divalent metal ions in the RNase H active site. The hydroxypyridonecarboxylic acid scaffold has been implicated in inhibiting homologous HIV integrase (IN) and influenza endonuclease via metal chelation. We report herein the design, synthesis, and biological evaluations of a novel variant of the hydroxypyridonecarboxylic acid scaffold featuring a crucial N-1 benzyl or biarylmethyl moiety. Biochemical studies show that most analogues consistently inhibited HIV RT-associated RNase H in the low micromolar range in the absence of significant inhibition of RT polymerase or IN. One compound showed reasonable cell-based antiviral activity (EC50 = 10 µM). Docking and crystallographic studies corroborate favorable binding to the active site of HIV RNase H, providing a basis for the design of more potent analogues.

3-Hydroxypyrimidine-2,4-diones as Selective Active Site Inhibitors of HIV Reverse Transcriptase-Associated RNase H: Design, Synthesis, and Biochemical Evaluations.

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) remains an unvalidated antiviral target. A major challenge of specifically targeting HIV RNase H arises from the general lack of selectivity over RT polymerase (pol) and integrase (IN) strand transfer (ST) inhibitions. We report herein the synthesis and biochemical evaluations of three novel 3-hydroxypyrimidine-2,4-dione (HPD) subtypes carefully designed to achieve selective RNase H inhibition. Biochemical studies showed the two subtypes with an N-1 methyl group (9 and 10) inhibited RNase H in low micromolar range without significantly inhibiting RT polymerase, whereas the N-1 unsubstituted subtype 11 inhibited RNase H in submicromolar range and RT polymerase in low micromolar range. Subtype 11 also exhibited substantially reduced inhibition in the HIV-1 INST assay and no significant cytotoxicity in the cell viability assay, suggesting that it may be amenable to further structure-activity relationship (SAR) for identifying RNase H inhibitors with antiviral activity.

Nucleotide Sugar Pucker Preference Mitigates Excision by HIV-1 RT.

A series of DNA primers containing nucleotides with various sugar pucker conformations at the 3'-terminus were chemically synthesized by solid-phase synthesis. The ability of wild-type (WT) HIV-1 reverse transcriptase (RT) and AZT-resistant (AZTr) RT to excise the 3'-terminal nucleotide was assessed. Nucleosides with a preference for the North conformation were more refractory to excision by both WT-RT and AZTr-RT. We found that DNA primers that contain North puckered-nucleotides at the 3'-terminus can also affect the translocation status of the RT/template/primer complex, which provides an underlying mechanism to avoid being excised. Together, these results point to a correlation between the sugar conformation of the 3'-terminal nucleotide, the precise position of HIV-1 RT on its nucleic acid substrate, and, in turn, its catalytic function. Nucleotide sugar conformation is therefore an important parameter in defining the susceptibility to RT-catalyzed phosphorolytic excision.

Structural integrity of the ribonuclease H domain in HIV-1 reverse transcriptase.

The mature form of reverse transcriptase (RT) is a heterodimer comprising the intact 66-kDa subunit (p66) and a smaller 51-kDa subunit (p51) that is generated by removal of most of the RNase H (RNH) domain from a p66 subunit by proteolytic cleavage between residues 440 and 441. Viral infectivity is eliminated by mutations such as F440A and E438N in the proteolytic cleavage sequence, while normal processing and virus infectivity are restored by a compensatory mutation, T477A, that is located more than 10 Å away from the processing site. The molecular basis for this compensatory effect has remained unclear. We therefore investigated structural characteristics of RNH mutants using computational and experimental approaches. Our Nuclear Magnetic Resonance and Differential Scanning Fluorimetry results show that both F440A and E438N mutations disrupt RNH folding. Addition of the T477A mutation restores correct folding of the RNH domain despite the presence of the F440A or E438N mutations. Molecular dynamics simulations suggest that the T477A mutation affects the processing site by altering relative orientations of secondary structure elements. Predictions of sequence tolerance suggest that phenylalanine and tyrosine are structurally preferred at residues 440 and 441, respectively, which are the P1 and P1' substrate residues known to require bulky side chains for substrate specificity. Interestingly, our study demonstrates that the processing site residues, which are critical for protease substrate specificity and must be exposed to the solvent for efficient processing, also function to maintain proper RNH folding in the p66/p51 heterodimer.